REPRODUCTIVE BIOLOGY OF THE MARE BASIC AND APPLIED ASPECTS Second Edition, 1992 by O. J. GINTHER, V.M.D., Ph.D. Professor of Veterinary Science Department of Veterinary Science University of Wisconsin—Madison Madison, Wisconsin 53706 Second Edition, 1992 Copyright © by O. J. Ginther Library of Congress Catalog Card No. 91-075595 (Copyright to First Edition, 1979 by O. J. Ginther) 2nd Printing: January 1993 No part of this book may be reproduced Without written permission of the author Neither the author nor publisher assumes any legal responsibility or liability for accuracy or use of the information herein Published and distributed by: Equiservices 4343 Garfoot Road Cross Plains, Wisconsin 53528 USA Phone or Fax: (608) 798—4910 «wins 41" i’; A'fi‘q‘i‘y Lax/l“: Jk‘ UNNERSW: OF PENNSYLVANlA UBRANFS Preface This text and reference book was written especially for research scientists, college students and teachers, and vet— erinarians. The orientation is toward physiology rather than pathology. Most chapters incorporate a discussion of the applied ramifications of the biology cov- ered in the chapter. Maximum assimila- tion of some of the sections would likely require the equivalent of a college level course in reproductive biology. The ref— erence aspects are augmented by the fol— lowing: 1) Parenthetical references to other pages that bear on a given subject are made throughout the text; 2) In addi- tion to the primary and secondary head- ings (listed in the Table of Contents), paragraph headings are also used to aid in locating information; 3) Conceptual diagrams and summaries are used peri— odically; and 4) The bibliography is extensive and appears in a single, alpha- betized list at the end of the book. The conceptual diagrams are shaded for rapid detection and are listed at the end of the Table of Contents. Some of them would be comprehended by individu— als who lack formal training in reproduc- tive biology. It is inherent in the acquisi- tion of knowledge that such conceptual summations will be modified as research proceeds, and some will be discarded. It is hoped that the misguided concepts will quietly fade away and will not remain as historical oddities like the famous "body in sperm" drawings of 300 years ago. In addition to the periodic conceptual diagrams and summations, all chapters are followed by a listing of highlights and, in most instances, a list of mile- stones. The milestones are not intended to be used as indicators of the laborato— ries in which studies have been done, but rather to provide an overall perspective on the chronology of progress in the sub- ject area of each chapter. The extent of the bibliography re— quired that in some instances only mini- mal information on a giVen reference appears in the text. The citations do pro- vide, at the very least, direct lead-ins to the literature. The inevitability that some reports have been overlooked is regretted. Unless by error, every refer- ence is cited in the text. Many potential lines of research, and in some cases spe- cific experiments, are suggested through- out the text. The intended thrust was that new graduate students could use the text to generate a list of suggested exper— imentation. The chapter format is similar to that used in the first edition with one out- standing difference. The previous edition devoted seven pages to reproductive effi- ciency, whereas this edition devotes an entire chapter of 64 pages. This change reflects the progress made in this area by many laboratories during the past decade, as well as an expansion of the interests of our laboratory into embryo loss and twinning. Unless otherwise stated, the vertical bars used in the figures to indicate varia- tion around the means represent stan- dard errors of the means. Also, unless otherwise stated, a directional word (e.g., lower, greater) used to describe research results represents a statistically signifi- cant difference or the stated conclusion of the research workers. Effort was made to avoid disseminating or perpetuating con- cepts or statements that did not seem adequately documented. A capital ”D" is used in day designations when the day of ovulation is the reference point (e.g., Day 0=day of ovulation, Day 10:10 days after ovulation). When the reference day is an event other than ovulation, a small "d" is used (e.g., day 0=day of parturition, day 10:10 days after parturition or other stated event). —- cont. — Appreciation is expressed to: 1) Lisa Kulick for preparation of standardized graphs; 2) Rick Gneiser for prepa- ration of many of the photographs; 3) Drs. G. Adams, D. Bergfelt, E. Carnevale, M. Garcia, and P. Griffin for technical reviews; 4) Karen Baucus, Anita Ginther, Matthew Ginther, and Ellen Porath for editorial reviews; 5) Melinda Hermenet and Veronica McGinnis for general assistance; 6) Jane Ginther and the staff of Equiservices for word processing and typesetting; and 7) Bill Howard and the staff of Imagesetter Inc. for preparation of elec— tronic prepress. Sources of unpublished photographs or data are noted through- out the book. This book is proudly dedicated to the one that I bedazzled, 32 years ago, into becoming my partner and wife. She handled most of those aspects usually handled by a publisher, including the computerized typesetting. She main— tained her composure and upbeat approach throughout 23 grueling months. Even with the barrage of last- minute changes, re-changes, and triple reversals she remained encouraging and cheerful—although toward the end a slight pause was sometimes detected before the cheerfulness became evident. Companion projection slides A companion slide set is available for this book. The slides will enhance oral presentations and are marked to designate the corresponding figures or appropriate pages. CONTENTS CHAPTER 1 REPRODUCTIVE ANATOMY .................................................. 1 1.1. Organization of the Reproductive Tract ....................................... 1 1.2. Suspensory Ligaments ................................................................... 6 1.3. Associations between Reproductive and Digestive Organs ......... 9 1.4. Pelvic Cavity and Perineum .......................................................... 11 1.5. Ovarian Bursa ................................................................................ 12 1.6. Ovaries ............................................................................................ 14 1.7. Oviducts .......................................................................................... 25 1.8. Embryonic Vestiges and Accessory Structures ............................ 26 1.9. Uterus ............................................................................................. 27 1.10. Cervix .............................................................................................. 29 1.11. Vagina ............................................................................................. 30 1.12. Vulva ............................................................................................... 31 1.13. Vessels and Nerves of the Reproductive Tract ............................. 33 1.14. Pituitary and Hypothalamus ......................................................... 36 1.15. Pineal .............................................................................................. 38 Highlights .................................................................................................. 40 CHAPTER 2 REPRODUCTIVE HORMONES .............................................. 41 2.1. Pituitary Gonadotropins (LH and FSH) ...................................... 41 2.2. Prolactin ......................................................................................... 47 2.3. Chorionic Gonadotropin ................................................................. 47 2.4. Gonadotropin Releasing Hormone (GnRH) .................................. 51 2.5. Opioids ............................................................................................ 54 2.6. Inhibin and Related Substances ................................................... 55 2.7. Oxytocin and Related Substances .................. . ............................... 56 2.8. Melatonin ........................................................................................ 57 2.9. RelaXin ............................................................................................ 57 2.10. Prostaglandins ............................................................................... 58 2.1 1. Steroids ........................................................................................... 59 2.11A. General ........................................................................... 59 2.11B. Ovarian Steroids in Nonpregnant Mares ..................... 62 2.110 Steroid Hormones in Pregnant Mares .......................... 66 2.11D. Adrenal Steroids in Mares ............................................ 71 2.12. Hormone Assay .............................................................................. 72 Highlights ................................................................................................... 7 3 Milestones ................................................................................................... 74 vi Contents CHAPTER 3 SEXUAL BEHAVIOR ................................................................. 75 3.1. Problems Associated With Studies of Sexual Behavior ................ 75 3.2. Behavioral Signs ............................................................................ 77 3.3. Incidence of Behavioral Signs ...................................................... 84 3.4. Sexual Behavior in Herds .............................................................. 84 3.5. Intensity of Behavior ..................................................................... 89 3.6. Overt and Covert Estrus ................................................................ 90 3.7. Causes of Diminished Estrus ........................................................ 91 3.8. Physiologic Control ........................................................................ 93 3.8A. Pheromones and Other Stimuli ..................................... 93 3.8B. Flehmen .......................................................................... 94 3.8C. Unseasonal Sexual Behavior ......................................... 95 3.8D. Hormones ........................................................................ 97 3.9. Teasing Techniques ....................................................................... 99 Highlights ................................................................................................. 104 CHAPTER 4 REPRODUCTIVE SEASONALITY ......................................... 105 4.1. Annual Distribution of Ovulations ................................................ 106 4.2. Annual Distribution of Periods of Estrous Behavior .................. 108 4.3. Reproductive Seasonality in Other Equids .................................. 108 4.4. Factors Affecting Reproductive Seasonality ................................. 110 4.4A. Length of Photoperiod and Type of Mare ...................... 110 4.4B. Latitude ........................................................................... 110 4.4C. Age ................................................................................... 111 4.5. Mechanisms for Control of Reproductive Seasonality ................. 112 4.5A. Experimentation on the Effects of Light ....................... 113 4.5B. Neuropathway from Eye to Pineal ................................ 117 4.5C. Role of the Pineal Gland ................................................ 118 4.5D. Role of Hypothalamus .................................................... 120 4.5E. Role of the Pituitary ....................................................... 121 4.6. Role of Other Extrinsic Factors ..................................................... 126 4.7 . Role of Intrinsic Factors ................................................................ 127 4.8. Hair Coat and Reproductive Seasonality ..................................... 128 4.9. Seasonal Infertility ........................................................................ 129 4.9A. The Physiologic Problem ................................................ 129 4.9B. The Official Birth Date Problem .................................... 131 Highlights ................................................................................................. 133 Milestones ................................................................................................. 134 Contents vii CHAPTER 5 ANOVULATORY SEASON ....................................................... 135 5.1. Overview ......................................................................................... 136 5.1A. Sexual Behavior .............................................................. 136 5.1B. Follicular Dynamics ....................................................... 136 5.2. Receding Phase .............................................................................. 140 5.2A. Follicular Dynamics ....................................................... 140 5.2B. Endocrinology ................................................................. 140 5.3. Inactive Phase ................................................................................ 141 5.3A. Follicles and Tubular Genitalia ..................................... 141 5.3B. Endocrinology of the Inactive Phase ............................. 145 5.4. Resurging Phase ............................................................................ 146 5.4A. Follicular Dynamics ....................................................... 146 5.4B. Endocrinology of Resurgence ......................................... 148 5.40. Late Resurging Phase .................................................... 155 5.5. Applied Techniques for Induction of Ovulation ........................... 158 5.5A. Use of Artificial Lights ................................................... 158 5.5B. Stabling ........................................................................... 161 5.50. Nutrition ......................................................................... 161 5.5D. Progestin Treatment ...................................................... 163 5.5E. Gonadotropic Preparations ............................................ 165 5.5F. GnRH Treatment ............................................................ 166 5.5G. Other Treatments ........................................................... 170 Highlights ................................................................................................. 17 1 Milestones ................................................................................................. 172 CHAPTER 6 CHARACTERISTICS OF THE OVULATORY SEASON .. 173 6.1. Estrous Cycles ................................................................................ 173 6.1A. Length ............................................................................. 173 6.1B. Seasonal Changes ........................................................... 174 6.10. Interovulatory Intervals................. ............................... 175 6.2. Follicular Dynamics ....................................................................... 176 6.2A. Underlying Follicular Activity ....................................... 17 6 62B. The Wave Phenomenon ................................................. 178 6.2C. Major Secondary Wave ................................................... 17 8 6.2D. Primary Wave ................................................................. 180 6.2E. Characteristics of a Major Wave ................................... 182 6.2F. Effects of Month ............................................................. 183 viii Contents 6.3. Selection of the Dominant Follicle ................................................ 185 6.3A. Atresia ............................................................................. 186 6.3B. Time of Selection ............................................................ 186 6.30 Methods of Study ............................................................ 186 6.4. The Preovulatory Period ................................................................ 189 6.5. Ovulation ........................................................................................ 190 6.5A. Inequality in the Side of Ovulation ............................... 190 6.5B Time of Ovulation in Relation to Estrus ....................... 190 6.5C. Nature of the Ovulatory Process ................................... 191 6.5D. Clinical Prediction of the Imminence of Ovulation ...... 192 6.5E. Follicle Evacuation ......................................................... 193 6.6. Luteal Glands ................................................................................. 195 6.6A. Detecting Ovulation ....................................................... 195 6.6B. Transrectal Palpation Characteristics .......................... 197 6.6C. Ultrasonic Morphology ................................................... 197 6.6D. Microscopic Characteristics ........................................... 199 6.6E. Gross Characteristics ..................................................... 200 6.7 . Tubular Genitalia .......................................................................... 207 6.7A. Histologic Changes ......................................................... 208 6.7B. Cyclic Changes Detectable by Transrectal Palpation .. 209 6.7C. Cyclic Changes Detectable by Visual Inspection .......... 211 6.7 D. Changes in Ultrasonic Morphology ............................... 212 6.7E. Cyclic Secretory Activity ................................................ 214 6.7F. Contractions .................................................................... 215 6.8. Ovarian Irregularities ................................................................... 217 6.8A. Multiple Ovulations ....................................................... 218 6.8B. Diestrous Ovulations ...................................................... 223 6.8C. Failure of Ovulation ....................................................... 224 6.8D. Hemorrhagic Follicles .................................................... 224 6.8E. Prolonged Luteal Activity .............................................. 227 6.9. Horses Versus Ponies ..................................................................... 230 Highlights ................................................................................................. 231 Milestones .......................................................... ‘ ....................................... 232 CHAPTER 7 ENDOCRINOLOGY OF THE OVULATORY SEASON ..... 233 7.1. Concentrations of Hormones ......................................................... 233 7.1A. Luteinizing Hormone ..................................................... 233 7.13. Follicle Stimulating Hormone ....................................... 235 7 . 1C. Progesterone ................................................................... 237 7 . 1D. Estrogens ........................................................................ 241 Contents ix 7 . 1E. Androgens ....................................................................... 242 7.1F. Cortisol ............................................................................ 244 7 . 1G. Prostaglandins ................................................................ 244 7.1H. Inhibin ............................................................................. 245 7.2. Regulation of Gonadotropins ......................................................... 246 7.2A. Temporal Relationships Among Hormones .................. 246 7 .2B. Experiments on Altering Circulating Levels of LH ...... 248 7.2C. Experiments on Altering Circulating Levels of FSH 250 7 .2D. Role of GnRH .................................................................. 252 7.2E. Interactions of Ovaries and Season ............................... 254 7.3. Regulation of the Follicles and Ovulation ................................... 257 7.3A. Preantral and Intrafollicular Controls .......................... 257 7.3B. Experiments on Follicle Suppression ............................ 258 7 .3C. Experiments on Follicle Stimulation ............................. 260 7.3D. Selection of Dominant Follicles ..................................... 262 7.3E. Temporal Relationships ................................................. 263 7.4. Regulation of the Corpus Luteum ................................................. 265 7.4A. Role of Gonadotropins .................................................... 265 7 .4B. Role of the Uterus ........................................................... 266 7 .4C. Local versus Systemic Uteroluteal Pathways ............... 268 7 .4D. Role of Prostaglandin onc .............................................. 270 7.4E. Mechanism of PGan Production ................................... 274 7.5. Regulation of Tubular Genitalia ................................................... 276 7.6. Artificial Control of the Estrous Cycle .......................................... 278 7 .6A. Induction of Ovulation during Estrous ......................... 279 7.6B. Delaying Ovulation until Release from Treatment ...... 282 7 .6C. Termination of Luteal Phase With Prostaglandins ...... 284 7 .6D. Implementation of Cycle Control Programs ................. 287 Highlights ................................................................................................. 289 Milestones .................................................................. ‘ ............................... 290 CHAPTER 8 MATERNAL ASPECTS OF PREGNANCY .......................... 291 8.1. Oogenesis ........................................................................................ 291 8.2. Natural Mating .............................................................................. 296 8.2A. Number of Mares Mated per Stallion per Season ........ 296 8.2B. Optimal Time .................................................................. 296 8.2C. Deposition and Transport of Sperm in Mare Genitalia 301 8.3. Transport of Ova in Oviducts ........................................................ 302 8.3A. Time of Entry into Uterus .............................................. 302 8.3B. Retention of Unfertilized Ova in Oviducts .................... 303 x Contents 8.4. The Uterus and Physical Embryo-Uterine Interactions .............. 305 8.4A. Embryo Mobility ............................................................. 305 8.4B. Fixation ........................................................................... 309 8.4C. Orientation ...................................................................... 312 8.4D. Uterine Contractions ...................................................... 313 8.4E. Uterine Tone ................................................................... 314 8.4F. Endometrial Histology ................................................... 317 8.4G. Uterine Secretions .......................................................... 317 8.5. The Embryonic Bulge .................................................................... 320 8.6. Estrous Behavior During Pregnancy ............................................ 321 8.7. Maternal Ovaries ........................................................................... 321 8.7A. Follicular Dynamics ....................................................... 322 8.7 B. Corpora Lutea ................................................................. 324 8.8. Gestation Length ........................................................................... 329 8.9. Pregnancy Diagnosis ...................................................................... 330 8.10. Equine Biotechnology: Gametes and Embryos ............................. 333 8.1OA. Artificial Insemination .................................................. 333 8.10B. Assisted Fertilization .................................................... 335 8.10C. Embryo Transfer ............................................................ 337 8.10D. Preservation and Transportation of Embryos .............. 339 8.10E. Predetermining Gender ................................................. 340 8.10F. Genetic Engineering ...................................................... 341 Chronology .................................................................................. 342 Highlights ................................................................................................. 343 Milestones ................................................................................................. 344 CHAPTER 9 EMBRYOLOGY AND PLACENTATION ............................... 345 9.1. Terminology: Embryo and Fetus ................................................... 345 9.2. Fertilization .................................................................................... 347 9.3. Cleavage or Oviductal Stage (Days 1 to 5) ................................... 348 9.4. The Blastocyst Stage (Days 6 to 10) .............................................. 350 9.5. The Capsule .................................................................................... 352 9.6. The Yolk—Sac Stage (Days 11 to 21) .............................................. 364 9.7. Transition From Yolk Sac to Allantoic Sac (Days 21 to 40) ......... 367 9.8. Endometrial Cups .......................................................................... 370 9.8A. Origin of the Endometrial Cups .................................... 370 9.8B. Morphogenesis ................................................................ 372 9.8C. Histology of Cups and Pouches ...................................... 373 Contents 9.9. Amnion ........................................................................................ 377 9.9A. Embryonic Amnion ......................................................... 377 9.9B. Fetal Amnion ................................................................. 37 7 9.10. Fetal-Maternal Attachment .......................................................... 380 9.10A. Early Attachment ........................................................ 380 9.10B. Microcotyledons and Absorptive Arcades .................. 381 9.10C. The Mature Microplacentomes ................................... 386 9.10D. The Absorptive Arcade ................................................ 391 9.11. Fetus ............................................................................................... 392 9.11A. External Reproductive Organs ................................... 392 9.11B. Other External Features ............................................. 393 9.11C. Fetal Gonads ................................................................ 397 9.11D. Fetal Ovary .................................................................. 400 9.11E. Tubular Genitalia ........................................................ 404 9.11F. Pituitary Gland .......................................................... 406 9.12. Fetal Mobility and Activity ............................................................ 408 9.12A. Overview of Conceptus and Fetal Kinetics ................ 408 9.12B. Earlier Studies ............................................................ 409 9.12C. Recent Studies ............................................................. 410 9.13. Other Aspects of Conceptus Development .................................... 413 9.13A. Factors Affecting Growth of Embryonic Vesicle ........ 413 9.13B. Other Aspects of Fetal Development .......................... 414 9.13C. Hippomanes ................................................................. 415 9.13D. Reproductive Immunology .......................................... 416 Highlights ................................................................................................... 417 Milestones ................................................................................................... 418 CHAPTER 10 ENDOCRINOLOGY OF PREGNANCY ................................. 419 10.1. Hormone Concentrations ............................................................... 419 10.1A. Chorionic Gonadotropin ............................... 419 10.1B. Luteinizing Hormone .................................................. 422 10.1C. Follicle Stimulating Hormone .................................... 423 10.1D. Progestins .................................................................... 424 10.1E. Testosterone ................................................................. 427 10.1F. Estrogens ..................................................................... 427 10.1G. RelaXin ......................................................................... 431 10.1H. Prolactin ....................................................................... 432 10.2. Survival Mechanisms Initiated by the Conceptus ....................... 432 xi xii Contents 10.3. Roles of CG ..................................................................................... 434 10.4. Sources and Roles of Luteal Progesterone .................................... 436 10.5. Regulation of the Corpora Lutea ................................................... 438 10.5A. First Luteal Response to Pregnancy .......................... 438 10.5B. Interval between First and Second Luteal Response .................................................................. 443 10.5C. Second Luteal Response to Pregnancy ....................... 443 10.5D. Third Luteal Response to Pregnancy ......................... 444 10.6. Sources and Roles of Estrogens ..................................................... 446 10.7. Regulation of Follicles ................................................................... 448 10.7 A. Before CG Production .................................................. 448 10.7B. During CG Production ................................................ 449 10.7C. Effect of Progestins on Follicles .................................. 450 10.8. Elective Induction of Abortion ....................................................... 450 10.9. Seasonal Effects on Ovaries .......................................................... 452 Highlights .................................................................................................. 454 Milestones .................................................................................................. 456 CHAPTERll PARTURITION, PUERPERIUM, AND PUBERTY ............ 457 11.1. Characteristics of Parturition ....................................................... 457 11.1A. Predicting the Imminence of Parturition ................... 457 11.1B. Nocturnal Parturition ................................................. 458 11.1C. The Three Stages of Labor .......................................... 459 11.1D. Myometrial Activity .................................................... 461 11.2. The Discharged Placenta ............................................................... 462 11.3. The Endocrinology of Parturition .................................................. 465 11.3A. Adrenal Cortical Hormones ........................................ 465 11.33 Progestins and Estrogens ........................................... 467 11.3C. Prostaglandin F20c ........................................................ 469 11.3D. Oxytocin ....................................................................... 470 11.3E. Prolactin ....................................................................... 471 11.4. Induction of Parturition ................................................................. 473 11.5. Puerperium ..................................................................................... 475 11.5A. First Postpartum Estrus and Ovulation .................... 476 11.5B. Effects of Nursing ........................................................ 478 11.5C. Postpartum Endocrinology ......................................... 479 11.5D. Uterine Involution ....................................................... 481 11.5E. Artificial Control of Postpartum Ovulation ............... 487 Contents xiii 11.6. Puberty ........................................................................................... 488 11.6A. Morphology of Developing Ovaries ............................. 488 11.6B. Age of Puberty ............................................................. 490 11.6C. Gonadotropins ............................................................. 492 Highlights .................................................................................................. 497 Milestones .................................................................................................. 498 CHAPTER 12 REPRODUCTIVE EFFICIENCY ............................................. 499 12.1. Terminology .................................................................................... 500 12.2. Measures of Efficiency ................................................................... 502 122A. Fertilization Rate ........................................................ 502 12.2B. Pregnancy Rate ........................................................... 503 12.2C. Foaling Rate ................................................................ 508 12.3. Pregnancy Loss .............................................................................. 509 12.3A. Overall Pregnancy—Loss Rate ..................................... 510 12.3B. Time of Occurrence ...................................................... 511 12.3C. Role of Salpingitis in Pregnancy Loss ........................ 518 12.3D. Role of Uterine Inflammation and Fibrosis ............... 519 12.3E. Luteal Progesterone and Pregnancy Loss .................. 525 12.3F. Pseudopregnancy ......................................................... 528 12.3G. Role of Embryo Defects ............................................... 536 12.3H. Other Aspects of Pregnancy Loss ............................... 538 12.3 I. Etiology of Late Fetal Loss ......................................... 545 12.4. Twins .............................................................................................. 546 12 4A Incidence ...................................................................... 546 12 4B Origin of Twins ............................................................ 546 12 4C Early Development of Twins ...................................... 548 12.4D. Embryo Reduction ....................................................... 550 12.4E. Management of Twin Embryos ................................... 554 12.4F. Fetal Stage Twms ............................... 558 Highlights .................................................................................................. 560 Milestones .................................................................................................. 562 BIBLIOGRAPHY ..................................................................................................... 563 xiv Contents CONCEPTUAL DIAGRAMS and SUMNIARIES Hypothalamic-Pituitary Portal System .......................................................... 52 Sources of Progestins during Pregnancy ........................................................ 70 Effects of Photoperiod ...................................................................................... 117 Pathway from Photoperiod to Ovaries ............................................................ 125 Endocrinology of Anovulatory Season ............................................................ 156 Practical Considerations of Lighting Programs ............................................. 162 Folliculogenesis ................................................................................................ 184 Selection Mechanism ....................................................................................... 188 Hormonal Control of Reproductive Organs .................................................... 233 Gonadotropin Control by Ovarian-Seasonal Interactions ............................. 256 Regulation of Follicles during the Estrous Cycle ........................................... 264 Luteal Regulation ............................................................................................ 272 Mechanism of PGan Release .......................................................................... 277 Temporal Relationships among Hormonal and Ovarian Events .................. 288 Continuity of Life in the Horse ....................................................................... 292 Embryo—Uterine Interactions .......................................................................... 319 Ovarian Dynamics during Pregnancy ............................................................. 328 Fertilization ...................................................................................................... 347 Outer Layers of Ovum and Conceptus ............................................................ 352 Mobility of Fetal-Amniotic Unit ...................................................................... 411 Species Differences in Conceptus-Endometrial Contact ................................ 439 Luteal Progesterone Output during Pregnancy ............................................. 445 Estrogen sources in Early Pregnancy ............................................................. 447 Endocrinology of Pregnancy ............................................................................ 454 Control of Parturition ...................................................................................... 472 Gonadotropins from Birth to Puberty ............................................................. 496 Time of Occurrence of Pregnancy Failures ..................................................... 517 Embryo Loss following Luteolysis ................................................................... 523 Causes of Embryo Loss .................................................................................... 535 Perpetuation of Uterine Events in Aged Mares ............................................. 541 The Deprivation Hypothesis ............................................................................ 553 The Twinning Tree ........................................................................................... 557 —— Cfiapter 1— REPRODUCTIVE ANATOMY Dynamic physiology demands dynamic morphology. Despite this fundamental principle of animal biology, the remark is sometimes made that anatomy is a dead subject. Such an attitude is attributable to static teaching techniques that depend too heavily on dead and fixed tissues. As a result, the dynamics of living anatomy, the interrelationships of form and func- tion, and the awesome beauty of biologic mechanisms are denied. This chapter will develop background conceptual material on the anatomy of the mare’s reproduc- tive system. Some of the dynamics of anatomy will be incorporated into subse- quent chapters, wherein changes in mor- phology will be associated with physiolog- ic function. The reproductive system of the mare may be divided into two groups of organs. The first consists of those organs that are intrinsic to the reproductive tract (ovaries, oviducts, uterus, cervix, vagina, and vulva). The second group of organs (pituitary and hypothalamus) is extrinsic to the reproductive tract, but plays an integral and profound regulatory role in mare reproduction. The extrinsic organs have essential regulatory relationships to other body systems in addition to the reproductive system. Other outlying organs may also be considered part of the reproductive system (e.g., mammary and pineal glands). Division of the body into systems and a system into organs may be used as a learning tool. The eventual goal, however, is to develop an overall concept of the overlapping and indispens- able interactions among the various body systems and organs. Many of the descriptions and illustra- tions in this chapter are based on speci— mens prepared from 15 mares (575). The anatomy textbooks by Sisson and Grossman (1488) and Nickel et al. (1150) were frequently consulted. Weights and dimensions of various organs of the repro— ductive system have been tabulated for horses (Table 1.1) and ponies (Table 1.2). 1.1. Organization of the Reproductive Tract The in situ reproductive tract on dorsal View varies from Y— to T-shaped, depend- ing on the extent of intermingling with the intestinal viscera (Figures 1.1 and 1.2). Each arm of the tract consists of ovary, oviduct, and uterine horn. The trunk consists of uterine body, cervix, vagina, and vulva. An ovary is located at the extremity of each arm and is contigu- ous with and partly attached to the end of the oviduct. The oviducts, uterus, cervix, vagina, and vulva form a continuous tube with adjustable constrictions at the utero- tubal junction, cervix, and labia; these organs are collectively called the tubular genitalia. The continuous lumen of the tubular genitalia opens internally at the cranial end of each oviduct and externally at the labia. The lumen of the female reproductive tract is the only channel in the body that extends from the abdominal cavity to the outside. The contiguity of the ovary with the tubular genitalia and the continuity of the lumen of the tubular genitalia provide an assembly line arrangement that commences with dis- charge (ovulation) of the ovum from the 2 Chapter 1 ovary into the oviducts and terminates TABLE 1.2, Size of Reproductive and With expulsion (parturition) of the foal Endocrine Organs in Nonpregnant Pony through the vulva. Well OVeI‘ half Of the Mares during the Ovulatory Season reproductive tract lies Within the abdomi- . . . . . No. Standard nal cav1ty, and the rema1nder lies Within Organ mares Mean deviation the pelvic cavity (Figures 1.2 and 1.3). Body weight (kg) . . . 115—300 Mammary glands (g) 39 173 154 Pituitary gland (mg) 38 1343 334 Pineal gland (mg) 26 98 48 Adrenal glands (combined; g) 39 17.6 "5.1 Th roid glands combined; g) 40 7.8 3.2 Uterine artery (diameter, mm) 39 3.7 0.8 TABLE 1.1. Size of Reproductive Organs in Uterus Nonpregnant Riding-type Horses during Left horn the Ovulatory Season Weight (g) 40 36.9 17.3 Length (mm) 40 76.2 17.5 Organ N0~ mares Mean Diameter (mm) 40 33.2 8.2 Right horn B ' _ _ 9d? weight (kg) 59 (227 614) Weight (g) 40 41.1 16.5 Pltultary gland (mg) Length (mm) 40 76 7 20 4 Anterior 47 1782 . ‘ l . Diameter (mm) 40 35.7 6.8 Posterior 49 615 Body Uterus Weight (g) 40 68.8 324 Tom (g). 50 689 Length (mm) 40 117.2 29.4 Horns (diameter/horn; mm) 34 59 Diameter (mm) 40 48 1 10 1 (length/horn; mm) 48 150 . i . Cervix ( ) 50 152 Cerv1x . g Weight (g) 39 39.2 15.6 Vanna (g) 50 200 Length (mm) 38 53.1 9.3 0V§30t§d Width (mm) 38 38.6 6.4 L OVL/uclég: 48 6'7 Ovaries (combined) 91.1534“ 0‘” PC (mm) 49 152 Total (g) 38 43.8 22.3 Ovaries (combined for both) Minced extraluteal Total (3) ‘ 50 123.0 tissue (g) 39 24.8 10.0 Minced extraluteal tissue (g) 50 72.6 Extraluteal fluid (g) 38 16.9 19.3 Extraluteal fluid (g) 50 41.6 Follicles 2—10 mm Follicles 10 to 20 mm (N0.) 50 5.7 (N0.) 40 7.8 3.9 Follicles 20 to 30 mm (N0.) 50 2.0 Follicles 10-20 mm . (N0.) 40 3.4 3.4 Folhcles >30 mm (N0.) 50 0.6 . . . Follicles >20 mm Diameter largest follicle (mm) 50 22.5 (N0.) 40 0,3 0.6 Dimensions of an ovary (mm) Largest follicle/mare Length 131 51.6 Diameter (mm) 39 18.8 10.3 Width 131 28.5 Volume (ml) 38 4.5 9.1 Height 131 32.7 Corpus luteum (g) 36 6.2 2.7 Adapted from (1732) for mares necropsied on Days Dimensions of the uterus changed considerably 2, 4, 7, 11, and. 17 after the beginning 0f estrus, after handling and removal. Length (mm, mean except. for ovarian d1mens10ns WhiCh were rectal iSD) of left and right uterine horns, respectively, palpatlon estlmates adapted from (1230). before handling was 126.5 $24.7 and 130.0 124.1. b bl CV cx g1 inf l 10 luh oa 0d of H bladder broad ligament constrictor vestibuli and vulvae cervix glans clitoris infundibulum labia left ovary left uterine horn ovarian artery oviduct ovulation fossa Reproductive Anatomy 3 plo tm ua ub uo ur va ve Vf vgo tf proper ligament of ovary transverse fold tubal membrane uterine artery uterine body urethral orifice ureter vagina vestibule vaginal fornix vestibular gland openings FIGURE 1.1. Dorsal View of reproductive tract. The tract has been reflected to expose the medial surface of the broad ligament and uterine horns. The left infundibulum has been retracted, whereas the right infundibulum has been placed over the ovulation fossa. A portion of the transverse fold has been retracted to expose the urethral orifice. Locations of vestibular gland openings are based on (1488). 4 Chapter 1 FIGURE 1.2. Lateral View of reproductive tract. Details of the arrangement of muscles around vulva and anus were adapted from (1488). an = anus k = kidney r = rectum as = anal sphincter l = labia rl = round ligament b = bladder 10 = left ovary mg = mammary gland cve = constrictor vestibuli luh = left uterine horn so = small colon cx = cervix 0d = oviduct ub = uterine body cvu = constrictor vulvae pb = pubic bone va = vagina il = ilium pf = pelvic fat Reproductive Anatomy 5 FIGURE 1.3. Midsagittal View of reproductive tract. Details of the clitoral area were adapted from (1150). an = anus gc = glans of clitoris uo = urethral orifice as = anal sphincter iom = internal obturator muscle ur = urethra b = bladder l = labia urm = urethral muscle bc = body of clitoris mlb = median ligament of bladder va = vagina cf = clitoral fossa ps = pubic bone or symphysis ve = vestibule ct = cavernous tissue rgp = rectogenital pouch vf = vaginal fornix cve = constrictor vestibuli sac = sacrum vgp = vesicogenital pouch cvu = constrictor vulvae st = symphysial tendon vp = venous plexus 'cx = cervix ub = uterine body 6 Chapter 1 1.2. Suspensory Ligaments Function. The abdominal portion of the reproductive tract is attached to the abdominal wall by two large ligamentous sheets called the broad ligaments. The ligaments serve not only as attachments to a relatively fixed surface (body wall), but also provide an avenue for blood ves— sels, lymphatic vessels, and nerves. The broad ligaments, therefore, provide both physical and functional attachment of the organs to the rest of the body. Furthermore, the ligaments contain a large amount of smooth muscle that is continuous with the outer longitudinal muscular layer of the uterus and oviducts. Research is needed to determine the role of the broad ligaments, especially the elaborate smooth muscle layer, in reproductive mechanisms (e.g., movement of gametes and conceptus in oviductal and uterine lumens, mobility of fetal- amniotic unit within the allantoic sac, and expulsion of fetus). Structure. The broad ligaments in the mare are long and sheetlike and originate from the sublumbar region (Figure 1.4). The right and left ligaments, which sus— pend the ovaries, oviducts, and uterine horns, converge over the body of the uterus and cervix. Although each broad ligament forms a continuous sheet, it is customarily divid- ed into three nondemarcated areas: the mesometrium, attaching to the uterus; the mesovarium, attaching to the ovary; and the mesosalpinx, projecting from the lateral surface of the mesovarium (1150) and attaching to the oviduct. The cranial limit of the mesometrium is delineated by the round ligament of the uterus (1122). FIGURE 1.4. Frontal View of suspended reproductive tract after removal of other abdomi- nal viscera. b = bladder bl = broad ligament inf = infundibulum llb = lateral ligament of bladder 10 = left ovary luh = left uterine horn r = rectum ruh = right uterine horn rgp = rectogenital pouch vgp = vesicogenital pouch There is, however, disagreement on termi- nology for the other two portions of the broad ligament. The portion cranial to the round ligament of the uterus has been designated the mesovarium, and the small lateral fold has been designated the mesosalpinx (1150). Other writers take the opposite approach and refer to the main sheet as the mesosalpinx and use the term mesovarium for a fold of the mesos- alpinx (1122). A broad ligament consists of two layers of thin serous membrane with vessels, nerves, and smooth muscle located between the two layers. The continuous relationship of the serous layers of the broad ligaments with the serous lining of the abdominal cavity is depicted diagram— matically in Figure 1.5. Embryologically, the abdominal reproductive organs devel- op in the mesodermal tissue of the body wall. As the organs enlarge and protrude. into the abdominal cavity, they carry with them a fold of peritoneum and the atten— dant vessels and nerves. The fold of peri- toneum becomes the broad ligament. The continuation of the layer that covers an organ is termed the visceral layer of the peritoneum. The visceral layer or serous membrane (serosa) which covers the uterus is called the perimetrium, and the line of attachment of the broad ligament to the uterus is called the mesometrial attachment. The portion of the serous membrane lining the abdominal cavity is the parietal layer of the peritoneum. The mesentery of the digestive tract develops similarly, and the fold of peritoneum (mesentery) differs from the broad liga— ment primarily in the absence of smooth muscle. The length of the mesometrium per- mits the late gravid uterus to be partially supported by the abdominal floor. The long broad ligaments permit partial exte- riorization of the ovaries, oviducts, and uterine horns through a midventral laparotomy in parous mares (Figure 1.6). The organs may be retracted to approxi— mately the level of the laparotomy. Reproductive Anatomy 7 I I. I If I; i a r l I, (3* I. I9 I I\ -— Peritoneum Body wall ----- Path of vessels Viscera and nerves FIGURE 1.5. Diagrams showing the relationships of layers of the peritoneum and broad ligaments at level of the uterine body (top panel) and uterine horns (lower panel). bl = broad ligament llb = lateral ligament rgp = rectogenital pouch ub = uterine body of bladder uh = uterine horn plp = parietal layer vgp = vesicogenital pouch of peritoneum vlp = visceral layer pm = perimetrium of peritoneum 8 Chapter 1 FIGURE 1.6. Photograph taken during surgery depicting the degree of exteriorization of uterus and ovaries through a midventral laparotomy. Both uterine horns are in the left hand, and the left ovary with bursa and oviduct are in the right hand. Experimental surgical procedures such as selective cannulation of ovarian and uter- ine vessels are possible but often must be done within the abdomen. Attachments. The attachment of the broad ligaments to the body wall extends from approximately the level of the third or fourth lumbar vertebra t0 the fourth sacral vertebra. The ligaments are, there- fore, quite long in the craniocaudal direc- tion and attach to the sublumbar area and the lateral pelvic wall. Attachment in the sublumbar region is broad and may involve considerable adipose tissue (Figure 1.4). The cranial edge of each of the two broad ligaments contains the ovarian artery and vein and associated structures. The visceral attachments of the liga- ment and the principal points of entry of vessels and nerves are on the dorsal aspect of the organs. Attachment to the dorsal surface in mares is related to the V-shaped form (hanging side view) of the abdominal reproductive organs. In cattle, the ligaments originate from the upper part of the flanks about 10 cm below the level of the tuber coxae (1488), rather than from the sublumbar region. The attach- ment of the ligaments in cattle, therefore, is toward the ventral aspect of the organs. The cervix, uterine body, and intercornual area ride in a ligamentous sling. These species differences are important Considerations in transrectal palpation of the reproductive organs; in mares the free surface of the uterine horns is ventral, whereas in cattle the free surface is dorsal. Round ligament. The round ligament of the uterus is a very prominent structure that courses along the lateral surface of each broad ligament (Figure 1.2) and, as noted above, demarcates the cranial boundary of the mesometrium. The round ligament forms a prominent, free, round projection at its cranial end near the tip of the uterine horn. The round ligament courses along the broad ligament and blends with the peritoneum near the inguinal ring. In some species (e.g., dogs), the round ligament enters into a narrow peritoneal evagination (vaginal process). The round ligament of the uterus is, therefore, homologous to the ligament of the tail of the epididymis (gubernacu- lum). Ligaments and pouches of the urinary bladder. The suspensory apparatus of the urinary bladder is formed by fetal rem— nants of the umbilical artery and urachus. A large peritoneal fold is attached to each lateral surface of the bladder and is called the lateral ligament of the bladder (Figures 1.4 and 1.5). The edge of the lateral ligament contains the round ligament of the bladder, which is a remnant of the umbilical artery. Ventrally, the bladder is attached to the pelvic floor by the middle ligament of the bladder, which in the fetus formed the urachus. These three ligaments (middle and two lateral) extend to the umbilicus in the fetus and newborn foal. The lateral ligaments are readily palpable through the rectum in the adult and are aids in distinguishing the bladder from a gravid uterus. When the bladder fills, the two ligaments become taut and therefore are readily distinguishable palpation land- marks. As the fetal abdominal organs and their respective peritoneal folds move embryologically into the abdominal cavi- ty, recesses or pouches form between the suspending ligaments. The pouches open cranially into the abdominal cavity and project caudally into the pelvic cavity. The pouch between the rectum and geni- tal organs is called the rectogenital pouch, and the pouch between the genital organs and the bladder is called the vesicogenital pouch (Figures 1.4 and 1.5). These pouch— , es, especially the rectogenital pouch, are of clinical importance since they may be entered surgically by puncturing the vagi- nal wall near the cervix. 1.3. Associations between Reproductive and Digestive Organs Organs of the reproductive and diges- tive systems are in intimate and active contact. The direct effect of intestinal motility on the uterus can be appreciated by continuous ultrasonic viewing. The motility and extent of impingement of the intestine upon the uterine horn affects the cross-sectional shape of the horns, especially When the horns are flaccid (e.g., during anovulatory season). Occasionally, even the early conceptus (e.g., Day 18) may be momentarily indented by a moving segment of adjacent intestine. m. Apparently, no consideration has been given to a possible physiologic role of the intimate, active association of the reproductive organs with the digestive organs. Because biologic economy is so stringent, one might hypothesize, for example, that intestinal temperature or Reproductive Anatomy 9 motility has an effect on reproductive function. At any rate, a full appreciation of reproductive anatomy should incorpo— rate the relationship between reproduc- tive and digestive organs. The intimate relationship between intestinal and reproductive organs hin- ders viewing the ovaries and uterus in the living, standing mare through a long viewing tube or laparoscope inserted through the abdominal wall. This prob- lem has been partially overcome, howev- er, by Withholding feed and by insuffla- tion of the abdominal cavity with a gas to push the intestinal viscera downward ( 710, 1795). Effect on uterine shape. The abdominal reproductive organs are not suspended as completely as Figure 1.4 might lead one to believe. The ovaries and uterine horns may float on the intestinal viscera, or they may hang down among the coils of the viscera and intermingle with them. The in situ shape of the uterus depends on the degree of uterine suspension by the broad ligaments versus the intermin- gling with or floating on the intestines. These relationships, in turn, are modified by mare age and reproductive status and the resulting effects on size of the uterus and length of the broad ligaments. Thus, at one extreme, the uterus on dorsal View can be Y-shaped when fully suspended; at the other extreme, the uterus can be T- shaped when riding upon the ‘other abdominal viscera. Similarly, on side View, the uterus may or may not be V- shaped depending on the extent of sus— pension. The distinct Y-shape on dorsal View and V—shape on side view will not be seen unless an exposure is prepared with the mare’s body in an upright position and other viscera removed (Figure 1.7). The T-shape is seen when the uterus is excised and lying on a flat surface (Figure 1.1) or when the in situ uterus lies upon the sublumbar region when the mare is in dorsal recumbency and the uterus is viewed through a midventral laparotomy (Figure 8.12). These varying relationships 10 Chapter 1 should be kept in mind during transrectal examinations (palpation and ultrasonic scanning). Some appreciation of the intimate and dynamic association between the alimen— tary and reproductive viscera may be obtained from Figure 1.7. The abdominal organs have been exposed by progressive removal of portions of the left abdominal wall in a fresh cadaver suspended in the upright position. The ovary did not come into View until a portion of the intestine was retracted through the opening; that is, the ovary in its undisturbed position was nestled among coils of the intestine and, except for the dorsal attached bor- der, was completely covered by the coils. As more of the intestinal coil was retract- ed, the uterine horn came into View. The uterus was also enclosed by intestinal coils. Before removal of the intestinal vis- cera, the round ligament of the uterus did FIGURE 1.7. Progressive exposure of abdominal portion of reproductive viscera to show relation- ships between digestive and reproductive organs. Exposure was made through an incision in left par- alumbar fossa with body suspended upright. A) A part of the intestine has been reflected with forceps (arrow) to expose the ovary. The cranial pole of the ovary is deflected laterally by the small colon. B) The intestine has been further reflected to bring the tip of the left uterine horn into View. C) The entire lateral aspect of left ovary and uterus has been exposed. Note the V—shape of the suspended uterus on side View with the caudal portion of the horn at the lowest point (arrow). cpo = cranial pole of ovary k = kidney 10 = left ovary luh = left uterine horn mes = mesentery r1 = round ligament so = small colon sp = spleen not appear to be bearing weight. When the intestinal viscera were completely removed, the uterus dropped and resulted in a stretched round ligament. Note also that the cranial pole of the ovary is directed laterally. The ovary was resting upon a coil of intestine and therefore would have moved in response to intesti- nal motility and changes in body position. The uterine body is located partly in the pelvis and often contacts the rectum dorsally. The body may also be deflected to either side depending upon the position of the colon and the fullness of the uri- nary bladder, which is immediately ven- tral to the uterine body. The position of the rectum is most fortunate for those who need to examine the reproductive organs by inserting an arm into the rec- tum. The opposing walls of the abdominal viscera are separated and lubricated by a thin film of serous fluid that is secreted by the serous lining and is contained Within the peritoneal cavity. This arrangement eliminates friction as the uterine horns and ovaries move and intermingle with the intestines. 1.4. Pelvic Cavity and Perineum Pelvic cavity. The pelvic cavity is of considerable clinical importance in repro- duction, particularly in obstetrics. It con- tains the rectum and anus, the caudal urinary bladder and urethra, and the caudal portion of the reproductive tract (Figure 1.3). The pelvic cavity is enclosed by the bones of the hip, the wide sacrosci- atic ligament, and the muscles of the thigh, gluteal region, and tail (Figure 1.8). The pelvic cavity in the mare may be divided transversely at approximately the level of the third or fourth sacral segment into peritoneal and extraperitoneal por— tions. The peritoneal portion, which rep- resents an overlapping of the abdominal and pelvic cavities, is lined by peri— toneum. The peritoneum invests the cra- nial portion of the pelvic organs and, as described above, forms the associated lig- Reproductive Anatomy 1 1 FIGURE 1.8. Transverse section through vagina showing collapsed vaginal wall (depicted by dashed line) and the location of the pelvic organs within a skeletal cage consisting of pelvic bones and spinal vertebrae. pb pf r ur va V pubic bone pelvic fat rectum with fecal material urethra vagina vertebra aments and pouches. The extraperitoneal or caudal portion is lined with loose fascia and adipose tissue (Figures 1.3 and 1.8) that are continuous with corresponding abdominal retroperitoneal tissues. The internal entrance to the pelvic cav- ity is a bony ring called the pelvic inlet that may be palpated through the rectum. This inlet represents a major impediment during parturition. The pelvic outlet is formed by the floor of the pelvis, by the coccygeal vertebrae, and on each side by a broad sheet called the sacrosciatic ligament. Although the outlet may be 12 Chapter 1 slightly smaller than the inlet, it expands during parturition. Perineum. The perineum or perineal region includes the tissues surrounding the anus and urogenital tract at the pelvic outlet. Internally, the perineum is demarcated by the pelvic outlet, and it extends externally from the base of the tail to the ventral commissure of the vulva (664) or the base of the udder (1150). Deep to the cutaneous bridge between anus and vulva is a mass of fibrous and muscular tissue called the perineal body. Interest in the perineum is far more than academic. During breeding and foaling, lacerations and rectovaginal fistulae may cause damage to the per- ineal region and, if deep, may involve the perineal body (1169). Such injuries, as well as problems associated with malforma- tions (e.g., vulvar deformities leading to pneumovaginitis), have encouraged detailed anatomical study of the equine perineum. Pelvic diaphragm. The term pelvic diaphragm has occasionally been used in the mare to describe the structures involved in the caudal containment of the pelvic organs (1150). Habel (664) indicates, however, that it is difficult to visualize a pelvic diaphragm in the mare comparable to that of man. The pelvic diaphragm in man is well developed and consists of a concave muscular plate and rudimentary tail that span the caudal portion of the pelvic outlet. Because of man’s upright position, the diaphragm supports the weight of the pelvic organs and to some extent the abdominal organs. Because of the mare’s horizontal position, the pelvic organs are supported by the floor of the pelvis. Nevertheless, there are periods of intense pressure against the “pelvic diaphragm” during defecation, urination, copulation, abdominal distention (in the latter part of gestation or with a full digestive tract), labored breathing, and parturition. A tremendous force is exerted against the caudal pelvic outlet during the birth of a foal. Much of this force is apparently borne by attachments of the vulvar muscles. The vestibule has several sphincter-like muscles that close the tract to the outside and are attached to the sphincter muscles of the anus and the last sacral and coccygeal vertebrae. 1.5. Ovarian Bursa The ovarian bursa is a membranous pouch which partly or, in some species, completely separates the ovary from the abdominal cavity. Mossman and Duke (1122) have presented an interesting account of the divergent modifications of the bursa among species. The bursa in mares is a distinct reproductive struc- ture, but little is known about its impor- tance. It is generally assumed that the bursa assists in passage of an ovum into the oviduct. However, a specialized struc- ture like the equine ovarian bursa may exist for physiologic reasons that have yet to be defined. Because the dorsal, medial, lateral, and much of the ventral (except for the ovula- tion fossa) aspects of the equine ovary are covered by visceral peritoneum, the ovary is embedded in the mesovarium. The exposed portion (ovulation fossa), howev- er, faces caudoventrally into the ovarian bursa and forms the cranial portion of the bursa (Figures 1.9 and 1.10). The lateral wall of the bursa is formed by the meso- salpinx and its suspended oviduct. The free margin of the mesosalpinx extends beyond the oviduct to form the lateral lip of the bursa. The term tubal membrane has been used for the free margin of the mesosalpinx (1122), although some authors consider the free fold as a portion of the mesosalpinx (1150). The medial wall of the ovarian bursa consists of a fold of the broad ligament with the proper or round ligament of the ovary on the free border of the fold. The round ligament of the ovary is a strong band of fibromuscu- lar tissue, connecting the tip of the uter- ine horn to the caudal pole of the ovary. The cranial portion of the uterine horn FIGURE 1.9. Lateral view of relationships of ovary, oviduct, ovarian bursa, and tip of uter- ine horn in normal position and after exposure of the ovari- an bursa and proper ligament of the ovary (medial wall of bursa). The forceps are on the infundibulum, tubal mem- brane, and uterine horn, expos- ing the ovarian fossa and bursa. Note the openings (exaggerated) 0f the ovarian and uterine ends of the oviduct. amp = ampulla inf = infundibulum ist = isthmus luh = left uterine horn mo = mesovarium ms = mesosalpinx r1 = round ligament tm = tubal membrane tuj = tubouterine junction forms the caudal aspect of the bursa. In the embryo, the round ligament of the ovary is continuous With the round liga- ment of the uterus. Together, the two lig- Reproductive Anatomy 13 aments are homologous to the gubernacu- lum testes since they develop from the gubernacular fold of the embryonic gonad (1122). 14 Chapter 1 FIGURE 1.10. Oblique lateral-ventral view of left ovary, oviduct, and bursa showing exposed (A) and covered (B) ovulation fossa. inf = infundibulum plo = proper ligament ob = ovarian bursa of ovary od = oviduct trn = tubal membrane of = ovulation fossa uh = uterine horn ms = mesosalpinx 1.6. Ovaries External Structure. Individual mature follicles, the corpus luteum, and the whole ovary are, on a body weight basis, larger in the mare (Tables 1.1 and 1.2) than in other farm species (Figure 1.11). For descriptive purposes, the equine ovary may be assigned two surfaces (lat- eral and medial), two borders (dorsal or attached and ventral or free), and two poles or extremities (cranial or tubal and caudal or uterine). The ovary is kidney shaped with a very prominent depression (ovulation fossa) on the free or ventral border (Figure 1.12). The convex dorsal border is often called the greater curva— ture. The poles are rounded, and the cra- nial or tubal pole is attached to a portion mm 3;} 3:“: KT: 3‘ ‘ ,Es’ as as 3:“: sit: as a); PRE OVULATORY FOLLICLES HILLOCK FIGURE 1.11. Upper panel: Difference in size of a preovulatory follicle in mares and cattle. Follicles were dissected from the ovaries. Scale in millime- ters. Lower panel: Hillock containing the oocyte (arrow) in a mature equine follicle as seen by translucent light after dissecting the follicle from the ovary. Specimens prepared by M. Del Campo and K. Tucker. of the fimbriae of the oviduct. The caudal or uterine pole is attached to a point just caudal to the end of the uterine horn by the proper ligament of the ovary. The surfaces of the ovary between the free (ventral) and attached (dorsal) borders are called the medial and lateral sur- faces. Location. When the ovary is completely suspended, as occurs after removal of intestinal viscera or in an excised repro- ductive tract, the medial and lateral sur- faces and the cranial and caudal poles are easily identified. As noted above, however, the ovaries in the nonpregnant mare may ride on the intestines, and the mesovarium may be loose. The orienta- FIGURE 1.12. A pair of trimmed ovaries taken approximately one month before the onset of the ovulatory season. Ovaries are shown before and after a midsagittal incision. After the incision, the ovaries were opened like a book. Note the irregular shape of the follicles and the prominent depression or ovulation fossa. tion of the ovaries is thus variable. It can be difficult to identify poles and surfaces through the rectum because of ovarian mobility, but the border areas are identi- fiable because of their characteristic forms (concave ventrally and convex dor- sally). Since the ovaries may be lifted by the intestines, their actual location in the body cavity is quite inconstant. According to Sisson and Grossman (1488), the right ovary is often about 15 cm behind the cor- responding kidney, but the distance varies from 5 to 30 cm. The left ovary is usually further back than the right ovary; however, ”the two structures are closer together On the left side because of the caudal location of the left kidney. The dis- Reproductive Anatomy 15 tance from ovary to uterine horn may vary from O to 5 cm. The noncoiled uterus in the mare is associated with a more cra- nial position of the ovaries (third and fourth lumbar vertebrae) than in the cow and sheep (sixth or last lumbar verte- brae). The ovaries and oviducts, there- fore, are in the cranial-most transverse plane of the reproductive tract in the mare, whereas in other large domestic species a portion of the uterus is cranial to the ovaries. Attachment. As in the other portions of the abdominal reproductive tract, the ves- sels and nerves reach the ovaries through the broad ligaments and enter each ovary at the dorsal convex border or greater curvature and spread over the lateral and medial surfaces. The convex surface, therefore, is the hilus of the ovary. The term hilus is defined as the part of an organ where nerves and vessels enter and leave. The hilus is depressed in other organs (e.g., kidney, spleen, lung), but not in the equine ovary. This difference has led to false analogies between the kid- neys, for example, and the equine ovary to the extent that it is not unusual to find reference to the “ovulation fossa at the hilus of the equine ovary.” This point of confusion has led to the publication (840) of diagrams in which the vessels are shown entering at the ventral or concave border rather than dorsally. The attachment of the mesovarium is very broad and extends for a considerable distance over the medial and lateral sur- faces. The visceral layer of the peri— toneum of the ovary is loosely attached to more than half the total surface of the ovary, and there is considerable fat and loose connective tissue between the Vis— ceral layer and the ovary itself. This area of loose attachment also contains a com- plex of arteries and veins. The vessels become more deeply embedded upon leav- ing this area, but very large vessels remain visible running in approximately parallel fashion from the convex to the concave borders (Figure 1.4). 16 Chapter 1 Internal structure. The relationship between cortical and noncortical areas of the ovary is unusual in the mare. By defi- nition, the cortex of an organ is the outer portion or external layer as in the kid- neys, adrenals, or brain, or ovaries of other species. The medulla is the softer, vascularized area in the center. The ovary of the adult mare, however, is structured so that the medullary or vas— cular zone is superficial, and the cortical zone, which contains the oocytes and folli- cles (parenchyma), is partly in the interi- or of the gland (Figure 1.13). The cortex reaches the surface only at the depression (ovulation fossa) on the free border. This is the only area from which normal ovula- tion occurs. The corpus luteum does not project from the greater surface of the Nonequine Surface germinal epithelium ovary as in other species. A projection (ovulation papilla) may be seen, however, in the ovulation fossa, especially in the newly-forming corpus luteum (pg. 202). In the newborn foal, the ovaries are in the form of an oval and the germinal epitheli- um covers a portion of the surface. The ovary later assumes a kidney shape, and the germinal epithelium becomes con- fined to the ovulation fossa. Limited examination of foal ovaries indicated that the kidney shape becomes marked prior to puberty and apparently as early as seven months of age (1727). It has been stated that a definite ovulation fossa can be observed by five months of age (1292). Details of the morphogenesis of the equine ovary and ovulation fossa have been reported (pg. 489). . o . u . . Medulla FIGURE 1.13. Diagrammatic comparison of relationships of cortical and medullary areas of ovaries in equine and nonequine farm species. Adapted from (1122). Function. The ovaries are the master organs of the mare’s reproductive tract. They produce the ova that justify the existence of the remainder of the tract. The care and fertilization of the dis- charged ovum and subsequent develop- ment of the embryo are assigned to the tubular organs, but the ovaries, through their hormonal role, largely integrate and control the functions of the tubular geni- talia. The complexity of form and function of the ovary is heightened by its dual role. It has gametogenic (development of gametes or ova) and endocrine (produc- tion of hormones) functions. Of the two principal ovarian components, the folli- cles play a dual role (production of ova and estrogens), whereas the role of the corpus luteum is endocrine only (produc- tion of progestins). Dynamic anatomy. The ovary’s role as the master organ of the reproductive tract necessitates a morphology more dynamic than for any other organ in the body. A very large structure, 50 mm in diameter (mature follicle; Figure 1.11), can be here today and gone tomorrow (ovulation). If a mare ovary is sectioned at a given time, there may be numerous vesicular (cavity- containing) follicles Visible to the naked eye, ranging in diameter from 2 to 30 mm or more. Some of these may be growing, and others may be undergoing regression (atresia). There may be a developing or regressing corpus luteum along with rem— nants of corpora lutea of previous cycles (corpora albicantia). If it were possible to section the same ovary 10 days later, the appearance may have completely changed. The large follicle may have ovu- lated and formed a corpus luteum, or it may have undergone atresia. The previ- ous corpus luteum may have regressed into a corpus albicans. The follicles of the mare may be classi- fied into primary, secondary, and tertiary structures, as described for mammals in general reproductive physiology books Reproductive Anatomy 17 (665). The oocyte with its surrounding cumulus is attached to the internal lining (granulosa) of the follicle, and the area of attachment is called the hillock. The author has not found a published pho— tomicrograph or description of the in situ hillock with its attached cumulus-oocyte complex from a mature equine follicle. In some species, the hillock is pedunculated and projects well into the antrum (1122). In mares, the relationship between the hillock and the wall of the preovulatory follicle is unknown. The anatomy of the hillock, location of the hillock relative to the ovulation fossa, and when and how the cumulus-oocyte complex is released in reference to ovulation are open equine research areas. Recently an oocyte-collec— tion project was done that involved peel- ing intact follicles from the ovary (395); it was observed that the hillock of a removed and trimmed follicle is visible to the naked eye (Figure 1.11). Therefore the anatomy of the hillock is quite amenable to investigation. Preliminary examinations indicated that the hillock of a mature follicle has a broad-based attachment in mares with little or no pedunculation (600); thus, in this species the hillock may consist only of a raised area in the granulosa. PHOTOGRAPHIC PLATES The series of plates on the following seven pages depicts scanning electron micrographs of the tubular genitalia (Figure 1.14) and colored photomicro- graphs of structures in and near the ovary (Figure 1.15), oviducts (Figure 1.16), uterus and cervix (Figure 1.17), and vagina and vulva (Figure 1.18). Also shown are videoendoscopic views of the oviductal papilla, uterus, and cervix (Figure 1.19) and a diagram (Figure 1.20) and photograph (Figure 1.21) of the uteroovarian vasculature. Figures 1.15 to 1.19 were prepared by GP. Adams. Chapter 1 g ; INFUNDIBULUM ISTHMUS r \ FIGURE 1.14. Scanning electron micro— graphs of the epithelial surface of infundibulum, ampulla, isthmus, uterine horn, and cervix. Note the ciliated and nonciliated cells. Magnification, x 3000. Courtesy of Arthur Wu. E _ _. L m m mmmmmmwa U % E rfiluupum 9 D T taallrro 1 O F Sldouoar N N U u.lele O L cmvodfieua & I S obnnhcs T U S u SO C A ,P hmcarmtam y H L .R pf)an m R W O. aTofiioem o 0 .C flanbdmmfl m w 0. .F. o.morMu n D . rrfiectde A N .macwacnh E Vfl tuat e R moummw)g .W. m te.J(esE.n c B mmmmhdnm U Prt.mteer d a 1ftwe. m eseObOd) D. 5nanummququ e ldhiub Du LHSWHIOWHLM Ea)1MeGWH Rmmmfimmlflo U ip fla wameeuce wrwmmmmemg chmgnNmumh § . G. GRANULOSA ADRENOCORTICAL NODULE A 20 Chapter 1 A. ‘NFUNDIB‘UL gWg 4: ‘ E. ISTHMUS F. iSTHMUs FIGURE 1.16. Low-power (left) and high-power (right) photomicrographs of the three segments of the oviduct. When proceeding from the ovarian end (infundibulum) to the uterine end (isthmus), the complexity and prominence of the epithelium decreases, Whereas the relative proportion of smooth muscle increases. The isthmus (low power; E) is delineated by arrows. Note the cilia in the high-power Views. 21 we Anatomy Reproduct MY OMETRIUM B UTERIN E WALL A END OMETRIUM D EN DOMETRIUM C § § 66.. nt mmmm fa cmomm up mmfim mama/k a r reme ghfiy OTeh r P C. r .laea m.nh]u. omotc tasr ovwd hdmd PnSn 0a)a Hx,®a, mey r a eul Ecfimm Rsaan Uumpm Gqu It .t Fummo tom). The last View (G) shows the cervical epithelium (below)vand vaginal epithelium (above). G. CERVIX AND VAGINA 22 Chapter 1 EBfVESTIEULE FIGURE 1.18. Photomicrographs of the epithelium of vagina and vulva. C. LABIA A. OVIDUCTAL PAPILLA B. ENDOMETRIAL FOLDS FIGURE 1.19. Includes facing page. Videoendoscopic views of cervix and uterus. A. Oviductal papilla (arrow). B. Endometrial folds after horn was dilated with fluid. Note bare area at tip of horn. C. Endometrial folds after removal of Day 14 conceptus. D. Entrance to uterine horns and a contraction in uterine body (arrow). E. Longitudinal arrangement of cervical folds. Cervix has been dilated. F. Straight View of cervix and surrounding vaginal fornix. Arrow = dorsal frenulum. G. Ventral View of cervix after lifting with finger (left). H. Lifted cervix to expose the ventral frenulum. Ive Anatomy Reproduct ORNUAL JUNCTION C CORPUS D ENDOMETRIAL FOLDS C CERVIX F CERVICAL FOLDS E H. VENTRAL FRENULUM G. CERVIX 24 Chapter 1 FIGURE 1.20. Drawing of dorsal overview of vascular system of reproductive tract. Arteries are depicted in red and veins in blue. From (611). 0a oboa obov 0d 0v ro ua ovarian artery ovarian branch of ovarian artery ovarian branch of ovarian vein oviduct ovarian vein right ovary uterine artery uboa ubov ubva ubvv uh uv uterine branch of ovarian artery uterine branch of ovarian vein uterine branch of vaginal artery uterine branch of vaginal vein uterine horn uterine vein FIGURE 1.21. Lateral View of arterial system of uterine horn and ovary. Vessels were injected with red latex and the tissues cleared. From (611). 0a oboa ovarian artery ovarian branch of ovarian artery ua uboa uterine artery uterine branch of ovarian artery 1.7. Oviducts Each oviduct (uterine tube or Fallopian tube) is a tortuous tube in the meso- salpinx. The oviduct serves as the initial home and transportive duct for the ovum and embryo. Orientation of the oviducts (Figures 1.9 and 1.10), magnified views of the epithelial folds (Figure 1.22), pho- tomicrographs (Figure 1.16), and scan— ning electron micrographs (Figure 1.14) are shown. External structure. The oviducts in horse mares are 20 to 30 cm long when extended (dissected free). From the ovari- an to the uterine end, an oviduct is divid- ed into infundibulum, ampulla, and isth— mus. The terms are descriptive: the infundibulum is funnel-shaped like a catcher’s mitt; the ampulla is expanded FIGURE 1.22. Magnified views of the exposed sur- face of the infundibulum showing the highly plicat- ed structure (upper panel) and the inner folded sur— face of the ampulla as seen through a longitudinal incision (lower panel). amp = ampulla inf = infundibulum Reproductive Anatomy 25 like an ampule; and the isthmus is a nar- row structure connecting two larger structures (ampulla and uterine horn) like the Isthmus of Panama. The funnel- shaped cranial portion of the oviduct is the infundibulum. Its margin consists of irregular processes called fimbriae (Figures 1.16 and 1.22). At the cranial edge of the infundibulum, the fimbriae are attached to the cranial pole of the ovary to form the cranial margin of the ovulation fossa. The abdominal opening of the oviduct is at the approximate center of the infundibulum (Figures 1.9 and 1.10). During ovulation the infundibulum covers the ovulation fossa to facilitate the entry of the ovum into the oviduct (Figure 1.10). The nature and mechanisms involved in covering of the fossa in the mare are unknown. The caudal demarcation of the ampullary portion is not distinct. For pur- poses of this discussion, it will be defined as extending from the abdominal opening to a poorly demarcated area where it gradually narrows into the isthmus in the general Vicinity of the lateral surface of the ovary’s caudal pole. The isthmus makes up the remaining portion of the oviduct and opens into the uterine horn slightly caudal to the blunt end of the horn. The junction of the oviduct with the uterine horn is called the uterotubal junc- tion. The lumen of the oviduct communi- cates with the lumen of the uterine horn through a small opening located in the center of a small papilla which projects into the uterine lumen (Figures 1.9 and 1.19,A). The papilla and uterine os of the oviduct deserve specific and detailed investigation. For example, research is needed on possible roles of the papilla in selective transport of the fertilized ova into the uterus and in preventing reflex of uterine contaminants into the oviducts. It is emphasized that a gradual tapering or funneling of the uterine horn into the oviduct, as occurs in some species, is not a feature of the uterotubal junction in mares. 26 Chapter 1 Internal structure. The external wall of the oviduct is covered by a peritoneal coating or serosa. The serosal layer is continuous with that of the mesothelium of the broad ligaments and the serosa of other abdominal reproductive organs (Figure 1.5). Fibrous supportive tissue or adventitia is present beneath the serosa and is continuous with the fibrous layers of the broad ligaments. Squamous serous cells of the serosa extend onto the exter- nal aspect of the fimbriae and join the mucosal lining of the internal aspect. The prominence of the muscular layer (tunica muscularis) and the mucosa varies considerably from one end of the oviduct to the other (1075), providing important indications of functional diver— gence of the various portions (Figure 1.16). The thickness of the muscularis increases from ovarian end to uterine end. The fimbriae of the infundibulum contain very few muscle fibers, and the ampulla contains only a thin layer. The isthmus, however, has a well developed muscularis. The muscularis consists chiefly of an inner circular layer of smooth muscle. The outer layer is longi- tudinally or obliquely arranged, and the muscle fibers are continuous with those of the broad ligament. The mucosa decreases in complexity from the ovarian end to the uterine end (Figure 1.16). The folds are chiefly longitu- dinal, but the folds in the ampulla are extremely complex with primary and sec- ondary folds. This folding is more pro- nounced in mares than in ruminants (1634). The mucosa is considerably less plicated in the isthmus. The epithelium is columnar and many of the cells are ciliated. Functional anatomy. Harmony of form and function is well exemplified by the oviduct. The funnel-shaped infundibulum and its fimbriated processes and ciliated cells (Figures 1.10, 1.16,A,B and 1.22) favor passage of the discharged ovum into the ampulla. The prominent mucosal folds of the ampulla are important in pro- viding a proper setting and adequate sur- face area for fertilization and initial development of the fertilized ovum. The mucosal fluid provides a fluid medium for movement and nourishment of sperm and ovum. At the same time, the thin muscu— lar layer suggests a relatively quiescent area for fertilization. The isthmus, how- ever, is dominated by muscle rather than mucosal folds. This portion of the oviduct, therefore, may function more as a pro— pelling channel for movement of sperm from uterus to ampulla and for the later passage of the fertilized ovum in the opposite direction. 1.8. Embryonic Vestiges and Accessory Structures Many forms of fluid-filled embryonic vestiges may be found in association with the oviducts. Information on the origin of such structures is available (1122). They are interesting because they serve as a test of one’s knowledge of the embryologic derivation of the reproductive organs. It has been suggested that large numbers of cysts may present an obstruction to ovu- lation (1292), and fimbrial cysts may be a cause of infertility (92). Fossal cysts. O’Shea (1189) has done an extensive histologic study of nonfollicular cysts in the region of the ovulation fossa of the equine ovary. Such fossal cysts, many larger than 1 mm in diameter, were found in 67% of 42 ovaries. On gross examination, the fossal cysts cannot read- ily be distinguished from small follicles. Histologically, the distinction is clearer, since fossal cysts are lined with ciliated cells. Confusion may develop, however, when degenerative changes occur in the cysts or follicles. One portion of the ovula- tion fossa is lined by ciliated columnar epithelium, and another portion is lined with cuboidal or germinal epithelium. O’Shea’s work indicates that fossal cysts may form from glandlike infoldings of the columnar but not of the cuboidal epitheli- um. They are consequently of fimbrial (i.e., Mullerian duct) origin and not com- parable to the germinal inclusion cysts of other species. The formation of such cysts is presumably favored by the disruption associated with ovulation. Fimbrial cysts are common in mares (94). Histologically, they are similar to fos- sal cysts in accordance with the conclu- sion that the two are of common origin and differ primarily in location. Tubal cysts. The tubules and ducts of the embryonal mesonephros give origin to most of the transport system of the male reproductive tract. In females, however, the mesonephric ducts regress, and the reproductive duct system is formed from paramesonephric or Mullerian ducts. CySts in the mesosalpinx and mesovari- um have been described (94, 1732, 219). In one study (1732) there were 1 to 12 cysts per mare with diameters of 2 to 50 mm in 81% of 28 mares. Many of the cysts in the adult mare are remnants of the mesonephric tubules and ducts. Cysts may form in remnants of the cra- nial group of mesonephric tubules (epoophoron) or the caudal group of tubules (paroophoron). Such cysts are reportedly more common in young mares and tend to disappear with increasing age (1488). Thus, the incidence of these cysts, which are of embryonal origin, decreases with age, whereas the incidence of uter- ine cysts (pg. 524) increases with age. The openings of the canals of Gartner may occasionally be found on either side of the urethral orifice (1488). Remnants of the caudal-most portions of the mesonephric ducts are found rarely in mares. Persistent median wall of the Mullerian ducts has been described (505, 220). Adrenocortical nodules. Adrenocortical nodules near the ovaries are common in mares and have been extensively studied by Ono and co-workers (1180). These nod- ules were found in 59% of 271 mares and were described as yellow, 1 to 2 mm in diameter, and arranged in grapelike clus- ters in the loose connective tissue over rithe surfacewoflthe ovaries. Histologically, the structures have the appearance of Reproductive Anatomy 27 adrenocortical tissue (Figure 1.15,A,B). The cells are large, and the cytoplasm has a foamy appearance. In some specimens, the cells are arranged in columns or cords separated by sinusoidal areas character- istic of the zona fasciculata of the adrenal gland. Cells similar to those of the zona reticularis, but with different degrees of cytoplasmic vacuolation, are seen in some specimens and are located mainly at the periphery of the nodule. The functional importance, if any, of these structures is not known. They would be interesting subjects for a research project. 1.9. Uterus External structure. The cranial half of the mare’s uterus consists of two uterine horns, while the caudal half is a single uterine body (Figure 1.1). The intercornu- al ligament is much less prominent than in sheep and cattle. A short uterine sep- tum marks the internal bifurcation of the uterine horns (Figure 1.19,D). The exter- nal configuration thus reflects the inter- nal configuration (form of lumen) Without the masking effect of a prominent inter— cornual area. Handling artifacts. Few people have observed the equine uterus in its fresh in situ state. If the uterus of the nonpreg- nant mare is observed immediately upon opening the abdominal cavity and, most importantly, before handling, its true form can be appreciated. The walls of an estrous or anestrous uterus are quite flac- cid and intestine-like. It is emphasized, however, that tone increases during diestrus (pg. 209) and remarkably during early pregnancy (pg. 314). The flaccidity of the nongravid uterus is so pronounced that the horns of a mare in dorsal recum- bency drape over the other abdominal structures (Figure 9.8, pg. 354). Handling causes the uterus to contract rapidly to a more sausage—like form with reduced length and increased diameter. Necropsy or slaughterhouse specimens show this” artifactual form. For example, the uterine 28 Chapter 1 horns of 10 nonpregnant pony mares averaged 123 mm in length in situ and before handling, whereas after removal the mean length decreased to 69 mm (575). In a transverse View of a horn, mean width and height before handling were 11 and 34 mm, respectively, compared to 37 and 37 mm after removal. Endometrial folds. The lumen in the natural state is nearly obliterated by the collapsed walls and the prominent endometrial folds. Perhaps the apposition among folds with obliteration of the lumen results in a capillary or surface tension phenomenon. Capillary action may be involved in movement of uterine secretions and the debris or cells associat- ed with inflammation. The endometrial folds are arranged longitudinally and are yellowish or reddish brown (Figures 1.1 and 1.19,B,C). The arrangement of longi- Capillary Duct Vein Mid gland Basil gland Artery tudinal folds of the uterus and cervix has been studied by the use of latex molds (913). The number of folds was the same for the uterine horns, uterine body, and cervix; the average number was seven (range: 5 to 10). The number was not affected by mare age or stage of the estrous cycle. The folds can be appreci- ated during viewing through fiberoptic endoscopes or videoendoscopes (Figure 1.19). Histologically, the folds are com- posed of a longitudinal, gland-free core of connective tissue covered on both sides by endometrium (864). The structure of the folds has taken on applied importance with the advocacy of uterine biopsy and transrectal ultrasonography for detecting pathologic changes and stage of estrous cycle (pg. 519). Histology. A diagram of the uterine wall (Figure 1.23), photomicrographs Stratum compactum Lamina Endometrium propria Stratum spongiosum Inner circular muscle Vascular layer Myometrium Outer longitudinal ‘ muscle EGin-x_______-—-——:_——]-Perimetrium FIGURE 1.23. Kenney‘s diagram of the equine uterine wall. The relative thickness of the myometrium was considerably reduced for ease of presentation. From (864). (Figure 1.17), and scanning electron micrographs (Figure 1.14) are shown. The serosa of the uterus (perimetrium) and the vascular layer and longitudinal mus- cle layer beneath it are all continuous with corresponding tissues of the broad ligament. The myometrium, like the mus— cularis of the oviduct, consists of a thick, inner, circular layer of smooth muscle and a thinner, outer, longitudinal layer. A prominent vascular layer lies between the two layers of muscle. The epithelial lining of the endometrium is secretory in nature. The cells are high columnar in contrast to the pseudostratified epithelium of other farm species (1634). Pseudostratification may occur during estrus, however. The surface of the epithelial cells may be cili— ated (Figures 1.14 and 1.17,C,D; 864), but ciliation changes considerably during the estrous cycle (pg. 208). The lamina propria is composed of two layers (Figure 1.23; 864): 1) the stratum compactum, which is approximately 1 mm thick and contains a high density of stellate stromal cells, and 2) the stratum spongiosum, which is a loose arrange- ment of interconnecting cells with consid- erable interstitial fluid. The lamina pro- pria of the uterus, unlike the oviduct, contains prominent glands opening on the surface of the endometrium and extend- ing the depth of the lamina propria. The uterine or endometrial glands are branched and tubular and are greatly coiled in mares; the coiling is altered con— siderably during the estrous cycle (pg.208). The glands may have 10 or more primary branches and fewer secondary branches (864). The epithelium is simple columnar. The mucosa also changes considerably during the anovulatory season with histo- logic indications of inactivity (cuboidal epithelium, dense stroma, straight glands; g. 143). 1.10. Cervix Structure. The cervix is thick walled and firm and identifiable by transrectal Reproductive Anatomy 29 palpation. During estrus, however, it becomes flaccid and may not be readily discernible (pg. 210). The caudal portion projects into the lumen of the vagina (Figures 1.1, 1.3, 1.19,F,G, and 1.24) and may be palpated and examined through the vagina, either digitally or by Visual inspection with the aid of a speculum and light source. Another distinctive clinical feature of the equine cervix is the dilata- bility of the lumen (Figure 1.19,E) and the lack of obstructing cervical rings. The uterine body, therefore, can be entered by a large instrument, unlike in other farm species; examination of the endometrium through the cervix by digital palpation or by hysteroscopy (944) has been described. The folds of the equine cervix are arranged longitudinally and are continu- ous with the endometrial folds of the uterine body (Figure 1.25). Each dorsal and ventral fold may continue onto the floor of the vagina as a prominent frenu- lum (Figure 1.19,G,H). Form and function. The cervix is a highly versatile organ capable of perform- ing seemingly incompatible feats. It can perform the following functions: 1) pro- duce a large amount of mucus that may act as a lubricant or a sealant, 2) occlude its lumen so that it is impermeable to for- eign material and bacteria, and 8) expand to a size that will allow passage of the foal. Expansion and contraction are func- tions of a thick layer of circular muscle rich in elastic fibers. The production of large amounts of mucus by such a small organ is favored by a highly folded, fern- like mucosa which is lined by epithelium containing mucus—producing cells (Figure 1.17 ,E,F). The cervix produces a slippery mucus that acts as a lubricant to facili- tate passage of the penis into the caudal portion of the tract. At other times (preg- nancy, diestrus), the cervix produces a sticky mucus which helps seal the lumen. The formation of secretions with diver- gent physical properties further demon- strates the versatility of this amazing organ. FIGURE 1.24. Inner surface of vagina and vulva. Incision was made longitudinally through dorsal wall, and sides were reflected. The urethral orifice is beneath the transverse fold. c = clitoris uo = urethral orifice co = cervical orifice va = vagina ex = cervix ve = vestibule l = labia Vf = vaginal fornix tf = transverse fold vu = vulva 1.11. Vagina Gross anatomy. The vagina extends from the cervix to a point just cranial to the urethral orifice. Its caudal extremity is marked by a prominent transverse fold that often lies over the urethral orifice (Figures 1.1 and 1.24). The fold in young mares is more distinct, forming the hymen that restricts the entrance to the vagina. The fold marks the line of merger between the cranial portion of the repro- FIGURE 1.25. Inner surface of cervix (ex) and adja- cent uterine body (ub) plus vagina (va). Incision was made longitudinally through dorsal wall, and sides were reflected. Folds of endometrium and cervix are continuous, and a fold of the cervix extends onto the floor of the vagina as a ventral frenulum (f). ductive tract, which is of mesodermal ori- gin, and the caudal portion, which is of ectodermal origin. The lumen is extreme— ly dilatable, apparently limited only by the pelvic wall. Except during passage of penis or foal, however, the lumen is col- lapsed dorsoventrally and would appear on cross section as a transverse slit (Figure 1.8). Under usual conditions, the lumen is not dilated, as often depicted in illustrative material. The degree of col- lapse is partially dependent on the amount of fecal material in the rectum (Figures 1.3 and 1.8), and distention can occur due to urine pooling or aspiration of air. There is no external demarcation between the vagina and the cervix or vulva. The caudal portion of the vagina and all of the vulva are covered by fibrous tissue and a large amount of intramuscu- lar connective tissue. The remaining cra- nial portion of the vagina is covered by the visceral layer of the peritoneum to form the caudal extremities of the abdom- inal pouches (Figure 1.3). The fornix is an annular cavity around the projecting cau— dal portion of the cervix. The structure of the fornix and the surrounding anatomy (abdominal pouches) external to the vagi- na are of applied importance for surgical entry into the abdominal cavity through the cranial vagina (colpotomy). Histology. Photomicrographs of the vagina and vulva are shown (Figure 1.18). As in the other organs, the muscu- lar coat of the vagina consists of a thin, outer, longitudinal layer and a thick, inner, circular layer. The lumen is lined with stratified squamous epithelium that consists of a basal or germinal layer, a middle zone of several layers of polyhe- dral cells, and a superficial layer of flat- tened squamous cells (Figure 1.18,A; 85). The middle zone makes up the bulk of the epithelium. Cornification of the surface cells is not nearly as prominent as in some other species. The characteristics of the layers and cells change during the estrous cycle and are described elsewhere (pg. 209). The mucosa is highly elastic, per- mitting the accommodating dilation asso- ciated with parturition. There are no glands in the vagina (1488). 1.12. Vulva Terminology. The term vulva is used in English and French literature to encom- pass all of the reproductive tract caudal to the vagina (1488). In German works, however, it is applied only to the labia and other structures around the external orifice, while the portion internal to the orifice is termed the vestibule. Likewise, in common American vernacular the term is used for the labia. In this text, vulva will refer to all of the tract common to the terminal reproductive and urinary tracts. This area extends from the transverse Reproductive Anatomy 31 fold of the vagina, which is just cranial to the urethral opening, through and includ- ing the labia and associated structures. The term will thus be used for that por- tion of the reproductive tract that is ecto- dermal and originates from the embryon- ic urogenital sinus. It will be subdivided into vestibule (tubular portion, excluding labia), labia or lips, and the clitoris (Figure 1.24). Gross anatomy. The vulva is related dorsally to the rectum and anus, ventral- ly to the pelvic floor, and laterally to the sacrosciatic ligament and semimembra- nous muscle (Figure 1.26; 1488). The external orifice, a vertical slit 12 to 15 cm FIGURE 1.26. Transverse section through vestibule. as = anal sphincter cvu = constrictor vulvae r = rectum With fecal material sm = semimembranosus muscle ti = tuber ischii 32 Chapter 1 long in horse mares, has been termed the vulvar cleft. The cleft is bound by two prominent labia which form an acute angle above (dorsal commissure) and a rounded junction below (ventral commis- sure). The vulva hangs over the ischial arch of the pelvis (Figures 1.2 and 1.3) so that the ventral commissure is about 5 cm below the arch. This is an important consideration in attempts to pass a specu- lum or other instrument into the vulva and vagina. Thus, instruments should enter the vulva at an upward angle; this is the natural angle of intromission. The mucous membrane of the vestibule is rusty brown, and submucosal venous plexuses can be seen through the mucous membrane. The folds of the vestibule are arranged longitudinally. Histology. The vulva is lined with stratified squamous epithelium (Figure 1.18,B,C). This feature likely is an accom- modation to the abrasive effects of copula- tion and parturition. The epithelium of the mare vestibule often contains tubular evaginations (1634). The mucosa is rich in elastic fibers and contains numerous lymph patches. The epithelium is often infiltrated by leucocytes. The lymph patches and leucocytes may represent defensive mechanisms necessitated by the direct opening into the external envi- ronment in a heavily contaminated region of the body. In this regard, the joint reproductive and urinary roles of the vulva may also be important in that uri— nation may serve to periodically cleanse the area of fecal debris. The mucous membrane of the vestibule has rows of papillae on its ventral and lateral walls, including two linear series which con- verge toward the clitoris (Figures 1.1 and 1.27). The papillae mark the openings of the minor vestibular glands, which are branched and tubular and homologous to male accessory glands. The vestibular FIGURE 1.27. Exposed floor of vestibule showing rows of papillae which contain the openings of vestibular glands (A, arrows) and the glans clitoris (gc) after reflection of the prepuce of the clitoris (B). glands presumably contribute to the mucous secretions of the caudal portion of the reproductive tract. The wall of the vestibule contains cir- cular and longitudinal striated (skeletal) muscle (constrictor vestibuli) which is incomplete dorsally (Figures 1.2 and 1.26; 1634). There is some smooth muscle inside the constrictor, which is a continuation of the vaginal musculature. In addition, the constrictor is joined on either side by the band-like suspensory ligament of the anus which also consists of smooth mus- cle. The vestibular bulb is a flattened, oval body of erectile tissue located in the lateral wall of the vestibule between mucosal and muscular layers and just cranial to the labia. The vestibular bulbs and muscles contribute to the copulatory act by accommodating and securing the penis. Labia. The labia are covered by thin, smooth skin that is richly supplied with sebaceous and sweat glands (1488). The constrictor vulvae are layers of skeletal muscle located just beneath the skin that fuse with the sphincter ani above and surround the clitoris below. During copu- lation, the constrictors of the labia proba- bly function in concert with those of the vestibule. They serve to close the lips when necessary, yet are capable of extreme expansion during parturition. As this latter role suggests, the labia also contain elastic tissue. The constrictor vul- vae also function in the periodic eversion or winking of the clitoris in response to the passage of urine or mucus during estrus. Clitoris. The clitoris is the homologue of the penis and is located in a cavity (fossa clitoris) at the ventral commissure (Figures 1.3 and 1.27). It may be everted from the fossa by digital pressure and parting of the labia. The glans clitoris (the visible portion) is round, very irregu- lar or wrinkled on the surface, and about 2.5 cm in diameter. A thin fold (frenulum of the clitoris) attaches the dorsal central area of the clitoris to the roof of the fossa. Reproductive Anatomy 33 The glans clitoris is considerably more prominent in the mare than in other domestic species. The body of the clitoris (corpus clitoris) is about 5 cm long and is attached to the ischial arch by two crura (Figure 1.3). The integumentary fold enclosing the glans is the prepuce of the clitoris. The clitoris contains a prominent cor- pus cavernosum clitoris, which is homolo- gous to the corpus cavernosum penis. The equine glans is richly innervated and con- tains erectile tissue. The function of the equine clitoris has not been defined, but its prominent winking role during estrus (pg. 81) and its rich neural innervation suggest that it has been too long ignored. It is large and well developed in mares, and it is likely that it has developed in response to a physiologic need. The anatomical features of the clitoris have been receiving close attention in recent years because its sinuses support the growth of the causative organism of con- tagious equine metritis (1037, 1036, 1). 1.13. Vessels and Nerves of the Reproductive Tract The vascular, lymphatic, and nervous systems bring the reproductive system into functional relationships with other body systems. The vascular system of the reproductive tract performs the indis- pensable function of delivering products of the digestive, respiratory, and endocrine systems to the reproductive tract and of carrying reproductive tract products to the renal, respiratory, and endocrine systems. The vascular system thus complies with metabolic needs and also transports regulatory substances (hormones) through the systemic circula- tion from one organ to another. The ves- sels of the uterus undergo an expansion during pregnancy unequaled anywhere else in the vascular system. The vascular anatomy of uterus and ovaries has been described along with a review of earlier work (611). 34 Chapter 1 Arteries of Uterus and Ovaries. The uterus is supplied on each side by three arteries: the uterine branch of the vaginal artery (caudal uterine artery), the uterine artery (middle uterine artery), and the uterine branch of ovarian artery (cranial uterine artery; Figures 1.20 and 1.21). The uterine artery is the main arterial supply, since it is by far the largest in diameter. The uterine branch of the vagi- nal artery passes along the lateral side of the cervix and uterine body. The uterine artery follows the course of the uterine vein and supplies many small branches to the mesometrium. The uterine artery forms a caudal and cranial branch approximately 5 cm (in ponies) from the uterine horn (Figure 1.21). The caudal branch supplies most of the caudal por- tion of the uterine horn and forms anasto- moses with the uterine branch of the vaginal artery. The cranial branch is smaller and supplies the cranial portion of the uterine horn and forms anasto- moses both with the caudal branch and with the uterine branch of the ovarian artery. In older, parous mares, the main uterine artery follows a tortuous course, presumably as a result of involution of the broad ligament after parturition. The uterine branch of the ovarian artery in ponies begins approximately 6 cm from the tip of the uterine horn. The uterine branch of the ovarian artery provides a minor supply to the tip of the horn, but it has many branches supplying the oviduct and mesosalpinx. The three uterine arter- ies form prominent anastomoses with each other, contributing to arterial arches near the mesometrial attachment. There are also anastomoses of the arterial net— works between the two sides. The ovarian artery is located in the cranial portion of the broad ligament and follows the course of the ovarian vein and the uterine branch of the ovarian vein (Figure 1.20). The artery is not closely attached or applied to the ovarian vein; this is an important functional considera- tion (pg. 269). The ovarian artery bifurcates into a uterine branch and an ovarian branch (Figure 1.21). The ovarian branch forms many spirals of unknown function and is closely applied to a vein which runs from the ovary directly to a tribu- tary of the uterine branch of the ovarian vein. The surfaces of the ovary and the mesovarium are supplied with an exten- sive arterial network which forms anasto- moses with tributaries of the uterine branch of the ovarian artery (Figure 1.21). The internal vascular anatomy of the equine ovary (904), follicles, and cor- pus luteum (707) was described recently. Veins of Uterus and Ovaries. The uterus is drained on each side through three veins: uterine branch of vaginal vein, uterine vein, and uterine branch of ovarian vein (Figure 1.20). The course and area drained by the three veins are comparable to the course and area sup- plied by the corresponding arteries. However, the main venous drainage is through the uterine branch of the ovarian vein (cranial), whereas the main arterial supply is through the uterine artery (mid- dle). All three veins form extensive anas- tomoses with each other and with the veins of the opposite side. The main uterine vein (uterine branch of ovarian vein) is short (2 cm in ponies) and formed from two or three prominent branches approximately 7 cm long (Figure 1.20). The branches in some spec- imens appear to be approximately equiva- lent in diameter, whereas one of the branches appears to be dominant in other specimens. The uterine branch of the ovarian vein in ponies joins the ovarian branch approximately 9 cm from the uterus and 7 cm from the ovary to form the ovarian vein (uteroovarian vein). The ovary is drained through several veins that form the short (2 cm in ponies) ovarian branch of the ovarian vein approximately 5 cm from the ovary (Figure 1.20). The veins from the ovary form prominent anastomoses with each other and with branches of the uterine branch of the ovarian vein. In addition, a vein from the ovary passes through the spirals of the ovarian branch of the ovari- an artery and drains directly into a branch of the uterine branch of the ovari- an vein. The main uterine venous drainage and the ovarian venous drainage, therefore, form an extensive uteroovarian plexus of interconnecting veins. The ovarian vein and the main tributaries in the uteroovarian venous plexus contain prominent valves spaced approximately every 2 cm. Vessels of Vag'na and Vulva. Much of the caudal portion of the reproductive tract is supplied and drained by the inter- nal pudendal vessels that run caudally close to the ilium inside the sacrosciatic ligament (664). The pudendal vessels form the middle hemorrhoidal vessels Which, upon reaching the lateral surface of the vagina, divide into a cranial branch, the caudal uterine artery (previously described), and a caudal branch supply- ing the vagina and rectum. The corre- sponding vein drains an extensive plexus on the lateral surface of the vagina and vestibule. The pudendal artery also gives off a ventral branch, Which accompanies the nerve of the clitoris along the ventral surface of the vestibule and contributes to the arterial supply of the vestibule and labia. The internal pudendal artery ends as the artery of the vestibular bulb. A branch of the obturator artery contributes to the arterial supply of the labia and cli- toris. There are extensive venous plexus- es of large veins in the labia, especially at the ventral commissure around the cli- toris. The labial venous plexuses are drained by the perineal veins, the vein of the vestibular bulb, the middle hemor- rhoidal vein, and a large vein that runs cranially between. the thighs to the exter- nal pudendal vein. meh Vessels. Although the reproduc- tive organs are drained primarily by veins, the lymphatic system is also impor- tant. It has been estimated, for example, that approximately 10% of the ovarian steroids of sheep leaves the ovary through Reproductive Anatomy 35 lymph. Testicular lymph is an important route for secretion of the conjugated steroids in stallions (1422). Similar stud- ies apparently have not been done in mares. The lymph vessels in the broad ligament may be clearly visible and reach several millimeters in diameter in the pregnant mare (Figure 1.28). This com- plex arrangement of very large lymphat- ics is not generally appreciated, probably because the lymphatics are not apparent in the excised tract or in an in situ tract after it has been briefly handled or exposed. They are quite prominent, how- ever, immediately upon exposure of the broad ligaments, particularly in late ges- tation. Interstitial fluid enters the lym- phatic vessels, and the lymph passes through lymph glands and eventually reaches the thoracic duct and empties into the venous system near the heart. Unfortunately, no adequate description of the lymphatic drainage system of the mare’s reproductive tract was found; this must be considered a neglected research subject. The lymphatic system of the FIGURE 1.28. Lymphatic vessels (IV) in uteroovari- an area in a pregnant mare. Forceps is on uterine horn, and the ovary is indicated by an arrow. 36 Chapter 1 mare’s reproductive tract should be given research attention, including the role of the lymphatic vessels in the development of uterine cysts. Histologically, a rich net- work of lymph vessels can be demonstrat- ed in association with the endometrial cups (pg. 375). Nerves. The innervation of the genital tract is autonomic to some portions and voluntary to others. Parasympathetic fibers come from the sacral area Via the pelvic nerves, whereas sympathetic inner- vation comes from the caudal mesenteric ganglion and plexus and reaches the organs via the hypogastric nerves and pelvic plexus (1150). The caudal mesenter— ic ganglion is located on the origin of the caudal mesenteric artery. Two pairs of nerves proceed caudally from it. The internal spermatic nerve accompanies the ovarian artery and supplies the ovary, oviducts, and cranial portion of the uter- ine horn (similar to distribution of the ovarian artery). The other pair of nerves from the ganglion follow the aorta and enter the pelvic cavity. They anastomose with each other and with branches of the sacral nerves (3rd and 4th) and ramify on the pelvic organs. The pudendal and cau- dal rectal nerves from the sacral plexus carry sensory and motor fibers, the latter for the striated muscles of the vulva. Only the caudal portion of the tract (vulva) contains sensory fibers; incising the vulva will inflict pain, but incising the uterus will not. The innervation of the pelvic vis- cera in the mare has been described (547). 1.14. Pituitary and Hypothalamus Those with a weak background in embryology, anatomy, and physiology of the pituitary—hypothalamic area may wish to consult any of several contempo- rary endocrinology and reproductive physiology texts (665). It may be assumed that the general principles of pituitary- hypothalamic biology that have been established in many species also apply to the mare. This section will present anatomical features which have been demonstrated in the horse. Discussion of the physiology of this area is given else- where, particularly in Chapters 4, 5, and 7. A photomicrograph (Figure 1.29), mid— sagittal views (Figure 1.30), and dia- grams (Figures 1.31 and 1.32) are shown. Gross anatomy. The anatomical classi— fication of the various portions of the pituitary gland has evolved along lines consistent with embryologic origin. The gland results from a confluence of an embryonic rudiment from the brain and a rudiment from the epithelium of the oral cavity. The portion of the adult gland originating from neural tissue is called the neurohypophysis, and the portion originating from the oral cavity is called the adenohypophysis. The adenohypoph- ysis consists of the pars distalis, pars tuberalis, and pars intermedia. The pars distalis and pars tuberalis constitute the anterior lobe of the pituitary which is commonly called the anterior pituitary (Figure 1.31). The neurohypophysis con- sists primarily of the neural lobe and is FIGURE 1.29. Photomicrograph of adenohypoph- ysis. FIGURE 1.30. Midsagittal section through skull and brain showing overview (A) of exposed pituitary (pit) and closer view with additional dissection (B) to expose the pineal (pin). Nose is to the right. commonly called the posterior pituitary. The neural stalk of the neurohypophysis is of special importance because it attach- es to the median eminence Where the pituitary portal system begins. This por- tal system receives releasing or inhibitory factors from the hypothalamus and car- ries them to the anterior pituitary. The following description of the anato- my of the equine pituitary is principally based on published reports (682, 1488, 1706) and on examination of fresh heads by dis- section and frozen heads by slicing (575). The equine pituitary is flattened dorso-ven- trally and is horizontal in its long axis to the base of the hypothalamus (Figures 1.30, 1.31 and 1.32). It is approximately 2 cm wide. The pituitary is attached to the base of the brain by a delicate tubular stalk (infundibulum or neural stalk), which converges toward the rostrodorsal Reproductive Anatomy 37 h. // FIGURE 1.31. Diagram of components of equine pituitary and a portion of the hypothalamus. Adapted from (682). h = hypothalamus (striated) hr = hypophyseal recess mb = mammillary body me = median eminence nl = neural lobe (striated) pd = pars distalis (course stipple) pi = pars intermedia (black) pt = pars tuberalis (fine stipple) aspect of the gland. An inlet of the third ventricle is located within the neural stalk. Thus, substances secreted into the third ventricle by other portions of the brain (e.g., pineal) could have direct access to the neural portion of the pituitary. Unlike some other species (e.g., cow), the equine neurohypophysis is almost completely sur- rounded by the adenohypophysis; the pars tuberalis surrounds the neural stalk, and the pars distalis and intermedia sur— round the neural lobe. A small area of the neural lobe at the caudal pole is the point of entry of the caudal infundibular artery and is the only portion not cov— ered by adenohypophysis. The pituitary as a whole is covered by and adhered to a fibrous capsule derived from the dura mater. Grossly, the neural portion (neurohypophysis) is brain-like in color (light), whereas the glandular portion (adenohypophysis) is brown. The two are readily delineated. Contrary to what one might infer from its, name, the median eminence is not grossly distinguishable. It is a portion of the neural stalk and is surrounded completely by pars tuber- alis. The median eminence extends ros- troventrally to the optic chiasma (1706). The pars distalis forms the bulk of the 38 Chapter 1 pituitary. The hypophysial cavity is not present postnatally in the horse. Function. The pituitary and hypothala- mus interplay in complex fashion with the organs of the reproductive tract to provide reproductive rhythmicity. Through the hypothalamus and the pineal gland, the mare is brought into functional relationship with the environ- ment. The location of these three organs as an integral part of the central nervous system, therefore, facilitates general inte- gration of the reproductive tract with other body systems and with the external environment. Internal and external stim— uli are received by the hypothalamus and the pineal gland. These organs, in turn, influence the organs of the reproductive tract by way of the pituitary. The pitu- itary is located at the base of the brain in close physical and functional association with the hypothalamus (Figure 1.30). Since the role of the pituitary in repro- duction is that of an endocrine gland, proximity to the reproductive tract is not necessary. The anterior lobe of the pitu- itary is linked reciprocally to the repro- ductive tract by way of the ovaries. Gonadotropic hormones are released by the anterior pituitary into the blood and are carried through the circulatory sys- tem to the ovaries where they exert a reg- ulatory function. Similarly, hormones are released into the circulatory system by the ovaries. These hormones influence the anterior pituitary, primarily by way of the hypothalamus. In this manner, reg- ulatory messages are passed between the two distant organs. Blood vessels and portal system. The vascular system of the hypothalamus and pituitary is of special importance. The discovery of the vascular portal system between the two organs and the elucida- tion of the production of gonadotropic— releasing factors by the hypothalamus have resolved much of the mystery about the control of the anterior pituitary (non- neural tissue) by the hypothalamus (neu— ral tissue). Fortunately, extensive studies of the vascular system of this area have been done in horses by Vitums (1706) and have served to dispel earlier reports that the horse lacks a portal system. Vitums’ diagram of the blood supply of the equine pituitary system is shown (Figure 1.32). The primary capillary plexus of the equine median eminence is supplied by the rostroventral and rostrodorsal infundibular (hypophysial) arteries, which are branches of the internal carotid arteries and the rami communicantes caudales, respectively. Releasing hor- mones, which are secreted by neural tis- sue in the hypothalamus, pass into the capillary plexus of the median eminence and are then transported to the adenohy- pophysis by portal vessels. Two groups of portal vessels, the ven- tral and dorsal groups, can be distin- guished in the horse. It is possible that the ventral and dorsal groups are func- tionally related to specific zones of the median eminence and, in turn, to cytologi- cally differentiated areas of the pars dis- talis. Further study is needed to answer this important question. Vitums was unable to find a direct arterial supply to the equine pars distalis, and so it appears that the portal vessels in this species not only deliver messages from the hypothala- mus but are also the sole arterial supply. The embryonic development of the vascu- larization of the equine pituitary gland has been described (1707, 1708). The portal vessels were the sole blood supply to the developing gland, raising the question whether a functional portal system oper- ates in the early equine fetus. Further details of the hypothalamic-pituitary por- tal system are discussed in Chapter 2 (see especially Figure 2.3, pg. 52). 1.15. Pineal Although the equine pineal plays a pro— found role in reproductive seasonality (pg. 118), the dynamics of its anatomy have been neglected. Available information on the gross anatomy is included in this 39 Reproductive Anatomy FIGURE 1. 32. Diagram by Vitums showing the blood supply to the equine pituitary gland. From (1706). a — optic chiasma b = ventral wall of the median eminence dorsal wall of the median eminence : infundibular recess = neural lobe = pars tuberalis pars distalis = ' pars intermedia = mammillary body 0 ll H-U‘UQ HgCD 9-4 II chapter. There apparently have been no detailed histologic and ultrastructural studies of the equine pineal. The equine pineal has been described as a small, ovoid, red-brown structure about 10 to 12 mm long and 7 mm wide (1488). It is locat- ed deep between the cerebral hemi— spheres and a few centimeters dorsal to the pituitary (Figure 1.30). More specifi— cally, it is in a deep central depression between the thalami and corpora quadri gemina. It can be observed at necropsy by raising the caudal portion of the cerebral hemispheres away from the cerebellum. The pineal, like the pituitary, is attached by a stalk which contains a small recess of the third ventricle. The pineal is enclosed by a fibrous capsule from which numerous trabeculae pass inward, divid- ing the organ into spaces occupied by round epithelial cells (Figure 1.33). 1 0:01.th (DOD-<1 = a branch of the rostroventral infundibular artery = a branch of the rostrodorsal infundibular artery = caudal infundibular artery = ventral primary capillary plexus = ventral group of portal vessels = sinusoidal capillaries of the ventral region of the pars distalis = dorsal primary capillary plexus = dorsal group of portal vessels = sinusoidal capillaries of the dorsal region of the pars distalis FIGURE 1.33. Photomicrograph of pineal. 40 Chapter 1 HIGHLIGHTS: Reproductive Anatomy 1. The in situ reproductive tract varies from Y—to T-shaped, depending on the extent of floating upon or intermingling with the intestinal viscera. The broad ligaments attach the organs to the body wall and serve as an avenue for vessels and nerves. They contain large amounts of smooth muscle that is con- tinuous with the outer longitudinal muscle layer of the uterus and oviducts. The uterine body is located partly in the pelvis. It may be deflected, depending upon the fullness of the colon dorsally and the urinary bladder ventrally. The ovaries are concave on the ventral or free border (ovulation fossa) and convex dorsally at the attachment of the mesovarium (hilus). Much of the cortex is locat— ed internally unlike in other species. The oviducts are divided into infundibulum (funnel shaped), ampulla (expanded like an ampule), and isthmus (narrow structure connecting two larger struc— tures). From ovarian to uterine end, the mucosa of the oviducts decreases and the muscular layer increases in prominence. The myometrium is the longitudinal outer layer and circular inner layer of smooth muscle between the perimetrium (serosal covering) and endometrium (mucosa) of the uterus. Prominent glands open into the uterine lumen and extend to the depth of the endometrium. The folds of the cervix are highly branched (fernlike) and are continuous with the folds of the endometrium. The cervix projects into the vagina, and the area around the projection is called the vaginal fornix. A transverse fold lies over the urethral orifice and marks the separation of the caudal portion of the tubular genitalia into the vagina (mesodermal origin) and the vulva (ectodermal origin). The vulva originates from the embryonic urogenital sinus and may be divided into vestibule (tubular portion), labia (lips), and clitoris. The vestibular glands are homologous to male accessory glands and open in rows of papillae on the ventral and lateral walls of the vestibule. The clitoris is a homologue of the penis and is located in a cavity (clitoral fossa) at the ventral commissure of the labia. The clitoris is prominent in mares. The ovarian artery and veins from the uterus are not in close apposition, as in other farm species, accounting for functional differences among species. The posterior pituitary gland is almost completely surrounded by the anterior pituitary unlike in some other species (e.g., cow). The pineal gland is a distinct ovoid structure located between the cerebral hemi- spheres and a few centimeters dorsal to the pituitary. —-Cfiapter 2— REPRODUCTIVE HORMONES The principal reproductive hormones in the mare, as in other species, can be grouped into gonadotropins and steroids. The gonadotropins are luteinizing hor- mone (LH), follicle stimulating hormone (FSH), and chorionic gonadotropin (CG). The anterior pituitary is the source of LH and FSH, and cells of trophoblastic origin in the endometrial cups of pregnant mares are the source of CG. Gonadotropin releasing hormone (GnRH) is involved in LH and FSH regulatory mechanisms. Prolactin (PRL) has been purified from equine pituitaries, but its function in mares has not been defined. The repro- ductive steroids are numerous but may be subdivided into the estrogens, progestins, and androgens. The source of these steroids is less discrete than for that of gonadotropins and includes the ovarian follicles and corpora lutea, adrenal cortex, placenta, and fetoplacental unit. Other hormones that have important functions in female reproduction include inhibin, oxytocin, melatonin, the prostaglandins, and relaxin. A list of equine reproductive hormones and their sources and actions is given in Table 2.1. Modern endocrinology encompasses sig- naling mechanisms functioning within cells (autocrine) and between cells Within a tissue (paracrine), as well as between organs (endocrine). Little has been done on autocrine and paracrine messengers in the mare. This chapter will be concerned primarily with general chemical charac- teristics, sources, and synthesis of the interorganal hormones. It will be assumed that equids utilize those mechanisms that are generally considered to apply, in con- cept, to all mammals. Such fundamental concepts will be integrated with the cur- rent status of knowledge gained specifical- ly from equids. Readers seeking detailed biochemical information, especially at the intracellular and molecular levels, should consult recent sources; some will be sug— gested. Subsequent chapters Will cover regulatory mechanisms, concentrations in blood during various reproductive status- es, and other physiologic aspects. 2.1. Pituitary Gonadotropins (LH and FSH) The pituitary gonadotropins are ubiqui- tous among vertebrate species. When it is important to indicate species of origin of gonadotropins, a lower-case prefix letter is used (e.g., d = donkey, e = equine, h = human, 0 = ovine). Thus, in the equine species the pituitary hormones may be designated eLH and eFSH. The terms lutropin and follitropin are sometimes used for LH and FSH, respectively, espe- cially by biochemists (231, 232). The biologic activity of the gonadotropins is dependent on an initial binding to a specific receptor on the surface of target cells. A chain of events, initially involving cyclic AMP, then occurs and results in a response typi- cal for the hormone (1575, 691, 231, 1031, 1345). Purification. The structural and func— tional relationships of the gonadotropins in various species have been reviewed (1377, 858). The research area involving iso— lation and characterization of equine pitu- itary gonadotropins has a long history. The challenging aspects of this area of 42 Chapter 2 TABLE 2.1. Sources and Actions of Some Reproductive Hormones and Hormone-like Substances in the Mare Hormone Source Principal actions . Gonadotropins _ LH Anterior pituitary Luteal stimulant: progesterone Follicle stimulant: androgens FSH Anterior pituitary Follicle stimulant: estrogens . CG Endometrial cups Luteal stimulant: progesterone _. and estrogens Prolactin Anterior pituitary Mammary development GnRH Hypothalamus LH/FSH release Opioids Brain LH depression Inhibin Follicles FSH inhibition Melatonin Pineal Reproductive seasonality GnRH inhibition Relaxin Uterus-placenta Preparation for parturition Oxytocin Hypothalamus- Smooth muscle contractions posterior pituitary Reproductive steroids Estrogens Follicles, corpus luteum, conceptus Progestins Corpus luteum, conceptus Androgens ; Follicles, adrenals Prostaglandin F20: Widespread Endometrium Sexual characteristics and preparation of tubular genitalia Same as above FSH regulation Smooth muscle contractions Luteolysis investigation are attributable to the similarities in structure of eLH and eFSH, particularly because both must be extracted from the same gland. In addi- tion, purification attempts were ham- pered, at least initially, by poor assay specificity. Early studies (713) on the gonad—stimulating activity of equine pitu- itaries were based on the response of ovaries of immature rats to injections of pituitary extract. It was noted that the concentration of gonad-stimulating mate- rial was much higher in horse pituitaries than in pituitaries of other domestic ani- mals, probably because, as we have since learned, eFSH is less species specific. Much of the foundation of our current knowledge on the structure of eLH and eFSH is attributable to the persevering research of McShan and associates in the 1960-70s. Brazelton and McShan were the first to prepare highly purified eLH and eFSH (247). The hormones were diffi- cult to separate and required elec— trophoresis procedures that resulted in considerable loss of LH activity. Several laboratories continued to improve on purification and testing techniques, resulting in four- to-sixfold increases in yield by the mid-19808 (232). Preparations are now of sufficient purity and quantity for physiochemical characterizations. The earlier work used in vivo rat assay sys- tems that since have been found to show FSH activity in response to injections of eLH (232). More recent purification proce— dures have utilized in vitro bioassays that can distinguish eLH from eFSH (e.g., equine and avian testes receptor sys- tems). A recent study on FSH purification procedures was made by Mexican work— ers (949). Stewart and associates (1561) have initiated a program to isolate the genes for eLH, eFSH, eCG, and eGH (growth hormone) using recombinant DNA technology (genetic engineering). General composition. The pituitary gonadotropins (eLH and eFSH) are glyco- proteins with molecular weights of 33,500 and 33,200, respectively (1449). The hor- mones consist of 24% carbohydrate; the remainder is protein. Saccharides are linked in complex chains that are con- nected to polypeptide chains (1449). The high sialic acid content of the equine gonadotropins is noteworthy. In contrast, LH from many species does not contain sialic acid but FSH from various species does (1030). The presence of sialic acid has been long considered important for stabil— ity (half-life) in plasma; however, the amount of sialic acid in a given glycopro- tein cannot be the sole determinant of its plasma half—life (12). For example, eCG and hCG contain similar amounts of sial— ic acid but have much different clearance rates. Studies involving removal of sialic acid have suggested that it plays a role in in vitro activities of equine gonadotropins and affects the immunologic reactivity (12). Subunits. The subunit characteristic for eLH and eFSH, as well as for eCG and the gonadotropins from other species, is an important feature. The two glycopro- tein subunits are dissimilar and are referred to as alpha (0L) and beta ([3). A review of the amino acid sequence of the gonadotropin subunits of various species, including horses, is available (1729). The oc-subunit of the various gonadotropins has an identical amino acid sequence within species; it is apparently the prod- uct of a single gene (1563, 1561). The other pituitary glycoprotein hormone, thyroid stimulating hormone (TSH), also has the same a-subunit. The apparent amino acid sequence of the equine oc—subunit, howev— er, differs from that of other species and may be related to the unusual receptor binding ability of equine gonadotropins when administered to other species (1563). The B-subunit is unique for each hormone and determines the functional roles (231, 1563, 1449). Isolated B-subunits are Virtual- ly inactive; they must be bound to the Reproductive Hormones 43 oc—subunit to be biologically active. Combinations of an a-subunit of one hor- mone with the B-subunit of another hor- mone result in biologic activity consistent with that of the hormone from which the B-subunit was obtained (1449). Structure. Detailed structural studies of the equine gonadotropins have been done by Ward and associates and Papkoff and associates. The B—subunit of eLH (eLHB) consists of 149 amino acids, and the oc—subunit contains 96 amino acids (1729). The molecular weight of the oc-sub- unit was 12,500 and that of the B-subunit was 23,000. The immunoreactive proper- ties of eLH have been investigated (402, 943, 1309). Chemical studies, half-life determinations, and development of assay systems for eFSH have lagged behind those for eLH. The protein and carbohydrate analysis of eFSH is similar to that of FSH from other species (1166). The molecular weights of the oc- and B-subunits were similar (approximately 16,000). The amino acid sequence of eFSH has been reported (858). Isoforms. An active research area for many species during the 19808 centered on the multiple forms (isoforms, isohormones, microheterogeneity) of the molecules of the pituitary and placental gonadotropins. A recent compilation of reviews on the isoforms or microheterogeneity of the gly— coprotein hormones has been published (858). Other recent reviews are available (691) reflecting the current activity and interest in this area. More research in this area is inevitable because the ratio of iso- forms released by the pituitary, as well as the ratio of LH/FSH, can affect function at the target organs. In horses, the iso- forms of eLH (6) and eFSH (1031, 25) vary during the estrous cycle, and the chemical (247) and biologic properties (1030) of all the gonadotropins (eLH, eFSH, eCG) depend on isoform makeup. The different forms of LH and FSH could be attributed to various stages of biosynthesis in the pituitary, and perhaps the various forms are released into the circulation (1032). 44 Chapter 2 Changes in isoforms over time are indicated by changes in the ratio of in vitro bioassay results to immunoassay results (B:I ratio; Figure 2.1). The B11 ratio for eLH changed during the estrous cycle and during treatments with pro— lactin and gonadotropin releasing hor- mone (GnRH; 1089, 20, 21). The ratio was high when eLH concentrations were high (estrus) and low when eLH concentra- tions were low (diestrus). The ratio or biologic potency of circulating eLH appears to be due to an interaction of estrogen and GnRH (21). Several forms of eFSH were indicated by the results of a comparison of different assay systems (eFSH, hFSH, and oFSH antisera; 25). The eFSH concentrations were highest for the oFSH system when folliculogene— sis was expected to be slow (anovulatory season, estrus) but lowest when folliculo- genesis was expected to be rapid (transi- tional period, diestrus). Since the pres- ence of isoforms affects immunoactivity, selection of type of RIA system for a par- ticular goal is challenging. An interesting discussion by Irvine (800) on the applied ramifications of eLH isoforms could also apply to eFSH. Microheterogeneity and, therefore, bio- logic activity was attributed to the differences in the amount of sialic acid in the gonadotropin molecules of a given preparation or sample. It was also suggested that an increase in the ratio of desialylated to fully sialylated mole- cules may increase immunogenicity. This may account, at least partly, for the differences in assay results among laboratories using antibodies against various purified preparations. As another example, Irvine notes that results of half-life studies may be affected according to the proportion of molecules with various amounts of sial- ic acid. Two recent reviews comment on the complexities of interpretation of pituitary gonadotropin assays, espe— cially in regard to bioactive and immunoactive isoforms (791, 1728). eLH microheterogeneity 100 00 O O) 0 Concentration (ng/ml) J:- O M O _4 o 4 8 12 16 Number of days from ovulation FIGURE 2.1. Concentrations of eLH measured by radioimmunoassay (RIA) and in-vitro bioassay throughout the estrous cycle. Differing profiles are attributable to microheterogeneity of eLH. Means that differ between assays within a day are indicat- ed by a star. Adapted from Alexander and Irvine (20). Cross—hormonal activity. Despite their common and closely related chemical structure and nature, LH and FSH from most mammalian nonequine species show little, if any, cross-hormonal activity (1345). However, eLH is an exception; it has intrinsic FSH activity as well as potent LH activity when injected into nonequine species. Furthermore, eLH may both enhance and inhibit FSH action in other species. Papkoff and associates have reported that eLHa appears to bind to FSH receptors in nonequine species, thereby inhibiting FSH stimulation by competitive antagonism (1345). Curiously, it appears that the oc-subunits of eLH pos- sess inhibitory intrinsic FSH bioactivity even though the amino acid components of the oc—subunits of the gonadotropins are believed to be identical. Experiments that used in vitro stimulation of rat sertoli cells have indicated that eFSH stimulato- ry action lasts much longer than for eLH or eCG or for FSH from nonequine species (658). Donkey LH (dLH) has been found to possess little intrinsic FSH activ- ity, unlike eLH (1355). That is, eLH behaves much like eCG, which also has both LH and FSH activity, but dLH and dCG both possess minimal FSH activity. The target tissues of eLH and eFSH con- tain specific receptors for FSH, but the receptors for eLH and eCG are identical (231). The pituitary gonadotropins of zebras (zLH, zFSH) were recently described for the first time and compared to the gonadotropins of horses and don- keys (1029). Homologous RIAs for eLH and eFSH did not cross-react in a similar or parallel fashion with gonadotropins from the donkey and zebra. Clear intrinsic FSH activity was shown by eLH, but not by dLH and zLH. Half-life of eLH. The rate of disappear- ance of eLH from the blood of mares has been studied (321). The calculated disap- pearance rate (half-life) was approximate- ly one hour during the first hour after injection and 4 to 5 hours during the second and third hourly segments of the disappearance curve (Figure 2.2). Progressive decreases in the rate of disap- pearance of LH and FSH have been reported in other species and attributed to differences between rate of equilibra- eLH half-life M Concentration (ng/ ml) —L 0 25 50 75 100 125 150 175 Number of minutes after injection FIGURE 2.2. Disappearance rate of equine LH as depicted by the regression of LH on time after an intravenous injection of a low dose of a pituitary fraction. Adapted from (618). Reproductive Hormones 45 tion among body compartments and dif- ferences in rate of metabolism. The disap- pearance time of eLH is long when com- pared with the disappearance time of LH in other species, whether injected into the homologous species (equine) or a heterolo- gous species (nonequine). For example, the disappearance time for eLH when injected into the rat is more than four times longer than for hLH and approxi- mately 18 times longer than for ovine LH (1235). Study is needed on the extent to which the slow disappearance rate con- tributes to the slow decrease in circulat- ing endogenous eLH that occurs subse- quent to ovulation in mares (pg. 234). Release of hormones in pulses. Oscillating or pulsatile phenomena were first recognized in 1958 in simple inor- ganic chemical reactions (review: 1313). In biochemical reactions, oscillations are ubiquitous. For example, autonomous glucose oscillations (involving minutes) occur when glucose is added to the gly- colytic pathway. One View is that in the evolutionary process pulsatile phenomena were accepted because they did no harm and were too difficult to suppress. Episodic secretion of hormones has been known since the 1970s when serial mea— surements of LH and FSH revealed dif- ferences of about twofold between peaks and nadirs (308). Statistical methods of cross-correlation between the two hor— mones are used to determine if the same pulse oscillator controls both hormones. The pulsatile discharges of LH and FSH are in turn attributable to pulsatile dis- charges of GnRH and have led to the concept of a GnRH “pulse generator” involving synchronous activation of neurons (453). The most parsimonious hypothesis on the nature of the pulse generator is that a single cell or a small cluster of cells entrains other participat- ing cells in the resulting neuroendocrine cascade (459). Ovarian hormones also may involve pulsatile release (308). The degree to which periodic signaling (changes in frequencies and amplitudes of 46 Chapter 2 pulses) has been adapted for conveying regulatory messages is not clear. It now is known, for example, that eLH and eFSH pulses change in character as mares move from the anovulatory to ovulatory seasons. Does the nature of the pulses convey a message that is distinct from the message due to the cumulative level of circulating LH (e.g., as determined daily)? This question is of importance in experimental design. Frequent sampling (e.g., every 10 minutes for 2 hours) may be necessary to monitor the pulses, but one sample per day may be all that is nec- essary if accumulative signaling is of principal importance. Similarly, to what extent are previous descriptions of hor— monal interactions that were based on daily sampling negated by the discovery of pulses? From a statistical viewpoint, pulses would contribute experimental error to daily sampling techniques, but this could be overcome by providing for an adequate number of observations or animals in each test group. On this basis, the discoveries made by daily sampling remain valid, but frequent sampling may provide additional information. The prac- tical question is whether frequent sam— pling will provide enough information to warrant the added cost. Subsequent chapters will describe the nature of puls- es of various hormones and the manner in which they change during the various reproductive states. Pulsatile secretory patterns have been studied most extensively for LH and were described for many species in the 1970s. An attempt was made to document such a phenomenon during the summer in ovariectomized mares using sampling every 20 minutes; the attempt was not successful (552). However, episodic LH secretion was found during the equine estrous cycle by Evans and co-workers (484) and in association with seasonality (512) and postpartum events (511) by Fitzgerald and co-workers. Failure in the earlier study to detect pulses in ovariec- tomized mares in the summer (552) is attributable to the subsequent finding that when LH levels are high the pulse frequencies are too rapid to detect with sampling every 20 minutes (512). The duration of eLH peaks during GnRH treatment has been reported as approxi- mately one hour, regardless of reproduc- tive status (1393). Decreasing pulse ampli- tude of eFSH during the onset of the ovulatory season has been reported (731); the decrease in amplitude was associated with a decrease in mean daily concentra- tions. The task of monitoring hormone levels becomes complex when the phe— nomena of pulsatility, as well as half-life and microheterogeneity, are considered. Simultaneous peaks in LH and FSH were first detected in stallions by Thompson and associates (1608). In a sub- sequent study in mares (1602), these workers obtained sequential samples every 15 minutes for 24 hours during various reproductive states. It was con— cluded that mares in addition to stallions exhibit simultaneous peaks in the two gonadotropins in contrast to other domestic farm species. In ovariectomized mares, LH and FSH peaks were tightly coupled in both summer and winter. In cycling mares, samples were obtained during diestrus (Day 10 or 11) and dur- ing estrus. The LH and FSH peaks were coupled during diestrus but not during estrus. In a recent study involving daily sampling (184), synchronous surges of the two gonadotropins occurred during diestrus, but the synchrony waned and disappeared during the periovulatory LH surge (pg. 235). However, an experiment involving sampling of pituitary effluent (23) demonstrated that FSH and LH secretions are pulsatile and synchronous during the periovulatory period. These authors commented that failures to demonstrate such synchrony of the two gonadotropins in samples from periph- eral blood is probably due to long half- lives and the large pool of blood which leads to a damping of individual pulses. Concomitant LH and FSH pulses also have been detected during the transition between ovulatory and anovulatory sea- sons (731) and during the periparturient period (732). 2.2. Prolactin Prolactin (PRL) is produced by the anterior pituitary. It was originally termed a luteotropic hormone (LTH) because of its importance to progesterone production by the corpus luteum in rats (1128). Although there are receptors for PRL on the luteal cell membranes in many species, the support for a Vital luteotropic role for PRL across species appears to be waning. Equine prolactin (ePRL) has been isolated and character- ized (298, 959). The molecular weight is approximately 25,000 (298). The ePRL molecule consists of 199 amino acid units, as does PRL from other species (959). However, ePRL has two disulfide bridges in contrast to three bridges in other species. Antisera have been produced that do not cross-react with eLH or eFSH but do cross—react with sheep and cattle PRL (298). The pigeon-crop test is a traditional bioassay procedure. A heterologous RIA based on sheep preparations failed to detect changes in ePRL secretion rates in mares during pregnancy and lactation (1143) and did not detect changes in the response of ePRL to thryotropin releasing hormone (1603). However, homologous RIAs using ePRL or ePRL antiserum and radioidodinated canine PRL were devel- oped recently (1828, 1346, 828). These assays have shown that ePRL concentrations increase in late pregnancy, as occurs in other species. Development of a homolo- gous assay for equine growth hormone also has been reported (272). The role of PRL in mares has not been well defined. The increased ePRL proba- bly plays a role in the development of the mammary glands and in milk secretion (1828). In this regard, concentrations may be inadequate in agalactic mares (1613). Reproductive Hormones 47 Concentrations of ePRL in the blood (828, 830, 1601) and in the pituitary (1601) increase during the ovulatory season in relation to increasing daylength (pg. 124). A recent study (488) found a high positive correlation between prolactin concentra- tions and daylength. In addition, ePRL concentrations were greater during estrus of the ovulatory season than during the anovulatory season (1611). The patterns of ePRL concentrations and their role dur- ing the estrous cycle are unclear (828, 1827). A surge of ePRL sometimes has been found during estrus, perhaps in relation to estrogen production (1827); the authors noted that estrogen stimulates PRL production in other species. Release of ePRL occurs in pulses (1354), as does release of eLH and eFSH. The nature of the pulses changed during the estrous cycle, suggesting that ePRL may be involved in ovarian function. Recently (488), measurement of prolactin in the pituitary venous effluent showed pulses of 2 to 7 minutes duration. In a recent study (1149), treatment of anestrus lactat— ing mares with bromocriptine, a dopamine agonist that depresses PRL, caused a precipitous decline in PRL but did not stimulate ovarian activity. Bromocriptine has been used also to reduce ePRL and progesterone concentra- tions in late pregnancy as a model for studying the pathogenesis of fescue toxici— ty (pg. 471). 2.3. Chorionic Gonadotropin The endometrial cups of the equine species produce a gonadotropin of tro- phoblastic origin (pg. 370 and pg. 419). The gonadotropin was formerly called preg- nant mare serum (PMS) or pregnant mare serum gonadotropin (PMSG; 1558). Today, at least in the scientific literature, the preferred term in horses and ponies is equine chorionic gonadotropin (eCG) for balance with chorionic gonadotropins of other species (e.g., human, hCG; donkey, dCG). Chorionic gonadotropins occur in 50 Chapter 2 among the four days. The ratio of FSH:LH activities ranged from 2 to 96. Preparations with high FSHzLH ratios were more conducive to induction of ovu— lations in cattle. In more recent studies by this group (1012), FSH activity was greater on Days 71 and 104 than on Day 39; LH activity varied with sire but not day. Removal of sialic acid increased the LH bioactivity. It was proposed that variations in bioactivity of CG during ges- tation and among animals were a func- tion of differences in carbohydrate con- tent. In this regard, CG isolated from a low-titer versus a high-titer pool of sera differed in amino acid and carbohydrate composition and in assay assessment (9). Low—titer CG resembled CG from tissue, and high—titer eCG resembled eCG previ- ously isolated from sera. Cross activity in other species. Since eCG and eLH possess a common B-sub- unit polypeptide chain, both hormones exhibit dual LI-I/FSH activity in all sys- tems studied, except that they exhibit only LH activity in equine systems (330). However, eCG has only 4% of the binding activity of eLH to horse LH receptors. The dual LII/FSH effect of eCG in other species has been known for 50 years (325) and has been confirmed (960, 328, 233). Part of the FSH activity of eCG in nonequine species may be due to lack of specificity of FSH receptors (960, 328, 233, 1034, 1355). Even horse FSH receptors recognize some FSH—like structure in eLH and eCG (cited in 231). The FSH2LH activity ratio of eCG was reported to be 0.20 in the pig, 0.25 in the rat, and 0 in the horse (329). In the rat, the behavior of eCG was similar to that of eLH. Interestingly, eCG has an extremely low binding affinity for eFSH and eLH receptors in the equine ovary (1557, 1558). The eCG molecules were only about 10% as effective in binding to eLH receptors, compared to LH receptors of nonequine species. Furthermore, binding of eCG to eFSH receptors was negligible. These findings are compatible with the apparent refractoriness of equine follicles to exogenous eCG. In a preliminary com- munication, the receptor-binding activi- ties of zebra CG (zCG) were reported to be more like those of donkey CG (dCG) than eCG (1555). It is likely that zCG, therefore, is structurally similar to dCG. In this regard, a recent report (1057) described the partial purification and characterization of zCG; the authors indi- cated that zCG was bioactive as an LH and competed for LH but not FSH recep- tors. Apparently zCG, like dCG, dLH and, zLH has minimal FSH activity, unlike the high intrinsic FSH activity in eCG and eLH. Assay. The original quantitative mea- surements of eCG utilized bioassays based primarily on ovarian weight changes in immature rats. Potency was expressed in terms of ovarian weights or rat units. The First International Stan- dard for Serum Gonadotropin was subse- quently prepared, and potency was esti- mated in terms of International Units. The Second International Standard was prepared in the 1960s (153) and is now used as the reference standard. The first immunoassay for eCG was described in 1963 (1777) and used a rabbit anti-eCG serum-absorbed inhibition assay (41); the absolute eCG levels (43) were comparable to those obtained with a bioassay system (762). An LH radioimmunoassay system with high eCG cross-reactivity has also been used to quantitate eCG and was reported to be considerably more sensi— tive than the hemagglutination-inhibi— tion assay (1143). Equine pituitary gonadotropins cross-react with eCG radioimmunoassay systems, whereas those of other nonequine species give minimal or nonspecific cross-reactions (497). Whole-animal bioassay of eCG often utilizes weight changes in ovaries or uteri of immature mice or rats. Because bioassays are based on biologic response, they lack precision. The production of anti-eCG sera in rabbits and turkeys and the development of a sensitive hemag- glutination-inhibition assay have been described, and immunologic assay proce- dures for eCG have been reviewed (41). A method for quantitation of eCG by radioreceptor assay was reported by English workers (1559). The source of a standard can profoundly affect assayed concentrations of eCG. Some commercial producers of eCG use in—house standards for routine work, whereas others use an international reference. A comparison of two reference preparations of eCG was made recently (273). These authors noted that the commonly used Second Inter— national Reference Preparation (IRP2 obtained from WHO) was prepared with the use of only in vivo assay technology. It was concluded that IRP2 was a reliable standard for in vivo assay, but that a ref- erence preparation from NIH was more suitable for in vitro assays. However, in vitro assays do not take into account eCG half—life. Half-life. The half-life of eCG has been reported to be six days in geldings, 24 to 26 hours in rabbits (293), and 26 hours in rats (1234). The biologic half-life of eCG in rats is considerably greater than for any other gonadotropin (1320). It has been sug- gested that eCG may repeatedly stimu- late receptor sites during its stay in the plasma (1320). That is, eCG may bind to the sites, be released, and then bind again to give a repeated hormonal stimu- lus. The half-life of the endogenous eCG after removal of the source by hysterecto- my was approximately six days (319). Since these data were comparable to the findings in the gelding, it was concluded that mare ovaries do not play an impor- tant role in eCG metabolism. It is believed that eCG is destroyed within the animal body and, unlike hCG, is not excreted in the urine (293) or is present in urine only in small amounts (1406). An example is given of a 227-kg pony in which serum contained 170 iu/ml, urine 2 iu/ml, and milk 0.2 iu/ml on Day 67 of pregnancy. The total amount of eCG in the blood was estimated to be one million international units. It has been estimated Reproductive Hormones 51 that excretion accounts for about one-sev- enth of the eCG loss, and metabolism pre- sumably accounts for the rest (319). Recent preliminary studies indicated that approximately 1% of eCG is excreted in urine and that this amount is adequate to quantitate eCG production at various stages of pregnancy (1352); radioim- munoassay (RIA) or a commercial dip- stick enzyme-linked immunosorbent assay (ELISA) were adequate assay sys- tems. The assays used a high affinity monoclonal antibody (1353). The plasma and urine profiles of eCG were similar, demonstrating the presence in urine of eCG immunoreactivity. However, the antibody also detected apparent LH molecules in the urine of cyclic mares. Isolation of eCG from the urine and rais- ing a monoclonal antibody against it may result in a more specific assay. Late entries. A comprehensive review of 164 references on eCG has been pub- lished (1868) and is recommended. The review considers structure, secretion, bio- logic activity, and function of CG in equids and the biologic activity and half- life of eCG in other mammals. The pres- ence of immunoactive and bioactive eCG- like material has been described in full-term placentas of horses and zebras {1865). 2.4. Gonadotropin Releasing Hormone (GnRH) The hypothalamus, the true master gland, is the central transducer that con- verts neural signals to hormonal signals. One of its products, GnRH, is the pivotal regulatory hormone controlling reproduc— tive biology. Apparently every vertebrate species in its evolution included the GnRH gene in its genome. Environmental factors (e.g., daylength, pheromones) impinge upon the hypothalamus through neural or pineal signals, which are pro— cessed and converted to hormonal signals. The most profound such signal is GnRH. 48 Chapter 2 primates and possibly in sheep, rabbits, and guinea pigs (1575). However, equjds and primates are the only known mammals that produce chorionic gonado- tropins in massive quantities. Endometrial cups of mares also secrete a protein that resembles growth hormone (1560). As noted below, the discovery of eCG was a milestone in reproductive physiology, not only because of the important biologic role in the equine species, but also because of the widespread use of the hormone as a pharmacologic substance in other species. It has been used for many years to stimu‘ late ovulation and superovulation in cats tle and other species and for estrus syn‘ chronization in sheep, Its great utility for these purposes is related to its long half- life and its ability to stimulate both FSH and LH activities in other species, muggy, The premier report on the appearance of a gonadotropic sub- stance in the blood of pregnant mares was made by Cole and coworkers in California in 1930 (321), and a more limits ed report was made by Zondeh in Germany the same year (cited in 294).. The California work was initiated because of a finding two years earlier that such a sub stance is present in the blood and urine of pregnant women. it was found that blood serum of mares at certain stages of preg- nancy stimulated the genital system of immature rats and mice. The report included a characterization of the gonads- tropin curve and a proposal for use of the hormone in pregnancy diagnosis At that time the hormone was presumed to be of pituitary origin. During subsequent years the source of eCG was considered to be either the pitu! itary (321;) or the cells of the allantochori— on (294).. Based on quantitative studies of various tissues. it was eventually con—- eluded that the endometrial cups were the chief source of the gonadotropin (pg, W320) The cups contained 50 to 900 times more hormone/gram (ft to 12 iufmg)‘ than the chorion and t? to 2% times more than the endometrium between the cups. The cup secretion contained 10) to 35 times more hormone/gram (50 to 314: iu/mg) than the cup issues- The slight activity of the chorion found by earlier workers was explained by the adherence of endometrial cup secretion to the chori— on. An interesting account of the early debates regarding the source of eCG is available (320) W gflfiproducjng @112 cells. On the basis of histochemical staining for glycoproteim (PAS reaction), it was concluded that the epithelial cells of the cups, rather than the decidua—like cup cells, were the source of C G (311). The cup secretion, uterine epithelium, and glandular epithelium stained PAS positive, whereas the cup cells gave little if any reaction. On the basis of electron microscopy and immumo~ fluorescence techniques, other workers (724) also suggested that the epithelial gland cells within the cups synthesize CG. In contrast, others (I830) reported that the cup cells contained PAS-positive material. The classical studies of Allen and Moor ('62) resolved the questionofcup cell origin and eQG source (pg. 370). The origin of the cup cells fizom invathng tro~ phoblastic cells of the chorionic girdle of the conceptus was demonstrated In adds tion, the capability of the girdle cells to produce CG was demonstrated in— citro. Cultured girdle cells were morphologiods by similar to cup cells which, develooedin pivot and assays of? the ctdture media show ed high concentrations of? CG. Maximal concentrations in the culture fluid exceeded levels reached in the maternal circulation by awesomely fourfold. lt is not known, hQWKQVQfi how well the total productive capacity offan entire codtured chorionic girdle cowowas tothetotel output oftCGhy thesidows trial cups The cultured cells wels are ductive for about we dress loose that} them oioo celis $111in oultwesoflfstal skint eedozaetriow» sod allwuoohorios item outside ofttho girdle was did not produceCG Composition. Relatively simple tech- niques for preparation of eCG with high biologic potency have been reported (640, 641). Yields of the purified product were 50% to 80% of the amount initially present in serum. The equine glycopro- tein, CG, has been well studied by bio- chemists because of its intriguing make- up and its therapeutic value. The carbohydrate content of eCG was 47%, which is approximately twice as high as in eFSH and eLH (1449). A molecular weight of 53,000 has been found for the unreduced and unalkylated form of CG (640). Of the glycoprotein hormones from various species, eCG has been reported to be the most complex in carbohydrate makeup (653). The molecule is rich in sial- ic acid (14%), perhaps contributing to its long half-life and, therefore, to its effec- tiveness in other species even when given as a single dose. The half-life, however, is so long that regimens have been devised for cattle that terminate continued superovulatory activity by administra- tion of an anti-eCG. The relationships between hormone structure and function have been difficult to establish for this and other large glyco- protein hormones. One approach is to chemically modify a specific functional group and then study the resulting modi- fications in biologic or immunologic func- tion. The role of histidine in eCG has been studied in this manner (11, 1227). Results indicated that the histidine sites are important for both the FSH— and LH- like biologic properties of CG, but the his- tidine sites do not involve immuno- recognition. Subunits. A molecule of eCG consists of two subunits as do all placental and pituitary gonadotropins in all studied mammalian species. The two subunits of CG have been dissociated and character- ized (1225). The subunits were biologically inactive, but activity was regenerated upon recombination of the subunits. The amino acid sequence of eCGB consists of 149 units and has the same primary Reproductive Hormones 49 structure as eLH|3 (1575). It appears that equids, unlike primates, do not require different genes for expression of LHB and CGB subunits (1555). That is, equids apparently have a single B-subunit gene for LH and CG that is expressed in both the pituitaries and endometrial cups (1562; also see late entry 1872). Isoforms. As occurs for eLH and eFSH, various isoforms may be released accord- ing to biologic needs, and differences in composition between storage forms and circulating forms of the gonadotropins would be important in preparation of sub- stances for therapeutic purposes or for research (e.g., as standards in RIA). The eCG in endometrial cups or cultured tro- phoblastic cells has been purified and compared to the circulating form in sever- al studies by Papkoff and associates (10, 1228, 1226). A recent study (1033) con- firmed that the properties of eCG from cultured cells resembled those of CG from cup tissue. Considerable biologic and immunologic differences were found between the preparations derived from serum versus tissue. Cup CG was more potent with some assay systems but not with other assay systems. The authors suggested that various isoforms of CG may be under endocrine control as has been reported for the pituitary gonado- tropins (pg. 43). However, CG apparently is secreted in continuous fashion and is not under regulatory control like the pitu- itary gonadotropins (pg. 422,- 1604). Specific radioreceptor assays have been developed for FSH-like and LH-like activities of eCG using tissue receptors from rat testes (1559). The ratio of the two activities did not vary significantly among mares or stages of gestation. The CG had both FSH and LH activity, and the FSH-like activity in serum CG exceeded that of LH-like activity by a fac- tor of approximately 1.5. However, Murphy and associates (783, 637) reported that FSH activity was higher for CG col- lected on Days 60 and 90 than on Days 45 or 120; LH activity was not different 52 Chapter 2 Its action on the anterior pituitary leads to a cascade of hormonal events through— out the pituitary and reproductive sys- tem, complete with internal adjustments through hormonal feedback. Thus, GnRH releases both LH and FSH from the pitu- itary, but apparently the ratio of LH:FSH reaching the circulation is adjusted by GnRH pulse frequency or by feedback controls (e.g., inhibin, estrogens, pro- gestins; g. 252). The hypothalamic-pituitary portal sys- tfin. It will be recalled (pg. 38) that sub- stances produced in the hypothalamus are transported to the anterior pituitary through a system of portal vessels. Among the substances so transported is GnRH, which is elaborated by neurose- cretory cells in the medial basal hypotha— lamus (Figure 2.3). Axons of the neurose- cretory cells project into the perivascular space in the median eminence at the ori- gin of the pituitary stalk. In response to the appropriate hormonal and neuronal cues, the axons release GnRH in a pul- satile manner into the hypothalamic- pituitary portal system, which carries the GnRH to the anterior pituitary. A review of the detailed organization of the GnRH delivery system in mammals is available (1199). Sampling techniques. After the GnRH- laden blood from the median eminence bathes the gonadotropin-producing cells of the pituitary, a little GnRH presum- ably remains to pass into the pituitary venous drainage and general circulatory system. The resulting diluted circulating concentration of GnRH is miniscule, and, in addition, GnRH has a short half-life (5 to 10 minutes; 335); these factors have been a deterrent to assaying GnRH concentrations in the peripheral circula- tion (802). The assay problem has been overcome by measurement of GnRH in the pituitary-hypothalamic area (304) and has led to the discovery of the pul— satile nature of GnRH control (1 or 2 pulses/hour). Some of the sampling tech- niques include direct accessing of the por- Hypothalamic-pituitary portal system , ’ Gn RH \ ’ secreting \ neurons \ From hypophyseal arter y Fenestrated capillary plexus Gonadotropin secreting cells ;:(— Hypothalamus FIGURE 2.3. Conceptual illustration of the nature of the hypothalamic-pitu— itary portal system. \ (w Posterior Regulatory substances P'tU'tarY (e.g., GnRH) are produced - by neurosecretory cells in Anterior pituitary the hypothalamus. Axons of the cells project into the . median eminence which contains an elaborate com- plex of fenestrated capil- laries. The GnRH passes into the capillary complex and is carried by a system of portal vessels to a simi- lar capillary complex involving the anterior pituitary. The GnRH sig~ nals the pituitary cells to produce gonadotropins which in turn enter the complex of capillaries to be carried into the general To eneral , , g mrculation. circulation tal system in sheep, sampling of pituitary effluent in horses (803, 23), and push-pull systems for sampling the fluids of the median eminence or third ventricle. The push-pull technique has been used in mares (1434) and involves perfusing a fluid carrier medium and then aspirating the carrier. The retrieved carrier is assayed for GnRH from the hypothalamus. The technique of placing a cannula into the pituitary effluent apparently cannot be done in nonequine domestic species (803). It is a painless method, allowing sequen- tial sampling of unrestrained equids. It is anticipated that the technique will be widely used in the 1990s. Because of the distinct advantage that the technique brings to horses as research models, it is possible that we will soon know as much or more about hypothalamic-pituitary interactions in the horse as in any species. Horses could become the research model of choice for such studies. A recent study, presently available only as an abstract (1424), has used the technique to compare the microheterogeneity of eLH and eFSH in the pituitary. It was con— cluded that the isoforms of the gonado- tropins secreted by the pituitary at estrus are dramatically different from those stored in the pituitary. Structure and mechanism of action. The discovery, isolation, structural char- acterization, and synthesis of GnRH is one of the great milestones in reproduc- tive biology and was the subject of a Nobel Prize. It is a peptide containing 10 amino acids (decapeptide). Its composi- tion is identical in all species studied. The association of GnRH with granules sepa- rated from the equine hypophyseal stalk has been reported (811). The axons of the external layer of the equine hypophyseal stalk have vesicles of 60 to 250 mu diame- ter that are high in GnRH activity. Apparently GnRH, as well as other neu- rohypophyseal hormones, is contained in the vesicles of the median eminence in the horse. The distribution of GnRH in the neural structures of the equine brain Reproductive Hormones 53 has been studied by immunohistochem- istry using an antiserum prepared against GnRH conjugated to bovine or rabbit serum albumin (39], 1120). In this way, the nonantigenic property of GnRH (same molecule in all species) has been circumvented. Sites of GnRH storage in the equine hypothalamic area have been determined by assay of tissue sections (1570). The mechanism of action of GnRH, as well as other hormones, at the cellular and molecular level is beyond the scope of this text. For those interested in this important aspect of interrelationships between hormones and cells, other sources are available that review and describe results from nonequine species (775, 701, 307). These reviews consider the current concepts of the manner in which GnRH interacts with plasma membrane receptors of the gonadotropin—producing cells of the pituitary and the manner in which occupancy of the receptors results in a cellular response. The release of LH in response to GnRH occurs within sec- onds, and biosynthesis of additional LH occurs over hours and days. Pulsatility. The GnRH molecules are delivered to the pituitary in pulses (pg. 45; 23). The differential release of LH and FSH may depend on pulse frequen- cies as indicated by GnRH pituitary perfu— sion studies in rats (1400). In this regard, a preliminary report indicated that horses that were actively immunized against GnRH had a greater reduction in circulat- ing concentrations of LH than FSH (558). The lack of effect of GnRH immunization on FSH response to exogenous GnRH was taken as an indication that FSH stores in the pituitary were relatively independent of GnRH bioavailability. Down regulation. Because of the pulse phenomenon, prolonged or massive treat- ments with GnRH can be counterproduc- tive in most species. The effect of treat— ment with constant large doses has been called down-regulation and is so effective that continuous release systems have been advocated as contraceptives for 54 Chapter 2 humans (cited in 802). Chronic treatment in nonequine species suppresses the gonadotropin production and inhibits a wide spectrum of reproductive functions in the female, including follicular devel- opment, ovulation, luteal function, and pregnancy; cyclicity resumes after treatment is terminated (537, 1005). In horses, a constant (28 days) massive dose (1.3 mg/kg/day) of a GnRH agonist may suppress circulating LH concentrations in agreement with results for nonequine species (514). Maximal suppression (e.g., LH at 20% of pretreatment values) occurred a day or two after the end of the treatment regimen. Suppression, there- fore, may have reflected desensitization of the pituitary to endogenous GnRH. High doses (e.g., 10 mg/day) of a GnRH ana- logue to horses can result in reversible suppression on ovarian activity as indi- cated by lengthening of the estrous cycle (1218). Although ovarian and sexual sup— pression occurred in response to treat- ment with GnRH analogues (25 mg/day), mares required up to 30 days of treat- ment before suppression began (1112); a similar regimen in stallions was not effec— tive for suppression of sexual behavior. Exogenous GnRH. Exogenous GnRH stimulates the release of LH in stallions and mares during the ovulatory and anovulatory seasons (622) and stimulates the release of FSH in mares during the anovulatory season (492, pg.120). The initial GnRH ovulation—induction experiments in horses were designed on the assumption that the equine system was similar to that of other species, and therapeutic doses needed to be delivered in a pulsatile pattern (827). More recent trials, however, have obtained good practical results with systems for prolonged continuous admin- istration, suggesting that horses are not as sensitive to type of administration as are other species (pg. 166). In a recent study (1223), it was found that a low dose of a GnRH analogue stimulated LH release that was indistinguishable from those resulting from spontaneous pulses. In another study (1267), equine pitu- itaries were removed during estrus or diestrus and perfused with GnRH to induce LH secretion. Results indicated that a multiple dose, as opposed to a con- stant dose, elicits a GnRH self-priming phenomenon, and the response is differ— ent according to stage of the estrous cycle. The biologic and therapeutic roles of GnRH will be discussed in subsequent chapters. It will be seen that the poten- tial for regulation of seasonality by administration of GnRH analogues is considerable. Analogues that mimic the action of an endogenous hormone are called agonists, and those that block the action of an endogenous hormone are called antagonists. Antagonists to GnRH are now widely used in women in associ- ation with assisted-conception programs (1005). Stimulation tests for pituitary function have used GnRH. It has been pointed out that such tests must take into account the physiologic state of the horse (estrus, diestrus; 19). Information on therapeutic analogues and the man- ner in which they are prepared has been reviewed (335, 336, 1005). 2.5. Opioids The opioids, a family of peptides pro- duced by the brain, suppress gonad- otropin secretion. The following com- ments are based on recent reviews of the opioid modulation of the gonadotropins in nonequine farm species (700, 154). The gen- eral term, opioids, is used to distinguish them from opiates, which are plant- derived or synthetic alkaloids (heroin, morphine). Opioids and opiates bind to the same receptors in the brain, and both modulate the secretion of gonadotropins. Opioids suppress gonadotropin produc- tion, apparently by dampening the GnRH pulse generator. Some of the members of the opioid family are B-endorphins, enkephalins, and dynorphins. Naloxone is a potent antagonist of the opioids and therefore is used to investigate the physi- ologic role of the opioids. Because the opi- oids suppress gonadotropin production, injections of naloxone are expected to increase gonadotropin levels. Most of the research has centered on LH in primate, laboratory, and farm animals. In this regard, opioids were first isolated from pig brains in 1975. In farm species (excluding horses), some of the conclusions, sometimes equiv- ocal, of opioid/naloxone research (700) are as follows: 1. Opioids suppress the mechanisms involved in onset of puberty (equivocal); 2. Opioids are involved in LH modula- tion during the estrous cycle; administra- tion of antagonists (substances that block opioid action usually by competing for receptors) increases LH concentrations during diestrus; 3. Opioid agonists (substances that mimic opioid effects) reduce LH secretion and block ovulation; 4. Progesterone-induced suppression of LH secretion is mediated by opioids; 5. Naloxone counteracts the LH-sup- pressive effects of suckling; and 6. Opioids may be involved in LH sup- pression during seasonal anestrus. Although opioids have been implicated in these and other mechanisms through a depressing action on LH secretion, it remains unknown how important such opioid modulation is in physiologic terms. An experiment in horses tested the hypothesis that the opioids are involved in the seasonal decline in hypothalamic GnRH content and reduced LH secretion (1433). Naloxone failed to elicit GnRH or LH secretion and therefore did not impli- cate endogenous opioids in equine season- ality. Similarly, neither morphine (opioid agonist) or naloxone (opioid antagonist) affected estrus, ovulation, LH concentra- tions, or seasonality in mares (409). Recent studies indicate that immunoreactive B-endorphin is present in equine ovarian and endometrial tissues, and an apparent relationship exists with stage of the estrous cycle (1444); function is unknown. Reproductive Hormones 55 Considering the indications for opioid involvement in other species, continued search for opioid mechanisms in horses can be expected. 2.6 Inhibin and Related Substances Inhibin is a protein hormone that has a specific effect on the secretion of FSH but does not influence, or only marginally influences, LH secretion (393). Except for studies noted at the end of this section, information on inhibin is based on studies in nonequine species. In females, it is pro- duced by the granulosa cells and plays a profound role in regulation of FSH and, therefore, development of follicles. History. Inhibin and related sub- stances are currently undergoing inten- sive investigation, as indicated by the publication of many in-depth research reviews in recent years (e.g., 393, 1658, 1833, 1626, 394). The evolution of knowledge on inhibin followed a tortuous path with alternating periods of acceptance and denial during 1923 to 1976 (summarized by de J ong: 393). The subsequent flurry of activity on the effects of nonsteroidal fol- licular fluid on selective depression of FSH levels in female laboratory animals was first extended to female farm ani- mals in the late 1970s (cattle, ponies, sheep, 1093). Doubts about the existence of inhibin were removed with the isola- tions of pure preparations (393). The inhibin saga has removed some of the enigma regarding dissimilar LH and FSH circulating profiles, despite the common LH- and FSH-stimulating properties of GnRH. Structure. The molecular weight of inhibin is approximately 30,000 to 60,000, depending on species and isola— tion protocols. The development of satis- factory isolation procedures has been challenging, and the yield is probably less than 5% (1833). Two forms have been iso- lated, each consisting of two subunits linked by disulfide bridges. The precur- 56 Chapter 2 sors of the subunits consist of approxi- mately 300 to 400 amino acids (1833). Purification studies have involved human, porcine, bovine, and murine inhibin but apparently not equine inhib- in. Inhibin from follicular fluid selec— tively suppresses FSH release in pitu- itary cell cultures without affecting LH and other pituitary hormones (1833). The lag from exposure to initial action is much greater than for GnRH (4 to 8 hours versus seconds). Assays. Several types of in vivo bioas- says for inhibin have been developed. The in vivo assays are relatively insensitive and therefore in vitro bioassays are being developed. Assay systems have been described (1833). In recent years, several reports have appeared on assay of circu- lating concentrations of inhibin by RIA. One report considered the assay of plas— ma and tissues in many species, including horses (672). Another report (1696) described the use of an antisera against synthetic porcine inhibin a-subunit to measure inhibin in rat plasma. This assay system was used recently to quanti- tate circulating concentrations of immunoreactive inhibin during the estrous cycle of mares (pg. 245; 185). Another recent study (1272) found that equine granulosa-theca cell tumors secrete inhibin subunits; the inhibin could affect gonadotropin production and thereby account for the atrophied contralateral ovaries associated with such tumors. Unfortunately, the role of inhibin in mares based on exogenous administration has not progressed beyond experiments that involve admin— istration of steroid-free follicular fluid (discussed in subsequent chapters). Much activity involving assay develop- ment and treatment challenges with fol- licular substances in mares should occur during the 1990s. Related substances in follicular fluid. Studies in nonequine species have impli- cated avariety of other proteinaceous gonadal substances in follicular fluid that affect FSH secretion. The following brief discussion was abstracted from a review (1833). Activin and activin A are novel pro— teins that were isolated from the same follicular fluid as inhibin. These proteins have bioactivities similar to those of a protein transforming growth factor (TGFB) that is found in all tissues. Activins and TGFB promote basal FSH secretion and FSH—mediated estrogen synthesis by the granulosa cells. The activins and TGFB have biochemical structures similar to inhibin, but their biologic activities are opposite to inhibin. Another substance isolated from follicular fluid is follistatin. This is a peptide, struc— turally unrelated to inhibin, but having potent and specific properties for inhibi- tion of FSH release. At least two forms have been isolated. In addition to effects on FSH secretion, inhibins and activins may play an important paracrine/autocrine role (local or within ovary) in regulating the function of gonadal cells. Indications also seem to be emerging that follicles produce a sub— stance that directly suppresses other folli- cles without a direct effect on FSH levels (937). In a recent study (441), charcoal- treated equine follicular fluid was added to culture media in which sow follicles were incubated. Fluid from small folli- cles inhibited estradiol production, and fluid from large follicles stimulated estradiol production. The authors con- cluded that growth factors in the follicu— lar fluid may have affected aromatiza- tion and cell division. 2.7. Oxytocin and Related Substances Several octapeptides with biologic activity have been isolated from the pos- terior pituitary of various species (oxy- tocin, arginine vasopressin, lysine vaso- pressin, and arginine vasotocin). Of these, oxytocin is the most important hormone associated with reproductive function. Oxytocin is synthesized in the hypothala- l l mus and is stored in the posterior pitu- itary. A short communication (1045) describes the distribution of oxytocin immunoreactivity in the hypothalamus and posterior pituitary of horses. Immunoreactive granules were discerned in the axons. Oxytocin exerts its functional activity almost entirely on the reproductive tract and mammary glands. Functions include milk letdown and the stimulation of uter- ine contractions during the estrous cycle (pg. 216) and parturition (pg. 470). The release of oxytocin and vasopressin dur- ing parturition has been reviewed for all species (1733) and for farm animals, including horses (525). In all species stud- ied, oxytocin concentrations are low in late pregnancy and increase during labor. Little change has been noted in concen- trations of vasopressin. A role of oxytocin that has received considerable attention in the past decade involves the mechanisms for uterine- induced luteolysis. According to this con- cept, oxytocin stimulates the release of prostaglandin onc (PGan) from the endometrium, and PGan, in turn, stimu- lates further pulses of oxytocin (515). This concept has spurred a search for a similar mechanism in mares, and supportive results have been obtained (pg. 275). In domestic ruminants, recent studies sug- gest that the corpus luteum is a source of the oxytocin. Oxytocin production by the equine corpus luteum has not been demonstrated. Questions have been raised recently about paracrine (intraovarian) roles of ovarian-produced oxytocin in the regula— tion of ovarian steroidogenesis (999). In this regard, there appears to be a mount- ing scramble to find roles for misplaced hormones—that is, hormones found to be produced by other than the expected tis- sue. The effects of administration of vaso- pressin, as well as cortisol and corti- cotropin—releasing factor, on release of Reproductive Hormones 57 adrenocorticotropin and other substances in horses have recently been reported (972). In addition the release of oxytocin 2.8. Melatonin during parturition (37, 669) and suckling (1425) has been reported (pg. 470). An area of reproductive biology of mares that has been receiving much attention is the seasonality of reproduction. Melatonin is the best known substance produced by the pineal gland (pg. 38), an organ involved in the regulation of reproductive seasonali— ty. Melatonin was first isolated from bovine pineal glands in 1959 (review: 1291). Its chemical identity is N-acetyl-5- methoxy-tryptamine. It has been identi- fied in all vertebrates studied to date (1322). After release from the pineal, mela- tonin passes into the systemic circulation and probably also directly into the cere- brospinal fluid. Most (67%) of the circu- lating melatonin is bound to albumin (1291). Melatonin is degraded in the liver, and its conjugates are excreted in the urine (279). Melatonin synthesis is regu- lated by external photoperiodic cues and by internal hormonal feedbacks (pg. 118). Synthesis of melatonin occurs in the pinealocytes (pineal cells) and involves the uptake of tryptophan. After several enzy- matic steps, the tryptophan is converted to serotonin, which is then converted to melatonin (1291). One of the enzymes involved is hydroxyindole-O-methyltrans- ferase (HIOMT). Quantitation of HIOMT throughout the season in mares has been used to test the hypothesis that the pineal 2.9. Relaxin and melatonin are involved in equine sea- sonality (pg. 118). Relaxin is a water-soluble, polypeptide hormone present in the ovaries, especial- ly corpora lutea, placenta, and uterus of pregnant animals. The ovaries of preg- nant sows are an especially rich source, and relaxin appears in the blood and tis- 58 Chapter 2 species in which it has been studied increases during pregnancy, and high levels are maintained until parturition. Relaxin influences the growth, composi- tion, and distensibility of the uterus, cervix, and pelvic ligaments during pregnancy. Its role in relaxation of the cervix and other pelvic structures at the end of pregnancy seems to be extremely important. Despite its importance, relaxin did not attract much investigative attention fol— lowing its discovery by Hisaw in the 1920s, which preceded the discovery of progesterone (review: 1285). Hisaw detect- ed relaxin in pregnant mares by a bioas- say involving relaxation of the pelvic liga- ments in guinea pigs. A major advance was the development of methods of extracting sufficient amounts for purifica- tion and biologic studies by Sherwood and associates in 1974 (1450). Two subunits (a and [3) have been identified and are con- nected by disulfide bonds. The two chains have 22 and 26 amino acid residues, respectively, and the molecular weight is approximately 5,700 (1043). Little is known about the relationship between structure and function of relaxin. It has been noted that several of the actions of relaxin (myometrial relaxation, pubic sep— aration, and water inhibition) resemble those of estrogen, differing primarily in the faster rate of action of relaxin (1285). Perhaps, therefore, relaxin mediates some of the actions of estrogen. The previous edition of this text lamented that no reports were found on the profile of relaxin throughout pregnan- cy in the equine species. This informa— tional void has since been filled by California workers. Plasma relaxin was measured during the estrous cycle and pregnancy by RIA using a porcine—relaxin system (1552, 1553). In subsequent work, equine relaxin was purified (1551) and used for development of a homologous RIA (1547). The equine assay system gave similar relaxin profiles but at much high- er levels (pg. 431). The placenta was the only equine tissue containing large amounts of the hormone (1554). The purified preparation indicated that equine relaxin was heterogeneous with a major form and several minor forms. About 1.5 mg of the major form was obtained per kilogram of placenta at term. More recently, monoclon- al antibodies have been used for further purification (1549). 2.10. Prostaglandins Few realizations have rocked the foun- dations of basic and applied physiology as has the acceptance of the many roles of prostaglandins. The prostaglandins con- sist of a 20—carbon fatty acid incorporat- ing one five—carbon ring (Figure 2.4; 1266). Slight structural changes may result in distinct and divergent biologic effects. For example, prostaglandin E2 (PGE2) lowers blood pressure, Whereas prostaglandin F20: (PGan) raises blood pressure (1266). Yet, the two molecules differ only in that the former has an oxygen atom attached to one of its carbons, whereas the latter has a hydroxyl group (OH) at the corre- sponding site (Figure 2.4). Prostaglandins OH Prostaglandln F206 CO2H OH Prostaglandin E2 FIGURE 2.4. Structure of selected prostaglandins. l Prostaglandins can be synthesized in most body tissues and are considered to be autocrine/paracrine as well as endocrine messengers. Their effects are profound and diverse (571, 1266), and they are among the most potent of biologic substances. Administration of extreme- ly low doses produces marked effects on many physiologic processes. Prosta- glandins act as mediators in the male and female reproductive systems, in the spleen, skin, and eye, and in the diges- tive, cardiovascular, respiratory, and cen- tral nervous systems. Furthermore, prostaglandins play a role not only in many physiologic mechanisms, but also in certain pathologic conditions. For exam- ple, they are formed by the skin during contact allergies and by the lung during anaphylaxis. In general, the widespread effects may be attributed to regulation of blood flow and the activity of smooth muscle and secretory cells, including those of some endocrine glands. Apparently, they exert their diverse effects in connection with the formation of the important second messenger, cyclic AMP, which is a key factor in translating the messages of many hormones (1266). Because prostaglandins are found in so many tissues and because their effects are so diverse, one may wonder why the body is not in a continual state of chaos. The effects are very transient, and most functions are probably exerted at or near the site of origin. The prostaglandins that reach the general circulation are rapidly metabolized by enzymes. For example, when labeled prostaglandins are injected intravenously, 90% of the radioactivity appears in breakdown products within 90 seconds (1266). It appears that the prostaglandins tend to be produced as needed rather than stored for future use. The synthesis of the prostaglandins involves arachidonic acid and has been reviewed (879). The prostaglandins can be measured by RIA in horses in uterine flushings, uterine venous blood, and in other fluids Reproductive Hormones 59 and tissues, including tissue culture media (1700). A metabolite of PGFZoc (PGFM) has been used extensively for assaying prostaglandins in the peripheral circulation of horses by Kindahl and asso- ciates (e.g., 632, 880). The half-life of the metabolite is much longer than PGonc. Thus, similar profiles of PGan release can be obtained with PGFM, using less frequent sampling. In mares, PGFZa has a potent luteolytic effect, and the endoge- nous levels of PGFZoc increase in associa- tion with luteolysis (pg. 270). Native and synthetic forms of PGan are commer- cially available and are widely and exten- sively used in the control of equine repro- ductive mechanisms. 2.11. Steroids 2.11A. General Research obstacles. Research on the physiologic aspects of steroid hormones faces many obstacles traceable to bio- chemical similarities and common metabolic pathways. For example, study of the role of a given hormone by adminis- tration of the hormone in question is com- plicated by the possibility that the inject- ed hormone may enter the metabolic pathway and be converted to another hor- mone. Furthermore, the effect of a hor- mone is often dose-dependent, and a given hormone may carry the ability to mimic the effect of another, thereby adding greatly to research problems in this area. Study: of a hormone’s role by removal of the tissue of production (e.g., ovaries) also can be a misleading approach, since other tissues (e.g., adrenal gland and placenta) may also have the ability to produce the hormone, perhaps at an accelerated rate. In vitro approaches, such as tissue culture, have the advantage of simplifying the study since the cells in question can be isolated from the influences of a multitude of other factors. Advantage may turn to dis- advantage because the simplified system 60 Chapter 2 may have little resemblance to the in vivo situation. It is small wonder that progress has been slow in the study of the physiologic chemistry of steroid hormones in the equine and other species. Early studies. The first experiment to yield direct in vivo information on the biosynthesis of estrogens was done in a mare in 1954 by Heard and associates (705, 706); labeled estrone was recovered from the urine of a pregnant mare that was treated with labeled testosterone. This provided the first conclusive evi— dence that androgens were involved in the biosynthesis of estrogens. In 1960, Short (1461) and soon thereafter Knudsen and Velle (901) estimated the levels of estrogens and other steroids in equine ovarian follicles. Extensive studies on the role of various ovarian cell types in the formation of steroid hormones also have been done in mares. Noteworthy in this regard is the work of Short and associates (1464). Channing and others have used tis- sue culture of equine granulosa cells to study the formation of steroids from vari- ous precursors in the tissue culture medi- um (295). This approach is now being used with increasing frequency. The extensive use of the mare in these pioneering stud- ies can be attributed to: l) the appearance of species-unique estrogens in the urine of pregnant mares, 2) the availability of large amounts of cells and fluid from Cholesterol 21 12 18 mare ovaries, and 3) the ease With which granulosa cells can be separated from the thecal layers. Most of the early biochemi- cal work utilized material from very lim- ited numbers of animals, often only one mare; an interesting account of a series of such experiments has been made (1464). Furthermore, in much of the earlier work in this area the stage of the estrous cycle was estimated or unknown, and interpre- tations were necessarily based on incom- plete knowledge of ovarian morphology. The biochemical studies have forged far ahead of the studies of physiologic and morphologic aspects of steroid function in this species, causing difficulty in inter- preting and relating the biochemical find— ings to mare reproductive biology. Steroid biochemistry. The classical steroid hormones (excluding the ring-B unsaturated estrogens; pg. 67) are derived from a common precursor molecule, cholesterol, and share a basic structural feature of four rings—A, B, C, and D (Figure 2.5). The steroid hormones differ in the nature of attached groups and in the location of double bonds. Steroido- genesis is a complex mechanism involving many enzyme systems and is being exten- sively re—examined by the technologies of contemporary molecular biology (1097). The structure of some steroids that have been isolated from reproductive organs of mares is shown in Figure 2.6. 22 26 25 27 FIGURE 2.5. Basic structure of choles- terol. Note the four phenolic rings des— ignated A, B, C, D. The numbers are used to designate carbon atoms. Reproductive Hormones 61 Equine steroid reproductive hormones Cholesterol-derived hormones “1“3 0 OH C=0 Estrone Testosterone Progesterone 3% OH O C=0 /© Estrone ll ll Testosterone —> Estradiol FIGURE 2.7. Classical pathway for the biosynthe- sis of steroid hormones. Estrogens and progestins. A scheme for two alternate pathways for steroidogene- sis in the equine follicle has been pro- posed (Figure 2.8; 1462). It is generally believed that the major pathway for the ovarian synthesis of the reproductive steroids (progestins, estrogens, andro- gens) from cholesterol is through proges— terone and 17a-hydroxyprogesterone as shown (Figure 2.8). However, the alter— nate route involving 17a-hydroxypreg— nenolone and dehydroepiandrosterone (DHA) also may be important in the nor- mal follicle. More recently, detailed anal- yses have been done on equine follicles for identification of estrogen metabolites, progestins (40), and androgens (1469). The assay results for follicular fluid indicated that the follicular cells are capable of side chain cleavage, 17-hydroxylation, and aromatization (bioconversion from andro- gens to estrogens). The principal progestins in the fluid of preovulatory and viable follicles are progesterone and 1 7 or-hydroxyprogesterone. An earlier study (1463) failed to demon- strate estrogens in the equine corpus luteum. In retrospect, this failure may have been due to the use of a young cor- pus luteum. A pool of eight midcycle cor- pora lutea contained estradiol, whereas new corpora lutea (estimated as <72 hrs) did not (1834, 1835). Incubation of micro- somes from the equine corpus luteum With progesterone and NADPH resulted in 17a-hydroxyprogesterone and estro- gen production with a small yield of androstenedione (75). Equine granulosa cells seem to maintain aromatization abilities after luteinization, although this ability is usually diminished (76). The estrogen-producing abilities of equine cor— pora lutea were emphasized in recent studies; results indicated that in response to eCG the corpus luteum of pregnant mares produces estrogens in quantities that cause an increase in circulatory lev- , els, exceeding those of the preovulatory follicle (pg. 446). Reproductive Hormones 63 Steroidogenesis ...... Corpus luteum Acetate \ Follicle A _ . Cholesterol """""""" FO'llclea possnble alternate route \ BA Pregnenolone L" \ 3A 170c-Hydroxy- Progesterone pregnenolone .5 : x v v \ Dehydroepi- 17 oc-Hydroxy- androsterone progesterone ‘ 20 oc-Dihydro- 4 / progesterone Androstenendione / Estrone / Estradiol-17l3 FIGURE 2.8. Postulated pathways of steroid biosynthesis in the equine ovary. Sites of follicular steroido enesis. Various studies indicate that both layers of the follicle wall (granulosa and theca interna) are involved in steroidogenesis. The production of androgens by the thecal cells, followed by their conversion to estrogens by the granulosa cells, is known as the two-cell theory of follicular steroidogenesis (461). In mares, combined theca and granulosa cells were more effective than either cell source alone in the in vitro production of estradiol-17B (1008). However, the nature of the contri- butions of each tissue layer to total estra- diol-17B productivity in mares is unclear. Research approaches involving tissue cul- ture (1376, 297, 1414, 1642, 1486, 1487), in vivo injection with labeled precursors (1836), and histochemical staining techniques for enzyme systems (209, 696, 816) have result- ed in conflicting interpretations. Equine granulosa cells have considerable 3B- hydroxysteroid dehydrogenase (3B-HSD), 64 Chapter 2 an enzyme required for the conversion of pregnenolone to progesterone (Figure 2.8;, 696, 816). Tissue culture studies indi— cate that isolated granulosa cells from estrous follicles have an active aromatase system (816, 1642), indicating that estro— gens can be produced by isolated granu- losa cells; similar results have been obtained in many species. However, earli— er reports have concluded that a consider- able portion of the estradiol in the ovari- an venous blood of mares arose from other than the granulosa cells (1836). This early reported in vivo result has not been reconciled with the more recent in vitro tissue culture results. In one study (1642), isolated granulosa cells did not respond to gonadotropins. In another study (1486, 1487), however, both granulosa and attached theca tissues were cultured. Addition of combinations of eLH and eFSH to cells collected during late diestrus resulted in a much greater production of progesterone, androstene- dione, and estradiol than for cells collect— ed during estrus. Responsiveness of the follicular wall to gonadotropins dimin- ished between late diestrus and estrus; it was concluded that the preovulatory folli- cle undergoes a marked decrease in re- sponse to gonadotropins. The pituitary gonadotropins were more potent stimula- tors of in vitro steroidogenesis by the fol— licular wall than was eCG. The pattern of granulosa-cell steroid synthesis is influ- enced by the factors that regulate follicu- lar growth (1642, 816). Granulosa cells from estrous follicles were effective producers of estrogens in vitro, but later in estrus the cells secreted primarily progesterone (1642). This result indicated that proges- terone-producing capacity (luteinization) begins before ovulation, which is consis- tent with the rise in circulating levels of progesterone beginning just after ovula- tion (pg. 238). Apparently, after ovulation, the granulosa cells become fully luteinized, the conversion of progesterone to 17a-hydroxyprogesterone is retarded, and progesterone accumulates with some conversion to 20a-dihydr0progesterone (Figure 2.8). Androgens. Androgens are in the follic— ular pathway to estradiol, and the folli- cles can produce and accumulate andro- gens in the presence of gonadotropins. In contrast to the placenta, which cannot synthesize large amounts of cholesterol from acetate, both the isolated theca and granulosa cells of the ovary have this ability (1376). Testosterone is present in equine follicular fluid, and its concentra- tion is twice as high as in plasma (1468, 1469). Perhaps the follicles contribute to the production of circulating testosterone, and the biosynthesis of estradiol in the mare ovary also involves testosterone (1469). An experiment involving tissue cul- ture of thecal cells suggested that testos— terone is the principal steroid produced by the theca and that this effect may be enhanced by eLH (1643). It was also demonstrated (392) that 19-nortestos- terone, a widely used anabolic steroid, was an endogenous hormone in mares and often exceeded the concentration of androstenedione in follicular fluid. Another study indicated that equine granulosa may be a site for metabolism of dihydrotestosterone (816). Follicle structure and steroidogenesis. The extent of steroid content of follicles is associated with the degree of vasculariza— tion (1834). Granulosa cells from large vas- cular follicles had higher steroidogenic activity when grown in tissue culture than did those from nonvascular folli- cles (297). By definition, avascular follicles were those with a pale inner lining, and vascular follicles were those with an orange-pink lining and pronounced ves- sels. Mean follicular fluid concentrations (pig/100 ml) of estradiol-17B and estrone, respectively, in various reproductive states were reported to be as follows: anestrus, 7.5 and 0.5; proestrus, 7.1 and 0.9; estrus, 37.5 and 3.0; diestrus, 6.0 and 0.5 (901). Recently, Silberzahn and co- workers compared ultrastructure of the follicular wall with the androgen content of the follicular fluid (1469). Testosterone content remained constant during follicu- lar growth and early atresia, whereas androstenedione content increased up to the preovulatory stage. Excretion of estrogens in nonpregnant mares. The excretion of estrogens in the mare occurs primarily through the kid— neys (1698). In contrast, the feces are a major excretory route of estrogen metabo- lites in ruminants. The feces of mares do contain estrogen sulfates, allowing conve- nient hormonal monitoring in feral mares (1308). The levels of estrone sulfate in the saliva have been compared to serum levels (1491). In most cases the sali- va levels of estrone sulfate reflected the reproductive status of the mare, but false negatives and false positives were record- ed erratically. Estrone and estradiol-17B are the major urinary metabolites in the nonpregnant mare, whereas estradiol-17cc predominates in cow urine (1834). Estradiol-17B, as indicated above, is the major estrogen in mare follicular fluid, but approximately 10% estrone is also present. The presence of high estrone lev- els in urine of nonpregnant mares, there- fore, raises questions about its origin. Apparently, considerable estrogen meta— bolism occurs outside the ovary, perhaps in the liver, and this may account for the high urinary estrone levels (730). The extent and nature of equine extraovari- an metabolism of estrogens in the non- pregnant mare have received little attention. Urinary estrogen levels dropped approximately 10-fold within 24 hours in two mares ovariectomized near to or in estrus (730). The excretion rate of exogenous estradiol followed a well-defined pattern (730). Levels of estra- diol and estrone rose to a peak within 3 to 6 hours, depending on dose administered (0.5 and 5.0 mg), and then fell rapidly to preinjection levels within 48 hours after injection. The rapid fall in urinary estro— gens after ovariectomy and the rapid excretion of exogenous estrogens support the use of urinary estrogen as an index of Reproductive Hormones 65 ovarian secretion rate in nonpregnant mares. Variations in output of urine could cause variations in urinary estrogen con- centrations. Therefore, it has been sug— gested (1216) that creatinine concentration be used as a correction factor, as has been done in other species. Urinary estrogen conjugates are being used with increasing frequency for monitoring ovarian and fetoplacental function in both nonpreg- nant and pregnant mares under both domestic and nondomestic conditions (1109, 934). Late entry. Urinary estrogen conju— gates, estradiol-1’7B, and estrone were measured in the urine of nonpregnant mares (1860). Mean concentrations of uri- nary conjugates, plasma conjugates, and plasma estradiol-17B were parallel, and increases were shown during estrus. Following hydrolysis of urine, estrone predominated in both nonpregnant and pregnant mares. The sum of estrone con— jugate and estradiol conjugate in urine was less than the total conjugate, indicat- ing the presence of other estrogens. It was concluded that the assay of urinary or plasma estrogen conjugates provides higher resolution profiles than for the free estrogen forms. The authors noted that conjugated estrogens are stable in urine, and samples can be collected from voided urine on hard surfaces or even in the soil. They also noted that the low lev- els of circulating estradiol-17B in mares interfere with its usefulness for assessing follicular dynamics, even though estradiol is the most biologically active estrogen. The 100- to 10,000-fold higher levels of the estrogen conjugates in urine and plas- ma, respectively, provide an accurate determination of estrogen secretion. WWW The biologic half-life of exogenous progesterone in the mare has been studied (549). Radioactive progesterone was given intravenously to an ovariectomized mare. Three compo- nents of the disappearance curve were described, and the first two had half- times of 2.5 and 20 minutes, respectively 66 Chapter 2 (Figure 2.9). The third was greatly pro- longed and was not completely deter- mined. The same authors did another study concerning the interaction of pro- gesterone with specific steroid-binding proteins and concluded that the blood plasma of mares appears to lack proges— terone-specific binding proteins. Other workers (483) described a two-component disappearance curve of approximately 15 and 60 minutes, respectively, following cessation of infusion of labeled proges- terone. Metabolic clearance rates of pro- gesterone in anestrous, diestrous, ovariec- tomized, pregnant, and lactating mares were not significantly different (1573). The concentrations of progesterone in blood plasma of mares following daily intramuscular injections of progesterone in corn oil have been examined (764). Concentrations increased significantly within 30 minutes, and levels comparable to those of mid-diestrus (pg. 238) were maintained throughout treatment (Figure 2.10). The 100-mg dose appeared to close- ly approximate diestrous levels in ponies. Several other studies have been done on the levels of circulating progesterone fol- lowing administration of progesterone in oil or repositol forms (693, 411, 287, 35). Progesterone half-life _L 01 [3 H] progesterone —L 0 Rate (d/min x 104) 01 0 10 20 30 Time (minutes) 50 60 120 FIGURE 2.9. Disappearance of labeled proges- terone in ovariectomized mare. Adapted from (549). Exogenous progesterone (n=3/group) 10 c 8 100mg/day E / on \/\\\Q 5 6 C .9. E E 4 0“, d) 0 ~ : x-~__ 8 2 ° 0 -r-1—|—r——I—' Fr—Ifi—I—fi—fi 0 2 4 8 12 1 2 3 4 6 Hours Days FIGURE 2.10. Circulating concentrations of proges- terone following daily intramuscular injection of progesterone in corn oil in pony mares. Adapted from (764). 2.11C. Steroid Hormones in Pregnant Mares Steroids from the embryonic vesicle. The equine conceptus is capable of steroid production while it is still free within the uterine lumen (pg. 429). A wide range of hydroxysteroid dehydrogenases has been demonstrated in the equine trophoblast as early as Day 8 (1250) and Day 13 (521, 522), indicating that the trophoblast has a role in steroid interconversion. Hydroxy- steroid dehydrogenases were also present in the topical areas of the endometrium that were in contact with the trophoblast. In vitro culture of the Day 20 equine con- ceptus resulted in high estrogen concen- trations in the incubation medium, and further studies revealed a marked estro- gen—producing capability of the equine conceptus as early as Day 12 (1843). Furthermore, means (pg/ml) and stan- dard deviations for estrone and estradiol concentrations, respectively, in uterine flushings on Day 20 were 1442 i274 and 2651 i195 in pregnant mares and 41 1:50 and 36 i31 in nonpregnant mares. The early equine blastocyst (Days 10, 15, and 20) is capable of secreting progesterone in vitro (725). More recently, measurable amounts of androgens, progesterone, and estrogens were reported to be present as early as Day 8 (1250). The cells of Day 12 to Day 15 embry- onic vesicles have been dissociated and examined for steroid secretion by Canadian workers (1016). Cells from the endoderm secreted predominantly pro- gesterone, whereas cells from the ecto- derm secreted predominantly estradiol. The results indicated that both cell lay- ers and interactions between them are involved in steroidogenesis as occurs in the layers of follicles. One possibility, suggested by the authors, is that proges- terone is produced mainly by the endo- derm and then is transferred to the ectoderm for estradiol production. This suggestion was based on a more rapid decrease in estradiol production than in progesterone production by the respective cells. In a preliminary report, estradiol secretion decreased after manual rupture of the blastocyst, whereas removal of the capsule Without breaking the vesicle did not affect estradiol production (634). Another study indicated that the major metabolite synthesized from preg- nenolone was 17a-hydroxy-progesterone rather than estradiol, and that further metabolism occurs in the endometrium (942). Aromatase activity in the embryon- ic vesicle was used as an indicator of estrogen production and was present in the ectoderm (trophoblast) and endoderm at Day 28 (702). Yolk sac fluid was much higher than allantoic fluid in estrone and estradiol-17B concentrations. The authors commented that the mare and pig have appreciably higher aromatase activity in the early conceptus than do other placental mammals. Studies on steroidogenic capabilities of the early equine conceptus appear to be a fruitful and interesting research area from both the comparative and species-oriented viewpoints: Reproductive Hormones 67 Estrogens in urine of pregnant mares. It has been known since 1930 (cited in 346) that the urine of mares during the last two-thirds of pregnancy contains large amounts of estrogens (pg. 427). Urine from mares has been used as a source of natural estrogens in veterinary and medi— cal fields (e.g., as replacement therapy in menopausal and post—menopausal women). Sophisticated techniques for commercial collecting, processing, and determining the estrogenic content have been developed (527). A commonly pre- scribed commercial preparation is Premarin, which has been available for 40 years (cited in 207). The quantity of estrogens in the urine of pregnant mares is greater than for any other known species, and the pattern of excretion differs in that the rate decreas- es rather than increases during the last third of pregnancy (1303). At least nine estrogenic compounds have been extract- ed and identified in the urine of pregnant mares. Three of these estrogens are syn- thesized through the classical cholesterol pathway. Six are known as ring-B unsat- urated estrogens, and cholesterol is not involved in their synthesis. The ring-B unsaturated estrogens are equilin, equi- lenin, and the 170c- and 17B-dihydro forms of each (Figure 2.6). Equilin and equi- lenin were first described in 1932 (cited in 208) and are unique to the pregnant mare, although there is some indication that they may be produced in humans under certain pathologic conditions (208, 207). Estrone and equilin are the principal urinary estrogens. The other urinary estrogens, including the 17[3-hydroxy derivatives of estrone (estradiol-17B) and equilenin, are present in very small amounts (346, 207, 1399). The ring-B unsat- urated estrogens first appear in pregnant mares’ urine at about the fourth month and then increase; near term, they repre- sent 50% to 65% of the total urinary estrogens (207). Estrone and equilin have been chemically assayed in the peripheral plasma (346). Fluctuations in the concen- 68 Chapter 2 trations of urinary and plasma estrogens during gestation are discussed in Chapter 10 (pg. 427), A series of reports on the biosynthesis of estrogens that appear in the urine of the pregnant mare has been published by Bhavnani and associates (208) and has been recently reviewed (207). The biosyn- thesis of estrone and estradiol-17B and -170L followed the classical pathway of steroid biosynthesis with cholesterol as a precursor. However, the ring-B unsatu- rated estrogens were formed by a unique biosynthetic pathway that, unlike the classical pathway, did not involve choles- terol. A recent report has been made on metabolic studies of ring—B unsaturated estrogens (798). Source of pregnancy estrogens. Early workers postulated that the large amounts of urinary estrogens in late pregnant mares originated from fetal (323) or placental (689) tissues. The evidence marshaled in the 1930s in support of this View included the apparent elimination of the maternal ovaries as a source. The maternal ovaries were small without large follicles during the last third of pregnancy. Furthermore, ovariectomy of a mare at Day 180 did not result in abor- tion and did not appear to reduce urinary estrogen levels during the last 80 days of pregnancy (689). Raeside and co—workers postulated that the large amounts of estrogens in late pregnancy are formed by the fetal placen- ta with the assistance of enzyme systems in the fetus (1301, 1303, 1304, 1305). Current overwhelming findings that are consis- tent with the postulate include the follow- ing: 1. There was a strong temporal associa- tion between growth and regression of the fetal gonads and maternal urinary estro— gens (pg. 436; 689); 2. Estrogens in the urine of the mare and in the first voided urine of the foal were not detected within 24 hours after parturition (292); 3. Urinary estrogens decreased mark— edly after bilateral fetal gonadectomy, and unilateral gonadectomy caused an immediate decrease in estrogen values to one-half (1246, 1305), whereas progesterone levels remained unchanged (1246); 4. Large quantities of estrogens were present in the equine placenta (1305), whereas the content in fetal gonads was no greater than in other fetal organs (323); 5. Extracts of the fetal gonads con— tained large quantities of the estrogen precursor dehydroepiandrosterone (DHA), indicating that the fetal compo- nent in the fetoplacental production of the classical estrogens was the gonads rather than the adrenals (1301); 6. The DHA was in high concentration in blood flowing to the placenta from the fetus, whereas on both the fetal and maternal sides, estrogen concentrations were lower in blood going to the placenta than in blood leaving the placenta (1304); 7. When the artery of the fetal ovary was anastomosed to the carotid artery of the mare, the principal steroid secreted by the fetal gonad was DHA, based on analyses of maternal blood (1302); 8. Placental preparations of large domestic species, including mares, con- tain aromatizing enzyme systems for the conversion of androgens to estrogens (415) but seem to be incapable of forming estro- gens from progestins (14); 9. A wide range of hydroxysteroid dehydrogenases are present in the tro- phoblast (indicating it is capable of steroid interconversion), but much less so in the fetal gonads (522), indicating the gonads do not, in themselves, produce estrogens but may provide a source of androgens for further metabolism in the placenta; 10. Administration of a competitive inhibitor of 3B-hydroxysteriod dehydroge— nase reduced the concentrations of estro- gens in the uterine vein (528); '11. Based on fine structure, the inter- stitial cells of the fetal gonads have a fully developed steroidogenic apparatus (635); 12. The fetal gonads were found to be devoid of the enzymes that metabolize DHA, and DHA fell rapidly after fetal gonadectomy, confirming the role of DHA as a precursor of the classical estrogens (1247); 13. The ring-B unsaturated estrogens decrease in the urine after parturition and fetal death (207), and the fetal gonads have been implicated as a component of the pathway to their synthesis (1586, 1306, 1585); and 14. It was concluded from a recent in vitro study that the fetal gonads appear devoid of many of the enzyme systems associated with other steroidogenic tis- sues (1019). Bhavnani (207) acknowledges the indi— cations that the fetal gonads are involved in the pathway for the ring-B unsaturat- ed estrogens but suggests that it is more likely that the fetal adrenal and liver pro- duce the neutral steroids; these are then aromatized by the placenta, similar to the formation of estriol in human pregnan- cies. The contribution of the fetal organs to the precursors for placental synthesis of the ring-B unsaturated estrogens remains to be resolved. Testosterone. Androgen metabolites have been identified in equine placen- tal tissue preparations (1017, 1018). Testosterone levels in mares increase throughout pregnancy and do not return to basal levels until a week after parturi- tion (pg. 427,- 1472). These workers have sug- gested that initially the maternal ovaries, during the time of eCG production, and then the fetoplacental unit, are the sources. Progestins. The equine placenta is also a source of progestins. Progesterone has been demonstrated in the mare placenta (1458, 1459), and the formation of radioac- tive progesterone from labeled preg- nenolone has been demonstrated in vitro in mare placental preparations (15). The deficiency of 3B-hydroxysteroid dehydro- genases in the fetal gonads would explain the high concentration of pregnenolone in Reproductive Hormones 69 the fetal gonads, since this enzyme is needed for conversion of pregnenolone to progesterone. Discharge of progestins into the periph- eral circulation through the venous efflu— ent of the gravid uterus has been demon- strated (1506). Progestin levels were similar in the jugular and uterine veins on Days 24, 30, 40, and 50, tended to be higher in the uterine veins on Day 60, and were significantly higher in the uter- ine veins on Days 80 and 100. Thus, the gravid uterus or its contents secreted pro- gestins into the venous system from Day 80 to Day 220, which was the last day studied, and may have begun secre— tion as early as Day 60. Progesterone is extensively metabo- lized, primarily to saturated 5a-pregnane derivatives. In contrast, in sheep and cat- tle the principal metabolites are 5B-preg— nane derivatives (15). Radioactive metabo- lites (5oc-pregnanes) have also been identified following incubation of placen- tal tissue. Several of the 5a—pregnanes, which have been identified in vitro as metabolites of progesterone, have been isolated from the urine of pregnant mares. Information on a steroid conjugate (5a-pregnane sulfate) that has been iso- lated from the urine of pregnant mares is available (754). Both in vitro and in vivo studies indicate that the mare placenta contributes to the urinary output of pro- gestins. The 5a-pregnanes are also pre- sent in the blood of pregnant mares (113, 114, 760). The 5a-pregnanes cross-react with pro- gesterone RIA antisera, causing overesti- mates of the quantities of progesterone in the blood of pregnant mares (pg. 425). The fetoplacental unit is at least partly involved in the production of Soc-preg- nanes. In limited studies (760), the preg- nanes were detected only during pregnan- cy, whether or not the ovaries were intact, and the pregnanes appeared to be in higher concentration in umbilical ves- sels than in uterine vessels. However, other workers (113) reported that at least 70 Chapter 2 one of the 5a-pregnanes appeared in the maternal circulation before the develop- ment of the placenta. Determination of the concentrations of the various progestins in uterine and umbilical arteries and veins has been done (1121) and was recently reexamined using a more specific assay approach (761). Gas chromatography/mass spectrometry was used to quantify specific progestins and thereby greatly minimized the cross— reactivity problems that have plagued the immunoassay approach. The use of this methodology for identification of steroid metabolites in horses was discussed recently (766). Based on this technology, Holtan and associates (761) concluded that published results involving immunoassay of progesterone during the last nine months of pregnancy are questionable. Thirteen progestins were identified and eight were quantified. Four of these (pro- gesterone and three 5a-pregnanes) were previously identified (113, 762). One of the pregnanes attained near microgram con— centrations at term. The concentrations of various progestins reported by these workers are discussed elsewhere (pg. 426). The predominant tissue sources, based on concentrations in veins versus arteries, are depicted in Figure 2.11. The follow- ing points are especially noteworthy: Sources of progestins during pregnancy Days 60 to term _——_—_.——————— Days 0 to 40 Days 40 to 180 Primary and Primary CL supplementary CL Fetoplacental unit P4 5 oc-DHP? Soc—DHP? \NV 38—5P? 38-5P? Placenta V\N Soc-DHP 20 oc-5P Ba-Diol Maternal circulation Symbol Common name Structural name P4 Progesterone 4-pregnene-3, 20-dione P5 Pregnenolone 3B-hydroxy-S-pregnene—20—one 5 OL-DHP 5 oc—dihydroprogesterone 5 OC—pregnane—S, 20—dione Pregnanes 38—5P 38-OH-50c-DHP 3B—hydroxy—Soc-pregnane—20-one 20 oc-5P 20 oc-5 oc-DHP 20 or-hydroxy—S oc—pregnane-3-one Boc—Diol 3B, 20 oc—50c-DHP 50c- pregnane—SB, 200c—diol FIGURE 2.11. Postulated principle sources of progestins during pregnancy. Two of the pregnanes are shown with a question mark to indicate that small amounts are produced inthe maternal system in * early pregnancy, perhaps in the corpora lutea or as metabolites of luteal progesterone. Pregnenolone . (P5) and 5a—dihydr0progesterone (5a—DHP) are metabolized entirely Within the placenta and fetus, respectively. Note that progesterone (P4) does not enter the maternal circulation from the fetoplacental unit. Adapted from Holtan, et al. ( 7 61 ) and notes from personal communication with D. H; Holtan. 1) Progesterone metabolism in the feto- placental unit was so efficient that pro- gesterone did not enter the maternal cir- culation; 2) Pregnenolone from the fetus was rapidly converted into progesterone and 5a-DHP (a pregnane, Figure 2.11) by the placenta, as indicated by high concen- tration of pregnenolone in the umbilical artery, but not in the umbilical vein; 3) 5a-DHP was, on the other hand, metab- olized so efficiently in the fetus that it was not detectable in the umbilical artery (flow away from fetus). In regard to Point 1, pro- gesterone concentration was higher in the umbilical vein than in the umbilical artery (156, 761) and was seldom detected in the uterine vein (761). Some of the preg- nanes (5a-DHP, 3B-5P) also may be mater- nally derived (113, 762, 1121), perhaps from corpora lutea, since the concentration pro- files in early pregnancy were similar to the progesterone profile (761). All of the pregnanes, including 5a-DHP and 35-5P, are produced by the fetoplacental unit. Another recent study (677) indicated that some 5a-pregnanes may be derived from endometrial metabolism. 2.11D. Adrenal Steroids in Mares As noted above, the adrenal cortex is one of the structures with the appropriate enzymes for synthesis of steroid hor- mones. The equine adrenal cortex is capa- ble of synthesizing cortisol in vitro, and cortisol has been identified as the major corticoid in the peripheral plasma and urine of horses (815). Plasma concentra- tion of cortisol increases in response to administration of adrenocorticotrophic hormone. Corticosterone, cortisone, and deoxycorticosterone have been identified in addition to cortisol (1853), but there is disagreement on their concentrations in equine plasma (815). The release of corti— sol in horses in response to surgery, anes- thesia, exercise, and other stimuli has been reviewed (815), and the circadian pattern of cortisol and corticosterone in mares has been studied and discussed Reproductive Hormones 71 (228). Both hormones were in greater con- centration in the plasma during the morning hours. A description has been made of ultradian rhythms of cortisol (mean periods of 31 to 105 minutes) that were superimposed on the circadian rhythms (486). The concentration of plas- ma cortisol in the mare during the estrous cycle (pg. 244,- 104) and in the foal during the late fetal and early neonatal period (pg. 465,-1365) also has been studied. The equine adrenal cortex could con- tribute to circulating levels of androgens in mares as indicated by in vitro studies on adrenal cell production of androgens from pregnenolone (1471). The role of adrenocorticoids in repro- ductive function is poorly understood in all species. Perhaps, from the standpoint of this text, it is most important to recog- nize that the adrenals are a potential source of those steroid hormones that are generally associated with the ovaries (estrogens, progestins, and androgens). Clinicians and ovarian ablation experi- menters must be particularly aware of this possibility. The occurrence of estrous behavior during the anovulatory season and after ovariectomy in mares may reflect the ability of the adrenals to secrete sex steroids (pg. 96). In this regard, however, a recent study indicated that stimulation of the mare adrenals increas- es circulating concentrations of cortisol and testosterone, but not estradiol (1738). Perhaps the adrenal is not an important source of estradiol in the mare. It is clear that considerable research should be I directed toward the equine adrenals, not only because of the possible effects of adrenal steroids on reproductive func- tion, but also because abnormalities of ( adrenal function are being increasingly recognized. 72 Chapter 2 2.12. Hormone Assay A feat that should be forever admired was the ability of biologists, before the 1960s, to trick experimental models into divulging the fundamentals of interorgan hormonal regulation without a technology for measuring circulating hormonal con- centrations. Not until the late 1960s with the introduction of competitive protein— binding assays and, later, radioim- munoassays, were biologists able to con— firm much of what already had been postulated. The development of the high- ly sensitive radioimmunoassay is a land- mark in analytical chemistry. The theory and application of this technology result- ed in the Nobel Prize to the original developers (cited in 703). The technique provided the detailed background infor- mation we now enjoy on circulating levels of the various hormones in equids for both physiologic and pathologic processes. The early 1970S were the pioneer hor— mone-assay years in equine reproduction research. Competitive protein—binding (CPB) and radioimmunoassay (RIA) tech- niques, after initial development in other species, were adapted to equine plasma and serum samples. The first reports, as far as the author is aware, in reviewed journals of assay procedures and detailed descriptions of patterns of circulating con- centrations of the various equine hor- mones are listed in Table 2.2. During the 1980s considerable work was done to develop assays that are faster, do not require an elaborate labora- tory, and are not dependent on radioac- tive reagents (456). Progress has been so successful and rapid that the 1990s may become the age of dipstick assay technolo- gy. Progesterone kits are now available that are being touted as essential repro- ductive management tools. In the most recent report (980), commercial qualitative and quantitative “mare-side” proges- terone systems were evaluated. It was concluded that both systems were useful additions to other currently available diagnostic techniques. Commercial kits use assay technology known by a series of acronyms (e.g., ELISA, enzyme-linked immunosorbent assay; EAI, enzyme- amplified immunoassay; AELIA, ampli- fied enzyme—linked immunoassay; CELISA, competitive enzyme-linked immunoassay). The antibodies may be monoclonal and highly specific. The sys— tems involve enzyme-induced color changes and therefore do not require radioactive agents. This is especially important because it allows assays to be done without the necessity of meeting highly regulated requirements or han— dling and disposing of radioactive materi- al. The use of assay kits in farm manage- ment has been reviewed and discussed (68, 980, 467, 1515). Dipstick assays for qual— itative assessment at the farm are becom- ing available for progesterone (1639, 1515, 1595, 68, 468, 467), eLH (849, 848), and eCG (973, 849, 848). The more such capabilities and other technologic advances (e.g., ultrasonic imaging) become available, the greater the demand for knowledge in the fundamentals of reproductive biology. Thus, these technologies will likely stim- ulate, rather than suppress, the demand for professional services. TABLE 2.2. Apparent Year of Introduction of in vitro Assays for Characterization of Circulating Hormone Levels in Mares Hormone Year Ref. CG 1969 (43) Progesterone 1970 (1497) LH 1973 (1 768) Estrogens 1973 (1141) Oxytocin 1973 (3 7) FSH 1975 (490) Prostaglandin F 1976 (433) Relaxin 1981 (1552) Melatonin 198 1 (1435) Prolactina 1986 (828) Inhibinb 1991 (185) a Homologous assay b Immunoreactive Reproductive Hormones 73 HIGHLIGHTS: Reproductive Hormones Gonadotropins 1. The gonadotropins consist of two amino acid chains (cc and [3 subunits) that incor- porate about 25% (eLH, eFSH) and 47% (eCG) carbohydrates. 2. The oc-subunit is the same for all of the gonadotropins, and the B-subunit is responsible for specificity of the biologic activity. The B-subunits for eLH and eCG are the same and consist of 149 amino acids. 3. Each hormone is found in various forms (isoforms) that have different biologic and immunologic characteristics. The ratio of the various isoforms changes dur- ing various reproductive statuses. 4. In contrast to nonequine LH, eLH, as well as eCG, has potent LH and FSH activ— ities when injected into other species. 5. The equine ovaries are far less sensitive to eCG than are the ovaries of other species. 6. The equine gonadotropins have long half-lives, partly attributable to high sialic acid content. Reproductive Steroids 7. 10. 11. 12. 13. The major pathway for ovarian synthesis of steroids (androgens, estrogens, pro- gestins) is through cholesterol and progesterone. Estradiol-17B is the major estrogen in follicular fluid. Approximately 10% is estrone. ' Measurable production of androgens, progesterone, and estrogens by the embry~ onic vesicle occurs as early as Day 8. Urine from pregnant mares is a source of estrogens for therapeutic purposes. The quantity in the urine of mares is greater than for any other known species. Nine estrogens have been identified in urine of pregnant mares; three are formed through the classical cholesterol pathway and six through a noncholesterol path- way. The latter six are called the ring-B unsaturated estrogens (e.g., equilin) and are unique to the equine species. The fetoplacental unit begins to secrete estrogens, progestins, and probably androgens into the maternal circulation at about Day 60. Progesterone that is synthesized by the fetoplacental unit is metabolized primari- ly to pregnanes and seldom enters the maternal circulation. 74 Chapter 2 MILESTONES: Reproductive Hormones First report of a gonadotropic substance (later identified as eCG) in the blood of pregnant mares, including characterization of the gonadotropin profile (321). 1932 First descriptions of equilin and equilenin (review: 207, 208). 1943 Conclusion that the endometrial cups are the source of eCG (320). 1954 First experiment to yield direct information on conversion of testosterone to estrogens (705). 1957 First identification of progesterone in the equine placenta (1459). 1962 Initiation of studies on role of various ovarian cell types in formation of steroid hormones (1463). 1970 Preparation of highly purified eLH and eFSH (247). 1972-73 Series of reports demonstrating the fetal origin (chorionic girdle) of the eCG-producing endometrial cup cells (57, 62, 674). 1973 Initial study in a series that implicated the fetal gonads as an integral com- ponent in fetoplacental steroidogenesis (1305). 1974 Characterization of chemical and biologic properties of eCG subunits (1225). 1975 Demonstration of the discharge of progestins through the venous effluent of the gravid uterus beginning on Day 60 (1506). 1975 Identification of 5a—pregnanes in the blood of pregnant mares (760). 1979 First demonstration of pulsatile release of equine hormones (LH; 484). 197 9 Isolation and partial characterization of equine prolactin (298). 1982 Initial studies leading to the conclusion that the isoforms of eLH and eFSH vary during the estrous cycle and the biologic property is influenced by the ratio of isoforms (20). Purification and characterization of equine relaxin (1551). Structural studies of the amino-acid sequence of eCG (1575) and eLH (231) B-subunits Development of the technique for placing a cannula into the pituitary efflu- ent (803) with initial characterization of GnRH, eLH, and eFSH secretory patterns (23). Report that progesterone from the fetoplacental system is utilized or metabo- lized locally and is seldom discharged into the maternal circulation (761). —-Cfiapter 3— SEXUAL BEHAVIOR Considering the importance of the signs of equine sexual behavior to scien- tists, clinicians, and herdsmen and the spectacular beauty of equine courtship and mating, more research interest should be attracted to this area. Anyone who is launching an extensive research effort in behavioral characteristics under conditions of domestication should consid- er the information that has been obtained from feral herds (502, 896, 1652, 1649, 1040, 861). Reviews of ethology are available for farm species (362, 533). Reports and reviews have been published recently on other forms of behavior (nonsexual) in ponies and horses (e.g., communicative, eliminative, ingestive, investigatory, maternal, nocturnal, parturient, play; 355, 771, 862, 770, 769) and on social organization of feral horses (897). Reviews on sexual behavior in domestic mares (97, 1730) and stallions (1046, 1048, 1617) and a compila— tion of reviews on various forms of equine behavior are also available (354). 3.1. Problems Associated with Studies of Sexual Behavior Estrus is commonly determined by exposing the mare to a stallion (teasing). However, the reliability of various meth- ods of exposure and the interpretation of various behavioral signs during teasing have not been well defined. The decision whether a mare is in estrus usually has been made on a subjective basis depend- ing on the judgment of the operator. The occurrence of estrus behavior has been abused as an end point or reference point in mare research projects. The research scientist can be trapped into a compromising position in which the deci- sion whether or not a mare is in estrus may be affected more by bias toward the hypothesis under test than by objectivity. In many research reports the behavioral criteria used to define estrus are not ade- quately stated and standardized. Variation in behavioral response to teasing is an important consideration. An overall appreciation of the day-to-day, cycle-to-cycle, and mare-to-mare variabili— ty in response to teasing may be gained from Figure 3.1. The data were selected from an extensive graphic presentation of raw data (85, 1060). Note that in some cases the onset and termination of sexual receptivity were abrupt, and the level of receptivity during estrus and resistance during diestrus were pronounced and con- sistent (Mare A). The onset and termina- tion of some estrous periods (Mare D, 1st estrus) and diestrous periods (Mare C) were gradual. Some estrous periods were split by periods of resistance (Mare C, 2nd estrus and Mare E, 2nd estrus) or by a diminution in receptivity (Mare B, 2nd estrus). Other estrous periods were very short (Mare F) or silent (Mare G). Procedures for detecting estrus involve the teasing stallion, the observer, and the location or environment, as well as the mare. Critical evaluations have not been made on the effects of climatic (e.g., tem- perature) or diurnal factors on expression of estrus in mares. Apparently, the only formal attempt to measure the reliability of estrous detection using different stal- lions, locations, and operators was by Colorado workers (119). Independent 76 Chapter 3 0 5 10 15 20 25 3O 3 Very receptive 2 Moderately receptive 1 Mildly receptive 0 Phelmagic —1 Passively resistant -2 Mildy resistant -3 Actively resistant -4 Very actively resistant V - Ovulation MW 35 0 5 10 15 20 25 30 FIGURE 3.1. Illustrations of day—to-day, cycle-to—cycle, and mare-to-mare variation in behavioral response to teasing. A to G refer to individual mares throughout an estrous cycle and two ovulatory periods. Adapted from ( 85 }. determinations were made using a differ- ent stallion and operator at each of two isolated locations. The response of the mare or judgment of the operators was different in 8.3% of the determinations. The definition of an estrous response was not given, but perhaps (not stated) the two operators had reached a high level of agreement on how they would define an estrous response prior to conducting the experiment. Had this not been done, or if one of them had been influenced by bias pressures, the 8.3% disagreement might have been higher. Sexual behavior involves reciprocal factors between male and female (166). The interrelationships may be so com- plex that they may defy dissociation and characterization attempts. The possibil- ity that compatibility factors operate between certain mares and stallions may further complicate the picture. Furthermore, in group teasing (pg. 101) social interactions among mares can profoundly affect results. The principal considerations in study- ing mare sexual behavior involve the techniques for measurement or quantita- tion and evaluation. Sexual behavioral signs vary not only in kind, but also in frequency, intensity, and latency (interval of time from stimulus, such as approach of stallion, to response), in addition to patterns of onset and waning. Put all of these considerations together and one is left with a very complex situation. Sexual behavioral data have been organized into attractivity, proceptivity (maneuvers by mare to encourage the stallion), and receptivity (behavior that facilitates copu- lation; 166). Fundamental information for studying behavior has been summarized (398). In research projects, emphasis should be given to reliable, repeatable, and quantitative end points amenable to statistical handling. An example of gener- ating quantitative data in the assessment of sexual signs consists of assigning scores as follows (575): +3, standing for mounting with tail raised; +1, urinating; +1, flashing clitoris; +1, raising tail; 0, standing for mounting with tail held down; -1, kicking; -1, switching tail; -1, ears back; -1, moving; —3, not standing for mounting. The sum of these values can be used as an intensity index for each deter- mination. The resulting derived variable is considered to be a useful approxi- mation of a biologic end point (estrous intensity). 3.2. Behavioral Signs Sheer beauty and dynamism character- ize equine courtship and display. The demeanor of the stallion. varies from indifference to extreme aggressiveness. The response of the mare varies from active submission, highlighted by an accommodating posturing of the body, to vigorous repulsion. As a generality, sexu- ally receptive mares exhibit signs of serenity and accommodation, whereas nonreceptive mares exhibit signs of ner- vousness often accompanied by repelling maneuvers. In most teasing programs, the stallion is isolated from the mares between teas- ing sessions. Therefore, when exposed to a mare, the stallion may immediately and aggressively attempt mounting without prior direct contact and interplay between the sexes. More often, however, the pair will enter into precopulatory interplay. This may consist of sniffing and nuzzling about the head, lower flank and groin, and perineum of the mare, pre- sumably in search of olfactory cues (Figure 3.2). Often the stallion will bite or nip the mare. Biting may be Vigorous, consisting of prolonged grasping and tug— ging of skin folds or the vulva. The extent to which biting and other actions by the stallion (e.g., mounting, nuzzling) pro- mote estrous behavior or test the level of receptivity needs to be investigated. Frequently, the stallion will nibble at the mare’s rear leg, especially the area of the Sexual Behavior 77 hock; this results in a lowering of the mare’s pelvis and perhaps in some instances could facilitate intromission (Figure 3.3). Upcurling of the upper lip is another frequent behavioral manifesta- tion by the stallion and is called flehmen (Figure 3.3; pg. 94). The behavioral signs of the mare vary widely in kind and intensity, as discussed in subsequent sections. The extent to which proceptive behavior by the mare (e.g. nuzzling, following, posturing) encourage and excite the stallion requires study. When first approached by a stal- lion, an estrous mare may squeal and paw before turning her head to nuzzle and nibble the stallion; these preliminar- ies soon may be followed by frank signs of estrus. Posturing. Posturing is the most pro- nounced sign of sexual receptivity, since it is a whole-body response (Figure 3.4). This sign consists of positioning the body in a manner that is most accommodating to copulation. In addition, posturing as well as other behavioral traits may serve as an attractant to the stallion (1652). Posturing appears in many degrees and is characterized in its most exaggerated form by arched tail, flexed stifles and hocks, abducted rear limbs, and tipped pelvis with associated lowering of the per- ineal area. One rear leg may be raised and supported only by the tip of the hoof; the weight of the mare appears to be borne primarily by the front legs. This accommodating position of the body also has been termed lordosis and squatting. However, lordosis implies exaggerated convexity of the back, as occurs in rodents, but is not marked in mares. Squatting may be associated with the act of urination. It should be noted, however, that the body positions associated with posturing as a sexual response are quite similar to squatting associated with frank urination. 78 Chapter 3 M§x INSPECTION BY STALLION FIGURE 3.2. Mutual nuzzling and sniffing by stallion (black) and mare (upper panel). Nuzzling and sniffing of mare’s groin area by the stallion (lower panel); mare is in a moderate posturing position with tail raised. Sexual Behavior N IPPING REAR LIMBS FIGURE 3.3. Flehmen (upcurled upper lip) by stallion; mare in moderate posturing position with tail raised (upper panel). Biting of inner rear legs by stallion, causing mare to lower the pelvic area (lower panel). 79 80 Chapter 3 MODERATE POSTURING its FULL Pos URI G FIGURE 3.4. Moderate posturing position (upper panel) and full posturing position (lower panel). Note the string of mucus (arrow). Photographs prepared by M.J. Hermenet. Urinating. Other signs associated both with sexual receptivity and with the act of urination are raising and arching of the tail to expose the genitalia, passing of fluids through the vulva, and winking of the clitoris. These signs may occur during teasing and during mounting. Raising of the tail occurs in all degrees from barely perceptible to extremely exaggerated (Figures 3.2 and 3.4). Passing of fluids varies from a few drops of a Viscous sub- stance (Figure 3.4) to frank urination. This Sign is frequently termed urination, but it is emphasized that it has not been determined that all of the discharge is from the urinary bladder. The color and viscosity of the fluid vary widely from clear and thin with a yellowish tinge to cloudy and thick, but these variations do not appear related to the sexual state. To exemplify the variability in the nature of the fluid discharge, 350 determinations were described as dripping (39%), spurt- ing (2%), streaming (13%), combinations of these (10%), and not seen (36%; 575). Presumably, the passage of fluids leads to olfactory cues. Urinating or passing fluids may be persistent; one mare postured and passed fluids during 20 distinct periods in an hour. Apparently, when an estrous mare is first exposed to a stallion frank evacuation of a full bladder may occur. Thereafter, the passage of fluids may involve only Spurting or dripping or be imperceptible or missed. When it is not clear whether the mare is responding to a full bladder or sexual stimulation, it may be helpful to retease after the signs subside. Winking. Clitoral winking is character- ized by rhythmic eversion of the labia with exposure and projection of the cli- toris (Figure 3.5). This sign is also referred to as the clitoral or vulval flash. The winks may number from 1 to 100 or more in regular succession every few sec- onds. Nothing, apparently, is known about the role of the clitoral wink. One may speculate that it is part of the atten- tion-getting display of the mare. Sexual Behavior 81 In cattle, ovulation was hastened (1311) and pregnancy rate increased (1000) by various mating stimuli or digital clitoral massage. Attempts have been made to determine whether a similar phenomenon exists in mares, encouraged by the promi- nence of the clitoral wink. In a study involving pasture breeding (582), time of ovulation and growth rate of the ovula- tory follicle were not different between mares that were isolated from stallions and mares that were continuously pas- tured with a stallion. That is, there was no support for the hypothesis that ovu- lation would be hastened by frequent clitoral winking or the stimulation (physical, acoustical, Visual, olfactory) associated with a stallion. In a short report (1165), teasing with a stallion resulted in increased vaginal mucus fern— ing patterns and in vaginal pH, but it is not known whether clitoral winking was WIN KING FIGURE 3.5. Eversion of ventral portion of labia with exposure of clitoris. 82 Chapter 3 involved in the ferning and pH changes. An attempt was made to alter LH lev- els by continuous exposure of estrus mares to a stallion when the largest folli— cle reached 35 mm (600). A treated group (n=6) was continuously teased for one hour by alternating teasing stallions every 15 minutes so that winking occurred almost constantly. The control group (n=6) was not exposed to a stallion. Levels of LH immediately after the one- hour treatment and an hour later were not altered significantly. However, a recent short communication stated that a small but significant increase in LH con- centrations occurred in both stallions and mares during the first hour following cop- ulation (805). Another states that a mas— sive burst of LH was detected in the venous blood of the pituitary of a mare after teasing (1222). A recent study (804) in stallions found that after sexual arousal there was an increase in GnRH and LH in the pituitary blood but not in the peripheral blood. Perhaps further studies on the effects of winking in mares would be enhanced by collection of pituitary blood (pg. 113). These encouraging results, although sometimes contradictory, indi- cate the need for further study of the endocrinologic role of clitoral winking. Nonreceptivity. The signs of nonrecep- tivity of the mare include kicking, biting, holding back ears, switching tail, moving, shaking head, pawing, and other nervous and repelling manifestations (Figure 3.6). The incidence of these and other signs are discussed in the next sections. NONRECEPTIVITY FIGURE 3.6. Explosive repelling response by a nonreceptive mare. Mouth clapping. A sign known as mouth clapping, clamping, snapping, or champing occurs in foals and has been reported in one mare (1821). In foals, the behavior appears to indicate submission. Mouth clapping, however, is a distinct sign of estrus in jennies (1679) and zebras (895). In addition to the rhythmic mouth clapping, the lips are loose and the head is held low with the ears turned back, but not flattened as during aggression (Figure 3.7); all of these signs, collectively have been called mating face. In a study in jennies (1679), mouth clapping occurred consistently; there was no instance in which clitoral winking or posturing occurred in the absence of clapping. \ sssss‘ “ * \ \. Sexual Behavior 83 Furthermore, on the average, clapping began a day sooner and lasted a day longer than the other signs of estrus, and during individual determinations clap- ping was the first sign to appear. Mouth clapping was an especially useful indica- tor of estrus because it appeared consis- tently, often required minimal teasing, was readily observed, and often could be detected by sound alone. Other signs of estrus in jennies were similar to those described for horses and ponies. Pasture breeding behavior of donkeys was described recently (721). FIGURE 3.7. Sexual behavior in donkeys. Note the jenney’s open mouth with head down and ears back (mouth MOUTH CLAPPING clapping). 84 Chapter 3 3.3. Incidence of Behavioral Signs In a study of sexual signs (575), estrus was defined as standing firmly with tail up while being mounted, plus at least one of the following: 1) urinating at any time during teasing, 2) winking of clitoris at any time during teasing, or 3) raising tail before being mounted or after being dis— mounted. Determinations not meeting these criteria were defined as nonestrus (included diestrus, anestrus, and preg- nancy). A wider array of behavioral signs (Table 3.1) was then recorded and associ- ated with the conditions of estrus and nonestrus. Mares were teased individual- ly using a technique which included mounting by the stallion. The difference in incidence of behavioral signs between mares in estrus and nonestrus was signif- icant for each sign (Table 3.1). The most frequently observed signs during estrus in decreasing incidence were as follows: raising tail, remaining calm, winking cli- toris, posturing, urinating, and nuzzling. By definition, all mares in estrus stood with tail up while being mounted. The signs most frequently observed during nonestrus in decreasing incidence were as follows: moving, holding ears back, switching tail, issuing a vocal response, kicking, raising in rear, biting, pawing, shaking head, and raising in front. The stallion was significantly more active when the mare was in estrus, as indicat- ed by number of bites, mounts, and flehmen responses. 3.4. Sexual Behavior in Herds Feral herds: overview. Information on social organization of feral equids can be found in a review by Klingel (897). The fol- lowing brief account of sexual behavior in feral herds is based on a recent review (862). Feral horses most commonly live in bands or herds with the social unit being one stallion and 2 to 21 mares, although TABLE 3.1. Behavior of Mare and Stallion during Teasing when Mare was in Estrus or Nonestrus Incidence During During estrusa nonestrus Behavioral signb No. % No. % (No. determinations) 581 . . 2181 Mare Raised tail 569 98 266 12 Urinated 313 54 153 7 Winked clitoris 506 87 223 10 Remained calm 517 89 161 7 Nuzzled stallion 75 13 111 5 Posturing 420 72 40 2 Mounted 581 100 1121 51 Stood, tail up 581 100 35 2 Stood, tail down . . . . 157 7 Did not stand . . . . 929 43 Not mounted . . . . 1060 49 Kicked 63 11 1188 54 Bit stallion 19 3 745 34 Held ears back 100 17 1862 85 Switched tail 61 10 1795 82 Moved about 1 17 20 2037 93 Shook head 14 2 390 18 Pawed 17 3 602 28 Raised in front 5 1 171 8 Raised in rear 60 10 877 40 Vocal response 200 34 1433 66 Snorted 5 1 286 13 Squealed 195 34 1 147 53 Stallion Bit mare 533 92 1802 83 Mounted 562 97 1106 51 Flehmen 416 72 368 17 Activity score 2 0 71 3 30 5 731 34 273 47 1154 53 279 48 255 12 ODNHO a Estrus was defined as standing firmly with tail up while being mounted, plus at least one of the follow- ing: 1) urinating, 2) winking clitoris, or 3) raising tail. b For each behavioral sign, the difference in fre- quency of occurrence between estrus and nonestrus is statistically significant. From (575). multiple-male groups can occur. Harem stallions strive to maintain their band as a unit by herding the mares; the stallions ears are held back and the head and the neck are extended and held close to the ground and may be swung from side to side (Figure 3.8). This display occurs especially when the herd is threatened by the approach of another herd or stallion. Sometimes the stallion will leave the herd to confront an approaching stallion. The mares and foals do not show this activity. Harem stallions have 85% to 90% of the mating rights in their harem. In multiple—male harem bands, the majority of matings are done by a domi- nant male. Inbreeding does not occur often because offspring leave the herds before sexual maturity. The harem stallions urinate on the urine or feces of their mares, especially during the breeding season (e.g., 93% of eliminations were marked in May versus 1% in November). It has been suggested that marking of urine or feces by stallions Sexual Behavior 85 may serve to identify mares of a given harem. In this regard, it has been report- ed that stallions that invade a harem unprotected by the resident stallion may induce abortions (179); abortions were stated to occur in 90% of the mares that were less than 6 months pregnant and 80% of the abortions were associated with forced copulations. This observation needs confirmation. Domestic herds: overview. Groups of domestic horses or ponies show the same behavioral characteristics as feral groups. Pasture mating is sometimes done, espe- cially in the production of ranch horses (654, 1038). The mares usually are sent to their respective pastures for a period of social adjustment (e.g., 2 weeks) and then a stallion is introduced. A new mare introduced after the stallion is in place may not be accepted and may be severely attacked by the stallion. The characteris- tics displayed by a stallion in pasture mating of domestic mares seem similar to those described for stallions in feral HERDING FIGURE 3.8. Herding behavior of stallion under pasture conditions. Note that head is held down with ears back. 86 Chapter 3 herds. Domestic herds, however, may be moved by the rancher, sometimes fre— quently, to new locations. When this occurs or when the stallion senses an external threat or environmental change, herding of the mares by the stallion is likely to occur (600). For example, when the herd is moved into a new corral, the stallion may aggressively herd and con- fine the mares to a corner (Figure 3.9), especially if another stallion is nearby. The confinement sometimes continues for hours or a day. This can be alarming to an observer because the mares may be prevented from drinking or eating for many hours. The mares do not usually challenge the stallion in these situations. The mares seem to instinctively maintain their crowded position and usually face away from the stallion. After the stallion seemingly becomes accustomed to the new corral, the mares and stallion begin to mingle, and in an equilibrated herd the stallion is often inconspicuous. In an FIGURE 3.9. Herding by a domestic stallion in a corral. The stallion is herding the mares into a corner (upper panel). After herding is completed, the mares stand quietly in a tight group, and the stallion stands at a distance (lower panel). Photographs prepared by M.J. Hermenet. HERDING extreme example (600), a herd (25 mares and 1 stallion) that was accustomed to a corral was moved through a lane to a pas- ture. The stallion herded the mares into a compact group at the end of the lane nearest the corral and maintained the confinement for 30 hours. Occasionally a mare attempted to break away but was immediately returned by the stallion; during this time, the rest of the mares maintained the compact position. The operator terminated the phenomenon by holding the stallion overnight in the sta- ble. The behavior did not reoccur when the stallion was then released into the pastured herd. Much study is needed of herd behavior, and programs for optimal management for pasture breeding should be developed. Frequency of mating. Frequency of teasing and mating by a vigorous 6-year— old stallion during pasture breeding has been reported (250). As many as 16 mat- ings were recorded in one day (daylight hours). However, the matings were recorded using binoculars from a motor vehicle and perhaps were subject to inac- curacies. In addition, as noted below, stal- lions vary considerably in libido and the mating prowess of an individual cannot, of course, be taken as representative. Sexual behavior and interplay in domestic herds in which a rested (2 days) stallion was put into groups of 20 mares have been studied in two experiments (621, 582). In Experiment 1 (horses), a stal- lion (n=11) was introduced into a herd in a corral and the herd was observed for one hour every other day for a month. In experiment 2 (ponies), five mating groups were used, each consisting of a stallion and 20 mares. A rested stallion and herd were put into a two-acre lot for three hours every other day for observation. Length of intervals to first matings and between first and second matings and the number of matings for the hour (Experiment 1) or for the first hour (Experiment 2) are shown (Table 3.2). Sexual Behavior 87 There were significant differences among stallions for these end points, indicating variations in stallion libido. Sometimes a mare was mated without prior sexual interplay immediately upon introduction of the stallion into the group of mares. In 15% of the cases, a mare was mated before the observer detected estrus (pos- turing). About 50% of these occurred dur- ing the first three minutes after introduc— tion of the stallion, and in all cases, posturing of the bred mare was observed before the end of an hour. False mounts also occurred during the first few min- utes, often involving mares that were not in estrus. In two other herds (Experiment 3) in which the stallion was present con— tinuously, the mean number of breedings for the first observational period (first 3 hours after introduction) was 4.0. The number of breedings decreased during subsequent observations so that the mean number of occurrences thereafter was 0.5 per three-hour period. At that rate, the stallions that were present continuously would have mated four times per day, which is similar to the number of matings for a three-hour period in stallions that were rested for 45 hours between obser- vational periods (Experiment 2). It is not known, however, whether the three-hour observational period was representative of a 24-hour day. Pregnancy rates associ- ated with pasture or group mating are given in Chapter 12 (pg. 506). TABLE 3.2. Meansfor Mating Characteristics when Rested Stallions were Introduced into Groups of 20 Mares per Group. End point Exper. 1 Exper. 2 Interval (min) Introduction to mating 12 13 Between first 2 matings 17 34 No. matings during first hour 2.4 1.9 Number of observations for calculation of means was 46 to 62. Adapted from (621 ) and (582). 88 Chapter 3 Stallion’s choice of mares. The number of matings per hour was not altered by the number of mares in estrus (Exper— iment 1, 621; Experiment 2, 582). In both experiments, the number of times a stal— lion remated the same mare, when more than one mare was in estrus, was signifi- cantly greater than what was expected to occur by chance. Thus, the stallions apparently were not able to discern that a mare had already been mated. For exam- ple, when two mares were in estrus (posturing), the second mating occurred in the same mare 83% of the time, where— as remating of the mare was expected to occur 50% of the time by chance. Thus, neither the stallion nor mare avoided remating. Remating of mares occurred regardless of the number of posturing mares, taking into account the effect of increasing numbers on the decreasing likelihood of remating. The mechanisms involved in remating the same mare were not entirely defined. In this regard, how- ever, mares that were mated were gener- ally more active and exhibited more pro- ceptive behaviors during the hour of observation than mares that postured but were not mated (note results for Experiment 2 in Table 3.3). This was indicated by the greater number of follows (mare following the stallion) and rotations (mare rotating her rear toward stallion), as well as the greater number of postures per hour, in the mares that were TABLE 3.3. Incidence of Behavioral Characteristics by Estrous Mares that were Mated versus those Not Mated during Group Exposure to a Stallion Not Characteristic Mated mated Posturing 5.1 i0.4 3.5 i0.5 Following stallion 2.9 i0.4 1.5 i0.3 Presenting rear to stallion 1.1 4:02 0.4 i0.1 For each characteristic, means are significantly dif- ferent between groups. Incidence = mean number of observations/mare/hour. Adapted from (582) mated. Also, the interval from introduc- tion of the stallion to first posture was shorter for mares that were mated, although not significantly. The possibility that repeated mating of the same mare was related to availability or activity of the mare was further supported by the finding that the number of follows, rota- tions, and postures per hour were greater in mares that were mated more than once than in mares that were mated once. Mating did not always involve the mares that actively maintained close proximity to the stallion, however. Occasionally the stallion would leave a group of posturing mares and approach, inspect, and mate a distant mare. On Days —6 to -1 (ovulation = Day 0) the stallions did not give preferential treatment to estrous mares that were closest to ovulation. These results do not support the speculation that stallions can judge the imminence of ovulation. Significantly fewer estrous mares were mated when they were on Days -8, -7, 0, or 1 than when on Days —6 to -1; however, this was related to fewer number of pos- tures per hour on Days -8, —7, 0, and 1 (Figure 3.10). Estrous mares that were mated more than once per observational period (versus once) postured more fre- quently and more actively followed or positioned themselves near the stallion. The contribution of the stallion in deter- mining which posturing mare was to be mated was not clear. The stallion was not able to select the mares closest to ovula- tion or select against those that were already mated. Clearly, however, the stallion played a profound role in stimu- lating estrous behavior, since in the absence of a stallion, posturing was rarely observed (frequency not critically determined). Mare interference. In the experiments described above, mare interference to interplay between an interested pair was more frequently directed at the mare by keeping her away, Whereas aggression during mounting was more frequently Posturing & mating Mares that postured 60 (11) 40 Percent — —" 20 Number of days from ovulation directed at the stallion. Aggression dur- ing mounting resulted in apparent pre— vention of mating (ejaculation) in 64% of the aggressions. Mares in estrus were responsible for most of the interference. There was no difference between non- interfering estrous mares and interfering estrous mares in the proportion that was mated. When interfering mares were in nonestrus, their hostility was usually directed at the stallion (92%), whereas when in estrus the interference was more frequently directed at the mare (73%). Interference relationships were very com- plex, with considerable variation in their nature and outcome; such relationships will require much study. For example, when the mare with the greatest number of interferences (n=121) was in estrus, she interfered primarily (92%) by threat— ening or chasing other estrous mares and occasionally by chasing all mares away from the stallion; when not in estrus she seldom interfered, but when she did interfere, the aggression was directed at the stallion during mounting. Another mare’s mode of interference was to kick at the stallion during premounting inter- play or mounting, and another interfered by striking at the mating pair with her front feet. . Posturing mares —’ that were mated Sexual Behavior 89 ‘ FIGURE 3.10. Percentage of mares that postured and percentage of posturing mares that were mated during the periovulatory period. Groups of mares (n=20/group) were exposed to a free-running stallion in a corral for one hour. Numbers in parentheses are total numbers of observations from which the per— centages were derived. Adapted from (582). 3.5. Intensity of Behavior Most workers have distinguished only between periods of receptivity (estrus) and repulsion (nonestrus) and have not considered possible gradations of these two classifications. The intensity of behavioral signs is sometimes stated to increase progressively during estrus, reaching maximum intensity as ovulation approaches. This conclusion has been reached by at least two groups of investi- gators (85, 1153), but support for the con- clusion is weak. To obtain additional information in this regard, '70 estrous cycles were examined, excluding estrous periods of less than six days and the estrus associated with the first ovulation of the ovulatory season (575). Because ovu- lation occurred during the last three days of estrus and primarily on the second-to- last day in this series, the results (Figure 3.11) did not support earlier conclusions that there is a gradual increase in inten— sity from the beginning of estrus until ovulation occurs. However, the transition from diestrus to estrus and from estrus to diestrus occurred gradually and agrees with what has been reported (85, 1153). In a short estrus of 3 or 4 days, therefore, intensity does increase until ovulation 90 Chapter 3 Estrus intensity Estrus Diestrus O) A N Intensity of estrous behavior (coded) O -2 -4 -6 -3 -2 -1 Central Central day day Days FIGURE 3.11. Mean changes in intensity of behav- ioral signs during estrus and diestrus for 70 estrous cycles in ponies. Intensity index was derived by ‘ assigning negative and positive values to signs of estrus and nonestrus, respectively, as described in text. 1, 2, 3 refer to first three days of estrus or diestrus and -1, -2, -3 refer to the last three days. Means with different superscripts are different (P<0.05). From (575). occurs. If estrus is longer, the increase in intensity in early estrus is followed by a prolonged plateau. Therefore, when estrous periods of various lengths are averaged together, starting from the day preceding ovulation, a misleading, illu- sory effect is obtained; this may account for discrepancies in the literature. These results agree with the above described distribution of the incidence of posturing during the preovulatory period (pg. 88) and with a study in which a consistent pat- tern of increasing behavioral scores as ovulation approached was not found (1126). It has been stated that the relation- ship between stage of development of the follicle and the intensity of estrus was not significant (551). However, in a short report (1726), it was stated that the inter- val from exposure to a stallion to appear- ance of tail raise appeared to be associat— ed with maturation of the ovulatory follicle, indicating measurable increased receptivity as ovulation approached. Attempt to confirm this finding apparent- ly has not been made. 3.6. Overt and Covert Estrus Subestrus. Varying intensities of estrus, based on degree of uniformity and degree of response to teasing during the periovulatory period, are depicted in Figure 3.12. Overt estrous periods are those in which there is at least one day on which the behavioral signs fit whatever criteria the operator is accepting as estrus. Often the major portion, and sometimes all of an estrous period, is overt, such that the estrous period would be the same “length” in the records of operators using widely divergent teasing procedures. However, the response of some mares may be less obvious, causing considerable recording discrepancy among operators. This frequently occurs during the transition between stages of the estrous cycle as described above. It may also occur during all or much of the interval of time associated with ovulation. For this reason, the term subestrus is used to describe the level of behavioral response representing a change in a given mare’s behavior but not meeting whatever criteria the operator is accept— ing as estrus. As examples, subestrus would be indicated by standing for mounting with the tail down in mares that otherwise seldom show this sign, a pronounced decrease in the number of signs associated with nonestrus (kicking, moving, switching, biting), or the appear— ance of some of the signs of estrus but not enough to meet all of the criteria for estrus. Prolonged subestrus can cause dif- ficulty in estimating the stage of the estrous cycle and in discerning the peri- ovulatory period. Split estrus. Split estrus is character— izedby the occurrence of nonestrus or subestrus for a day or two during what is considered to be one full estrous period. The incidence of split estrus has been Estrus Diestrus Estrus Overt estrus I:- Manifest throughout period of subestrus Split by 1 or 2 days of subestrus Split by 1 or 2 days of nonestrous signs Covert estrus Subestrus throughout ------—. ------ Nonestrus signs, indistinguishable from diestrus reported as 5%, 5%, 10%, and 12% by var- ious investigators (review: 575). In Figure 3.12, the total 7% incidence has been divided into the incidence of estrus split by sub-estrus (5%) and by nonestrus (2%), reflecting, again, the dependence of inci- dence on the definition of estrus. Silent estrus. Ovulation in the absence of estrus (covert or silent estrus) occurs in mares as well as in the other large domestic species. The degree of dimin- ished estrus varies from partial (sube- strus) to complete (nonestrus; Figure 3.12). The incidence of silent estrus has been reported to be 6% (1139) and 7.5% (358) in horses, which agrees with the data for ponies in Figure 3.12. 3.7. Causes of Diminished Estrus The causes of diminished or weak estrous behavior in mares have been investigated in several laboratories. Follicular development (diameter of largest follicle, number of follicles >20 mm) the day before ovulation in mares Preceded or followed by 1 or 2 days of subestrus Sexual Behavior 91 Incidence (%) 47 22 Prolonged (>2 days) associated 17 FIGURE 3.12. Incidence of vari- 6 ous types of estrus from 1200 estrous determinations in 14 mares based on uniformity and 1 degree of response to teasing. From (575). with covert estrus and subestrus was compared to that of mares with overt estrus (575). There were no significant dif- ferences and not the slightest suggestion that gross morphologic development of follicles plays a role in the incidence of diminished estrus behavior. Hormonal associations. The concentra— tions of circulating steroid hormones also did not seem to be related to diminished behavior in one study (1126); plasma levels of estrogens were not different between overt and covert estrus. However, California workers noted that silent estrus sometimes (26%) followed induc- tion of luteolysis with prostaglandin treatment (874). Therefore, the relation- ships among estradiol concentrations, luteal regression, time of ovulation, and behavioral estrus were investigated fol- lowing spontaneous and induced luteoly- sis (1139). Silent estrus, compared to overt estrus, was associated with lower estradi- ol concentrations, a longer interval from maximum level of estradiol to ovulation, and a shorter interval from luteolysis to ovulation (Figure 3.13). 92 Chapter 3 Hormones during silent estrus Progesterone Overt estrus E 6 (n=22) O1 5 I: .9 4 E :15; ‘ ' Silent estrus 0 (n=5) 8 2 O 0 EstradioI-17B Concentration (pg / ml) -10-8-6-4-202 9 Number of days from ovulation FIGURE 3.13. Mean serum progesterone and estradiol-17B concentrations in mares showing overt estrus and silent estrus during the periovula- tory period. Decline in progesterone values occurred significantly closer to ovulation in the silent-estrus group. Estradiol-17B was significantly lower on Days -2, —1 and O in the silent—estrus group. Adapted from Nelson et al. (1139). Repeatability. Split estrus, subestrus, and covert estrus have not been convinc- ingly associated with breed or with any other internal or external factors. An analysis was done to determine whether diminished estrous behavior (covert estrus and the prolonged subestrus asso- ciated with very short estrous periods) tended to recur in individuals (575). The ratio of number of periods with dimin- ished estrous behavior to the total num- ber of estrous periods was significantly different among individual mares, indi- cating repeatability of occurrence within mares. To determine if the intensity of estrus was repeatable within mares, neg- ative and positive values were assigned to various behavioral signs as described (pg. 76). The intensity index varied Widely among individuals and Within individuals (Table 3.4). Measurable repeatability was obtained, as indicated by the intraclass correlation for the intensity indices. The difference among mares in mean estrus intensity index was significant, also indi- cating repeatability within individuals. Other data (85, 780) further indicate that diminished estrus response to teasing tends to recur in individuals. Silent estrus occurred in 6 of 11 Thoroughbred and Quarter Horse mares over two years; it occurred 32 out of 34 times in one mare and, in another, at all times during the TABLE 3.4. Repeatability of Intensity Index of Estrous Behavior within Individual Mares Mare No. estrous Intensity index identity periods (mean iSEM) 1 6 +0.0 i 0.6 2 5 +0.1 i 0.4 3 8 +0.8 i 0.7 4 5 +1.8 i 1.4 5 5 +2.2 i 0.9 6 7 +2.3 i 0.5 7 7 +2.8 i 0.9 8 8 +3.7 i 0.5 9 8 +3.8 i 0.4 10 5 +4.4 1- 0.3 11 7 +4.6 i 0.2 12 5 +4.9 i 0.3 13 9 +4.9 i 0.1 14 9 +5.0 i 0.2 Highest possible level of intensity is +6 and the low- est is —6. Intensity index is the sum of coded intensi- ty values divided by number of days of estrus. Mean intensity index is significantly different among mares, indicating repeatability of intensity of behavior within mares. Intraclass correlation = 0.57. From (575). first five months of observation (780). Thus, knowledge of a given mare’s behav- ioral responses (e.g., availability of a record of past responses) should facilitate detecting the periovulatory period. 3.8. Physiologic Control Pitifully little is known about the phys— iologic control of sexual behavior in the mare. Overt behavior, as determined for other species, includes a patterned motor output involving the central nervous sys- tem. Neuroendocrine mechanisms at the level of the brain are complex and beyond the scope of this text; interested readers are referred to a review (856). A hormonal component is interrelated with the neural component to account for the widely divergent behavioral responses in mares under different reproductive conditions (estrus versus nonestrus). Also, certain external stimuli play important roles, such as the reciprocal factors operating between the two sexes. 3.8A. Pheromones and Other Stimuli Knowledge of the nature of signals (Visual, tactile, olfactory, auditory) that cause a mare to express estrous signs has potential practical application, especially in artificially eliciting sexual responses in the absence of a stallion. Similarly, knowledge of the senses that arouse stal- lions could be useful in semen collection. Erection in stallions depends upon recep- tion of erotic stimuli associated with courtship (666). The sight of the mare (Visual stimuli) apparently elicits excitability in the stallion, since blind- folding decreases the percentage of responses. Presumably, Visual stimuli also contribute to the response of the mare. Tactile stimuli also operate in the stallion and presumably in the mare. Biting of the mare by the stallion and pinching and stretching of the vulva and folds of skin in the perineal area likely play a stimulatory role for both sexes. Sexual Behavior 93 Olfactory stimuli are also operative, at least on the part of the stallion. The stim- uli are apparently derived from different regions of the mare’s body, since much time is spent in sniffing the genitalia, muzzle, and inguinal area. Urine also provides olfactory stimulation; young stallions showed poor response to a phan- tom but appeared to respond after the phantom was sprinkled with urine from an estrous mare (666). However, experi- mental inhibition of the olfactory stimuli of adult stallions did not inhibit sexual behavior. It appears that young stallions utilize olfactory cues, whereas sexually experienced and conditioned stallions are not dependent upon them. Mares, as well as stallions, seem to respond to the smell of urine or discharged fluids. Urination by one mare in the approach aisle leading to the teasing area was often followed by urination in the same area by subsequent mares (575). Presumably, this occurred through olfactory cues. Although both estrus and nonestrous mares appeared to urinate in the area, a significantly greater proportion of the estrous mares (63%) urinated compared to nonestrous mares (16%). Pheromones. Sex pheromones are chemical substances that serve as a means of communicating information con- cerning reproduction between animals. These messengers originate from modi— fied skin glands and also may be present in urine (includes vulvar mucus) and feces. Presumably, it is these messengers that the stallionxis seeking when he sniffs the muzzle, groin area, and perineal region of the mare. Odors apparently have an arousing effect on male sex drive as manifested in the olfactory reflex of flehmen (curling the upper lip). Male dogs, as a comparative example, are attracted from great distances by an estrous bitch (1044). The substances involved directly in initiating or influenc- ing sexual behavior are called signaling or releasing pheromones. In this regard, dogs can be trained to detect the appar- 94 Chapter 3 ent pheromone in estrous cows (873), but, apparently, this has not been attempted as a method for locating estrous mares. Perhaps pheromonal communication by mares is aided by the repeated wetting associated with urinating and winking. Metabolic byproducts of bacteria have been implicated in olfactory communica- tion in other species (cited in 97). Integration of sensory stimuli. Of the nonequine farm species, the stimuli for reproductive behavior have been most thoroughly studied in swine (1466). In this species, every sense organ receives specif- ic information, but some information is more important than others. For exam— ple, smell and sound induce the standing reaction of the female. The various senso- ry stimuli have a combining effect, but in the absence of one signal, the behavioral sequence still may be elicited by the remaining stimuli. It would be valuable if the extensive specific information on the stimuli for sexual response available for swine was also available for mares. In a preliminary equine report (1697), the effects of the following stimuli were examined: recordings of acoustic expres- sions of a courting stallion; digital tactile stimulation of the neck crest, flank, and external genitalia; and a combination of the acoustic and tactile stimuli. Results suggested that these approaches may be useful for detecting estrus in the absence of a stallion. All three methods seemed effective, but the combination of acoustic and tactile stimuli elicited the most behavioral signs. Pinching the neck was ineffective, whereas rubbing the flank and especially the genitalia seemed effec— tive. Although these findings did not seem conclusive, they indicate the need for further study in this potentially fruit- ful area. In a preliminary trial (97), recordings of stallion vocalizations elicit- ed attention and approach by mares, but the effects were short-lived. Effects of pheromones of males and females on female physiology. Many of the pheromone phenomena were first dis- covered in mice and have been named for the discoverers (review: 1013). In mice, pheromonal cues in the urine accelerate puberty, induce estrus, and block preg- nancy in newly mated females (872). In humans, evidence has been accumulating that pheromonal effects may be exerted at the subliminal level (642). In boars (222) and humans (642), musk-smelling 15— androstene steroids have been proposed as candidates for the male pheromonal effect. These androgens are formed by the testes and secreted in saliva and seminal fluid. In the axillary region, they are linked with the action of bacteria. Among the farm species, it has been well docu- mented that the presence of a ram stimu- lates the onset of estrus in ewes (83). The effect of running a stallion with mares on the onset of the breeding season has received only meager attention. In South Africa, a stallion with a retroverted penis was run with a band of Thoroughbred mares beginning in midwinter (173). It was concluded that the presence of the stallion conditioned the mares to earlier breeding. However, the number of mares was limited, and in the absence of con- temporary controls, the conclusion is sub- ject to reservation. In another study, an attempt to hasten the onset of the breed- ing season by housing a stallion with mares was unsuccessful (573). This failure should not discourage searching for pheromone phenomena in horses, particu- larly in View of the biologic and applied importance of such mechanisms. 3.83. Flehmen The word flehmen was coined by a German worker in the 1930s (cited in 1652). This peculiar behavioral manifesta- tion is characterized by extension of the neck and raising of the head with curling of the upper lip to completely expose the upper incisor teeth (Figure 3.3). The sclera of the eyes are usually exposed. Flehmen occurs in many species but is especially prominent and noticeable in horses because of their long lips. A recent review on flehmen in farm animals is available (688). Equine flehmen. Flehmen behavior in horses was investigated recently (1014, 768, 353, 964). The phenomenon occurred espe- cially in stallions but occasionally in geld- ings and mares. The response was shown by 3 of 6 geldings, and the mean duration in geldings was 14% as long as for stal- lions and 50% as long as for mares (1014). Mares are likely to exhibit flehmen after sniffing the foal or discharged placental fluids; perhaps this behavior aids in maternal identification of offspring. There were no significant differences in frequency or duration of flehmen in stal- lions tested with urine and vaginal secre- tions from mares in estrus versus non— estrus (1014, 768). However, stallions showed flehmen during inspection of the mare and not during inspection of isolat- ed samples. Flehmen was done more for mares in estrus than for mares in non- estrus (768, 1538), probably because the estrus mares urinated more frequently, and the stallions exhibited flehmen after each urination inspection. It has been concluded that stallions can differentiate gender of a horse on the basis of inspect- ing feces but not urine (1538); a positive test consisted of a flehmen response when presented with various samples. These authors indicated that flehmen by the stallion was more frequently followed by marking behavior rather than courtship behavior. The fascinating development of flehmen in foals has been described (353); the flehmen rate decreased from birth to 20 weeks of age, and foals of either sex performed flehmen more than the mother. Noneguine species. The interrelation— ships among pheromones, the flehmen stance, and the vomeronasal organs have been the subjects of studies and postula- tions in nonequine species. An interesting hypothesis has been proposed involving an association between flehmen and the functioning of the vomeronasal organ Sexual Behavior 95 (480); flehmen may serve to activate the vomeronasal organ or to direct or trap pheromone-laden air into it by closing the external nares. The vomeronasal organs appear to be more sensitive than nasal odor receptors to low-volatility com- pounds and thus may function as recep- tors for pheromones (643). In hamsters, severing the nerves to the vomeronasal organ decreased the incidences of copula- tion, and lesions to the organs interfered with pheromone responses in mice (978). Equine vomeronasal organs. There are two vomeronasal organs in horses, one ventral to each nasal passage with a slit- like opening (1488) located close to the floor of the ventral nasal meatus (480). Each structure in mares consists of a blind, cartilaginous tube, approximately 12 cm in length and lined by mucous membrane (1488). The vomeronasal organs contain mucus-secreting glands and the mucus bathes the epithelium (768). Liquid may therefore drop from the nostrils after flehmen. The openings to the organs are 7 to 10 cm from the external nares. Interestingly, the organs open into the oral cavity just behind the incisor teeth in bulls; tongue manipulation just behind the incisors before flehmen appears to assist in forcing liquid into the ducts (814). It seems that horses would be a good research model; the vomeronasal organ is well-developed, flehmen is common, and pheromones are apparently involved in several dramatic behavioral responses in this species. 3.8C. Unseasonal Sexual Behavior An enigma in mare sexual responses is the propensity to show estrus signs after ovariectomy (mares, 575; fillies, 1760), dur— ing the anovulatory season when the ovaries are small (pg. 136; 573), before puberty (1760), and occasionally during pregnancy (pg. 321). Estrous behavior in mares with inactive ovaries has been called unseasonal (575) or paradoxical (699) estrus. Such behavior is not associated 96 Chapter 3 with ovulation and must be considered nonproductive in terms of establishing pregnancy. These periods may satisfy all of the usual criteria for identification of estrus, including standing for mounting and allowing copulation. That is, such periods may be indistinguishable from the behavioral display of mares during the periovulatory period. More often, however, the mare will show all the pro- ceptive and receptive signs but will not allow mounting. Incidence. The phenomenon of unsea- sonal estrous behavior has been studied during the ovarian-inactive phase of the anovulatory season and in ovariectomized mares (99, 102, 100). Daily observations were made of pony harem groups during the ovulatory season (intact mares) and anovulatory season (ovariectomized and intact mares). Estrus-type behavior was shown during the anovulatory season by both ovariectomized and intact mares. During the ovulatory season, such behav- ior was shown by estrous but not by diestrous, mares. No significant differ— ence was found between ovariectomized and seasonally anovulatory mares for the incidence of estrous behavior, mounts, intromissions, or ejaculations. However, negative reactions to mounts and intro- missions were greater in the ovariec- tomized and intact anovulatory season mares (2.9 reactions/mount) than in estrous mares in the ovulatory season (0.5/mount). The phenomenon occurred frequently in 2 of 3 fillies that were ovariectomized at four months of age (1760); estrous signs began in the ovariec- tomized fillies at the time that intact con- trol fillies entered the ovulatory season (13 to 14 months of age). The occurrence of the estrous signs in ovariectomized fil- lies indicated that the estrous behavior in mature anovulatory and ovariecto- mized mares was not due to a previously learned response. Adrenals and androgens. Dexameth- asone administration to ovariectomized mares was used to block adrenal synthe— sis of steroids (99). Data for days 5 to 10 following initiation of treatment showed suppression of sexual behavior in treated mares; there was a decrease in solicita- tions, mounts, intromissions, and ejacula- tions. The ability of dexamethasone to suppress estrous behavior in ovariec- tomized mares suggested that an adrenal source for the hormones (estrogens or androgens) is responsible for estrous behavior in seasonally anovulatory and ovariectomized mares. In a preliminary study (one mare; 1126), peak levels of dehydroepiandrosterone during estrus appeared to be reduced by dexametha- sone treatment. A recent study (1738) indi- cated that stimulation of mare adrenals increased the circulating concentrations of testosterone but not estradiol-17B. It has been noted that 5 of 7 mares treated with testosterone propionate in conjunc- tion with a progestin showed estrus behavior within 24 hours after injection (1600). Testosterone also has been shown to elicit estrous behavior in swine and ewes (cited in 1600). It is unknown Whether the effects were due directly to testosterone or to a metabolite (e.g., estradiol-17B). To summarize, it is likely that unseasonal estrous behavior in mares is due to androgens produced by the adrenal for the following reasons: 1. It occurs in ovariectomized mares; 2. It is suppressed when adrenal syn- thesis of steroids is blocked with dexa- methasone; 3. Stimulation of mare adrenals increased the circulating concentrations of testosterone but not estradiol-17B; and 4. Estrous behavior has been noted in mares receiving testosterone propionate. A specific test of the hypothesis that androgens elicit unseasonal estrous behavior in mares has not been done and is needed. m. The ability of the mare to display unseasonal sexual behavior is apparently unique among all subprimate species studied. It has been suggested (97) that such behavior may play a role in con- tributing to the cohesiveness of feral bands. Motivation to remain near the stallion may be especially important dur- ing the anovulatory season. 3.8D. Hormones Little is known about the role of the central nervous system and ovarian hor- mones in sexual behavior in mares. Therefore, we will consider briefly some aspects of knowledge gained from other species as reported in reviews (377, 835). Messages received by the detector or sen- sory organs are processed and pro- grammed through neural interconnec- tions. Responses are further influenced by experience of the animal. In addition, the circulating levels of certain hormones exert profound effects upon organization of neural impulses. It has been shown (1088) that estrogen increases locomotor activity in the female rat and produces a specific urge to seek sexual contact. It is generally accepted that estrogen acts directly on the brain and that estrogen receptor sites exist in the brain. In sever- al species, estrogen or testosterone implanted into the hypothalamus stimu- lates estrous behavior without producing a detectable effect on target tissues in the reproductive tract. Also, certain cerebral neuronal systems take up estrogens and androgens, as demonstrated by radio- chemical studies. The uptake and the induction of estrous behavior can be inhibited by estrogen antagonists. These and other studies show that estrogen is the important hormone for estrous behav— ior. Temporal relationships. Estrous behavior in mares does not begin until peripheral progesterone concentration has dropped to less than 2 ng/ml (1145) and, presumably, estrogen concentration is beginning to rise. Estrus usually ceases a day or two after ovulation at a time when progesterone is rising and estrogen concentrations are approaching low basal levels. ‘ Sexual Behavior 97 Estrogen treatment. Large doses of estrogens are needed to induce estrous behavior during diestrus when endoge- nous progesterone is present. However anestrous mares are unusually sensitive to small amounts of synthetic and natural estrogens (1153). The minimal effective dose of estrone for induction of estrus in anestrous mares was 50 mg. Estrus occurred as early as 4 hours (23% of mares) and within 8 to 10 hours after treatment in all mares. According to this report, the short interval from estrogen treatment to manifestation of estrus in mares contrasts to an interval of 2 or 3 days in similarly treated cows and sows. The duration of estrus from the single injection in mares was 3 to 10 days. Interestingly, it also was reported that continuous treatment with a synthetic estrogen in the presence of a functional corpus luteum resulted in male-like behavior. In a small trial, a single injec- tion of 0.5 or 5.0 mg of estradiol elicited estrous behavior in ovariectomized mares (730). Mares showed interest to a stallion Within three hours, stood within six hours, and showed maximal response within nine hours. Mares returned to pre— treatment behavioral patterns in 48 to 96 hours for the low and high dose, respec- tively. The rapid behavioral response to exogenous estrogens in mares (730, 1153) suggests a low threshold requirement, or perhaps endogenous levels are higher in ovariectomized mares than in other species. Some observations on estrous behavior were made during a study of the effects of ovarian steroids on LH concentrations in ovariectomized pony mares (cited in 575). Daily estradiol treatment (1 mg/day) elicited positive estrous signs. Exogenous progesterone (100 mg/day) resulted in negative estrous signs for as long as it was administered and also blocked the estrus-inducing effect of estradiol. As noted above, mares given progesterone showed more intense nonestrous signs than nontreated ovariectomized mares. 98 Chapter 3 Earlier work showed that behavioral estrus is blocked by daily administration of 100 mg of progesterone (996). Facilitatory effect of progesterone. There are indications in nonequine species that progesterone facilitates the effect of estrogen in eliciting estrous behavior. Progesterone given to an ani- mal that has been primed with estrogen causes a facilitory effect on receptive behavior. A sharp rise in plasma proges- terone coincides with or precedes slightly the appearance of lordosis behavior in rats and guinea pigs. In ewes, the appro- priate sequence for production of estrous behavior is progesterone followed by estrogen. There is no indication, however, that estrous behavior at the first ovula- tion of the ovulatory season is diminished in mares as it is in ewes. Sexual behavior has been studied by giving estradiol and progesterone to ovariectomized mares (101). Concurrent administration of both hormones first enhanced and then suppressed the estrous response (Figure 3.14). Thus, pro— gesterone plus estradiol produced greater response than estradiol alone but only for the first day of treatment. Estradiol alone stimulated and progesterone alone inhib- ited estrous behavior in ovariectomized mares within four hours after the first injection. It should be no surprise that progesterone can inhibit estrous behav- ior, since it is the hormone associated with the nonreceptive phase of the estrous cycle (diestrus). Thus, research in nonequine species indicates that proges- terone can have either a facilitory or an inhibitory effect, depending upon such factors as timing and the levels of estro- gen. That is, progesterone is involved in an initial facilitation in the estrogen— primed animal, but later in the cycle it causes inhibition. Progesterone effect on nonestrus signs. A comparison was made between the intensity of nonestrous signs during diestrus and the intensity of nonestrous signs for those periods in the anovulatory season when mares were not showing estrous behavior (575). The intensity of nonestrous signs was greater for diestrus (higher negative value) than for the peri- ods of nonestrus during the anovulatory season. It may be, therefore, that proges- terone has a stimulatory effect on the intensity of nonestrous signs, accounting for more pronounced nonestrous behavior during diestrus, and probably during pregnancy, than during the anovulatory season. Injections of progesterone Effect of steroids on behavior Mean frequency/ mare Day of treatment FIGURE 3.14. Mean frequencies of follow (mare following stallion) and urination for various hor- mone treatments. Stallion and mares were allowed to mingle and observations were made for a 20— minute period four hours after treatment each day. Within each day, means with no common super— script letters are different (P<0.05). C = control, E = estrogen, P = progesterone, EP = estrogen and pro- gesterone in combination. Adapted from (101). increased the intensity of nonestrous signs over those of ovariectomized con— trols (cited in 575). The possibility of a direct stimulatory effect of progesterone on nonestrous signs in mares should be further studied. 3.9. Teasing Techniques Teasing techniques vary widely among farms depending upon differing manage- rial conditions and personal preferences. Maiden and foaling mares should be given special attention. Time should be taken to introduce maiden mares to teasing procedures because of the possi- bility that bad habits may develop. In foaling mares, the maternal instinct may interfere with teasing procedures. In addition, a system should be used to pro- tect the foal from being injured. There may be advantage in mimicking certain natural conditions in developing teasing as well as mating procedures. In free-running ponies, it was noted that young, inexperienced mares showed signs of fear (snapping, kicking) even when in an obvious estrous condition (1652). Mutual grooming was noted between stallions and young mares as part of the precopulatory ritual, but not between stallions and older mares. It was suggested that mutual grooming initiat- ed by the stallions served to overcome the young mares’ fear. Such observa- tions, made in the natural state, can pro- vide guidelines for development of tech- niques that may improve teasing and mating procedures on farms. The obser- vations should be subjected to testing, however, for applicability to farm condi- tions. The report of Tyler (1652) on sexual behavior in free-running, semi-wild ponies is recommended for those interest- ed in such possibilities. Mares may show estrus signs when a stallion is not present. Also, they may vocalize frequently at horses in a distant pasture or show restlessness. However, such displays occur inconsistently and are Sexual Behavior 99 a limited aid in estrus detection. For this reason, the owners of small numbers of mares who do not have a stallion available are at a decided disadvantage in determin- ing when the mares are in estrus. This problem is therefore an obstacle to widespread use of artificial insemination. Recording responses. Regardless of the technique used for estrus detection, it is useful to develop a recording system that can be used uniformly on a given farm, even when different operators are involved. As noted previously, subestrus or very subtle changes during estrus are not uncommon. Fortunately, such behav- ior is often repeatable within mares. Availability of records of previous responses, especially for problem mares, can aid detection of the periovulatory period. A system of recording can be developed that is compatible with the teasing techniques used. Individual teasing. In individual teas- ing, each mare is exposed for a short time to a stallion. The mare may be separated from the stallion by a teasing wall or pen door of approximately vulval height (Figure 3.15). As a modification, the stal- lion may be led from mare to mare in an aisle. It is not unusual for a given mare to show nonestrous signs initially (kicking, biting) and then suddenly settle into frank estrus behavior. It is generally rec- ommended (1362) that the mare and stal- lion be introduced head to head over the teasing or trying board, allowing the stal- lion to nuzzle and nip the mare. Head-to- head introduction may simulate the nor- mal greeting behavior of free-ranging horses and ponies (1652). If the mare does not reveal her condition immediately, the perineal region can be made accessible. Mares that remain indifferent to teasing present a challenge to the operator. More prolonged or vigorous teasing (e.g., with another stallion) may be required or the operator may need to look for more subtle behavioral changes. Tail raise, clitoral Wink, and urination are not, by them- selves, reliable indicators of estrus, since 100 Chapter 3 ‘5 W s\ \k 1“ ‘ i . \IH INDIVIDUAL TEASING ACROSS A BARRIER Sexual Behavior 101 GROUP TEASING FIGURE 3.15. Includes facing page. Illustrations of several methods of teasing including individual teasing with mare and stallion separated by a wall, individual teasing in an aisle by leading the stallion from mare to mare, and group teasing. Courtesy of B. W. Pickett and J. L. V088, Colorado State University. these three signs also are associated with the act of urination at any time. Upon teasing, approximately 10% of diestrous mares show such signs. When there is question whether urination is in response to teasing, the stallion is removed until urination and winking stops. Allowing mounting. Mounting may be allowed, especially for research purposes that require critical determination of behavioral signs (575). When mounting occurs, the penis is deflected to prevent copulation if indicated. Mare response to mounting seems to be an aid in determin- ing estrus and occasionally may stimulate the mare to respond. However, mares apparently differ from other domestic species in that they may stand firmly for mounting by a male even when not in estrus. This is seen frequently in tranquil mares, very young mares, and at the beginning and toward the end of diestrus. Therefore, the mare is not considered to be in estrus unless she also shows urina- tion, clitoral wink, or tail raise before or during mounting or after dismounting. Group teasing over a barrier. Group teasing with the stallion separated from a group of mares by a barrier is a com— mon practice (Figure 3.15). A compari— son of the efficiency of group teasing ver- sus individual teasing has been made (575). Thirty mares were studied daily throughout a complete estrous cycle. The stallion was put into an outside paddock. The mares were allowed to congregate in a larger enclosure, which was exposed on two sides to the stallion’s paddock. If the stallion became inactive, another stal- lion was substituted. The mares were observed for 30 minutes. Individual teas— ing was used to define the stages of the estrous cycle, and the two techniques (group and individual) were compared. 102 Chapter 3 Only 50% to 60% of the mares that were in estrus (based on individual teasing) approached the stallion during group teasing and showed signs of estrus (tail raise, urination, or winking; Table 3.5). The remaining mares that were in estrus (based on individual teasing) did not approach the stallion and, with few exceptions, did not show signs of estrus while in the paddock. Urination occurred occasionally in mares that did not approach the stallion, whether the mares were in estrus or diestrus. When group teasing is used, it may be advantageous to drive the mares up to the stallion or to remove the indifferent mares and subject them to the more critical individual teas— ing. The failure of many (40%) mares to approach the stallion and display estrous signs during group teasing suggests that certain mares may require direct approach and stimulation by the stallion before they show signs of estrus. Social factors or dominance hierarchies also may be important under these conditions. Group teasing with stallion and mares in one corral. Turning a stallion in with a group of mares allows the animals to express a fuller repertoire of behaviors in a more natural setting. Therefore, this approach may be especially useful for sci- entific study of sexual behavior, including copulation. Domesticated mares tested in this situation display a wider range of proceptive behaviors and fewer negative behaviors than those tested by traditional teasing methods (102). Estrous mares pre- sent the perineal area and stand for inspection by the stallion, which includes sniffing, licking, nibbling, and nuzzling various body parts, particularly the neck, groin, and genital areas. The attractive- ness of the estrous mare to the stallion is evidenced by an increase in the number of times the stallion approaches and investigates. If in full estrus, the mare is receptive and stands for mounting, intro- mission, and ejaculation. Kicking and squealing, which might be interpreted as negative behaviors, are often displayed by TABLE 3.5. Percentage of Estrous and Diestrous Mares Showing the Indicated Signs during Group Teasing Stage of estrous cycle based on Behavioral signs individual teasing during group teasing Estrus Diestrus Approached stallion 60% 7% Raised tail 50% 3% Urinated 43% 0% Winked clitoris 40% 0% None of the above 3% 3% Did not approach stallion 40% 93% Urinated 10% 13% Did not urinate 30% 80% The stallion was put in a paddock adjacent to the mares’ paddock. n = 30 mares. From (575). estrous mares and may serve to excite the stallion. This kicking is, for the most part, distinguishable from the more Vigor- ous kicks of a nonestrous mare. If a small group of mares is used (e.g., 4 to 6), each mare and stallion is likely to interact during a 20 minute observational period. However, with large numbers of mares, the results may be similar to group teasing over a barrier. That is, some mares may not be tested adequately during the observational period. In this regard, when the stallion was allowed to interact with 20 mares (pg. 88), only 48%, 60%, 68%, and 71% of mares postured during a one—hour observational period on Days —4, —3, -2, and —1. However, 96% of the mares postured on at least one of the days before ovulation. Homosexuality. Teasing or mounting of females by other females is used routine- ly for estrus detection in cattle, but it occurs rarely in mares. The author has observed a mare mounting mares in estrus on only six occasions. This phe- nomenon must be quite rare, based on the isolated reports in the literature (543, 1362). Under certain abnormal conditions, however, mares may exhibit male-like behavior. These include androgenizing ovarian tumors (543) or the administration of estrogens (118, 1153), androgens (1804, 344), equine pituitary extracts (1815), or anabolic steroids (1042). Preparing teasers by hormonal treat- ment. An approach to estrus detection in cattle involves treating steers or heifers with an androgen. The androgen-treated animals are used to search out cattle in estrus while under observation by the operator. Alternately, the androgen-treat- ed detector animals may be rigged with a marking device to mark the females that stood for mounting. In a short communi— cation, it was indicated that a ZOO-mg testosterone implant in a gelding result— ed in an excellent teaser animal (1581). The gelding exhibited stallion behavioral characteristics after seven days and until removal of the implant five months later. More recently, several more experiments have been done on androgenizing mares or geldings. Treatment of mares with an anabolic steroid failed to produce useful teasers for across—the-fence estrus detec- tion; pasture exposure indicated that only 33% of estrus mares were identified (1047, 1049). In another study, testosterone propionate treatment (150 mg every other day) induced male behavior in pony mares that seemed adequate for estrus detection (1804). Mares so treated may rise in social hierarchy and begin to show male characteristics (344). After dis- continuation of treatment, the mares recovered their female hormonal balance but maintained their newly acquired social rank. Use of a marking harness. A mount- ing or inspecting marker fitted to a vasectomized stallion for detection of estrus in freely running mares has received only limited attention. In a herd of 9 mares, 65% of observed mounts were accompanied by marks from the marking apparatus (91). Each mare was marked an average of 2.9 times per day of estrus, and marking failed to occur during a complete day in only 3.1% of the occa- sions. Thus, such a scheme seems worthy Sexual Behavior 103 of further testing. Alternatives to teasing. Mares have not been teased as a routine part of the mat- ing program in our laboratory since 1983. Transrectal ultrasound scanners are used to estimate the stage of the estrous cycle and the suitability for mating (590). Three criteria are used: size of largest follicle, echotexture of the uterus, and nonde- tectable or regressed corpus luteum. During the follicular or estrogenic phase, the uterus becomes edematous and when Viewed ultrasonically has a characteristic echotexture (pg. 212). The incidence of fail- ure to display an estrogenic—like uterine echotexture is probably comparable (not critically determined) to the incidence of silent estrus. However, when natural mating is to be used, the operator may be reluctant to expose the stallion to a mare that has not been teased. Also, a scanner will not always be available. An alterna- tive reproduction management program that does not require detection of estrus has been proposed by Palmer (1203). The program utilizes treatments with pho- toperiods, progestins, prostaglandin and human chronic gonadotropin (pg. 286). The presence of fernlike patterns in smears of vaginal and cervical mucus was significantly correlated with behav- ior but did not appear to be as useful for detecting estrus as the use of a teaser stallion (412). The use of various aids for detection of the periovulatory period, including transrectal palpation (pg. 209) and visual inspection of genitalia (pg. 211), are discussed elsewhere. Only transrec- tal ultrasonic imaging, however, seems a reasonable alternative, as opposed to an aid, to estrus detection. Phantoms. Use of estrous mares as mounts for semen collection has on many farms been replaced by use of phantom mounts. A recent discussion with lead- ins to the literature on phantoms is available (1713). In this regard, semen can be collected in the absence of a mare or phantom by training stallions to respond to penile massage (357, 1050). 104 Chapter 3 HIGHLIGHTS: Sexual Behavior Sexually receptive mares exhibit signs of serenity and accommodation, whereas nonreceptive mares exhibit signs of nervousness often accompanied by repelling maneuvers. Posturing is the most pronounced sign of sexual receptivity and is accompanied by tail raise, passage of fluids (urine, mucus), and clitoral winking. Herding of mares by the stallion is common in feral harems and in domestic ‘ groups. _ . . . __ _ Mouth clapping occurs in foals and 1s a sign of submission Mouth clapping is a; useful and sensitive indicator of estrus in jennies but not 1n mares. * Recent studies indicate that stallions do not have the ability to select against mares that they have already mated or to favor mares that are closest to ovula- ti.on Estrous mares that interfere with mating usually direct their aggression against mares, whereas nonestrous mares usually direct their aggression against the _ stallion. Estrous intensity is reduced at both ends of the estrous period, with an intervenf __ ing period of constant intensity. W Silent estrus is associated with reduced estradiol levels at the expected time and i with less time after luteolysis for estradiol to stimulate estrous behavior. Diminished estrous intensity tends to be characteristic of individuals. Flehmen (upcurling of upper lip) traps pheromone-laden air in the vomeronasal ~ organs (blind tubes associated with the nasal passages). Estrous behavior is common in ovariectomized mares and in mares with inactive ovaries during the anovulatory season. One possibility is that the behavior is due to adrenal androgens. Estradiol stimulates and progesterone inhibits estrous behavior, but concurrent administration of estradiol and progesterone to ovariectomized mares first enhances and then suppresses estrous behavior. Mares are more sensitive to exogenous estrogens than other farm species, and display estrous signs as early as four hours after injection. Only 50 to 60% of mares that were in estrus (based on individual teasing) 1 approached the stallion during group teasing and showed signs of estrus. —Cfiapter 4— REPRODUCTIVE SEASONALITY Reproductive seasonality is one of a host of endocrine rhythms that has been described in various species. Many rhythms are approximately 24 hours long, whereas others, such as equine reproductive seasonality, are approxi- mately 12 months long. Many species in the temperate zones, including Equus caballus, mate during the time of year that offers the optimum food supply and environmental conditions for survival and development of the young. Unlike most other species, the breeding season and the parturition season of mares overlap because of the 11-month gestation period. Conception in late spring (the beginning of the ovulatory season) results in foaling during early spring beyond the climatic hazards of winter and at a time when potentially there will be many months of abundant feed ahead. The economic and biologic impact of seasonality on the horse industry and the equine species is immense. This chapter will include discussion of the characteris- tics of reproductive seasonality in non- pregnant mares and the environmental and physiologic factors controlling sea- sonality. Seasonal infertility and the physiologic factors and artificial factors (e.g., official birth date problem) that con- tribute to it will be considered. An introductory overview of the patterns and variation in equine season- ality in individual mares is given in Figure 4.1. Reproductive seasonality Jun WVO'IUIAQOMA IO 11 l2 l3 l4 FIGURE 4.1. Seasonal data for each of 14 pony mares showing estrous periods (black bars) and days of ovu- lation (arrows). Adapted from (573). 106 Chapter 4 A Research Challenge. Research in the area of reproductive seasonality is loaded with pitfalls. Effects of photoperiod are confounded by a wide array of nutritive, climatic, and managerial conditions. As an example of the problems inherent in attempts to define seasonal changes, con— sider the effects of month on length of estrus during the breeding season as determined from breeding records. As the months progress, some mares become pregnant and no longer contribute to the means and variations for subsequent months. Thus a selection factor operates to change the makeup of the pool of ani- mals under study, seriously confounding the results. An important principle of experimental design is violated—each mare does not have equal opportunity to fall into (randomized design) or to con- tribute data to (sequential design) each month under study. The practical necessi- ty to mate or to use mares for other pur- poses usually prevents detailed study of an intact group of mares throughout all seasons of the year. Seasonality studies are given secondary status, which is reflected in the reliability of the results and conclusions. Terminology. Terms used to describe the time of the year when mares are able to reproduce include breeding, active, estrous, and ovulatory seasons. Those used to describe the time of the year when mares are not able to reproduce include anestrous, dormant, anovulatory, acyclic, and nonbreeding seasons. Breeding season and anestrous season appear to be the terms most commonly used. It will become clear in the following sections that much of the confusion in ter— minology can be attributed to the mare herself. She is unconcerned with our attempts at standardization and classifi- cation. As shown in Figure 4.1, a mare may show all of the signs of estrus during the anestrous season. This is a paradox in terminology, since “anestrus” literally means “no estrus”. Furthermore, she will sometimes accept the stallion (mate) dur- ing these times even though she cannot reproduce because ovulation does not occur during these off-season estrous periods. So, according to our confused ter— minology, we have an estrous animal that is anestrous and an animal that breeds during the nonbreeding season. Mankind has also added a measure of confusion by imposing an artificial breeding season for some breeds, encouraged by the use of January 1 (Northern Hemisphere) or August 1 (Southern Hemisphere) as uni- versal birth dates. The terms operational breeding season and physiologic breeding season have, therefore, been used (867). Herein, the term ovulatory season will be used for the time of year when the mare is capable of reproducing, since the occur- rence of ovulation is the most precise, readily defined, and least confusing criterion for classification of reproduc— tive seasons in the mare. Specifically, the ovulatory season is defined as the interval from the first to the last ovula- tion of the reproductive year. The remain- ing portion of the year will be called the anovulatory season. This will be done to provide a clearly demarcated period of time of considerable physiologic and practical importance. 4.1. Annual Distribution of Ovulations Data on the monthly changes in occur- rence of ovulation, based on transrectal palpation, are summarized in Figure 4.2 for six groups of mares in three countries. Months are arranged in units of three, corresponding to the four climatic seasons of the year, according to popular usage in the Northern and Southern Hemispheres. Although the conditions of data collection detracted from meaningful statistical handling, the consistencies among herds from one climatic season to another were striking and certainly indicative. A gener- al pattern emerges wherein the incidence of ovulation was minimal or absent dur- ing the winter, transitionally increasing Reproductive Seasonality 107 100 —~————_—— , —:_-- Daylength 80 Z #6, 60 S /h\\ — I \ >§ / \ a .‘ / " 4° )4.’ a . 3 ----- Group A. Several breeds. 26°S 9: 20 _ _._ _ ----- Group B, Quarter Horses, 30°N ---------- Group C, Thoroughbreds, 30°N O 100 ...... . ‘:—I.. G O Daylength 0’ 0 Percent mares ovulating and PONIES A O 20 --—--—Group D. 33°N ' 1,7 ----- Group E. 43°N .......... Group F. 30°N FIGURE 4.2. Association between changes in daylength and percentage of mares ovulat— ing per month. _—_< \ Group A: \ 188 mares, initially (1665) =._ \ Group B: "‘-.__ ‘\ 1 to 33 mares/month (1427) ‘ Group C: "v...“ 12 to 78 mares/month (1427) Group D: 12 mares/month (1394) 0 """ Group E: N Hem Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov 14 mares/month (573) S Hem Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May _ Group F: WINTER SPRING SUMMER FALL during the spring, maximal during the summer, and transitionally decreasing during the fall. The reproductive seasons were rigidly delineated into ovulatory and anovulatory seasons in ponies; only the pony herds had months in which none of the mares ovulated. The percentage of pony mares ovulating seemed to decrease sooner for Group D, perhaps reflecting the presumably more primitive nature of the herd (semi—wild Korean ponies). In all groups of horses or predominantly horses, a zero percentage level was not reached for any month. Thus, the ovulatory sea- son was shorter in ponies than in horses, and more ponies than horses became anovulatory. The comparisons between ponies and horses were most meaningful for Group F (ponies) and Groups B and C (horses) and indicate that the differences between ponies and horses are intrinsic. 7 to 56 mares/month (1427) These three herds were research herds at the University of Florida and were fed and housed under similar conditions, and data are contemporary. The low incidence of ovulation for the winter months in horse mares likely reflected uninterrupt- ed ovulation throughout the year by a minority of the total population, com- bined with anovulatory seasons at differ- ent times and lengths among individual mares. There is no question that some horse mares in some years do ovulate throughout the year and some mares in some years do not. California workers did a major study on seasonal changes in seven Thorough- breds and four Quarter Horses over a period of two years (780). In general, the data seem to agree with those for horse mares shown in Figure 4.2. Half of the mare-years involved a two- to three- 108 Chapter 4 month winter anovulatory period, and the other half involved continuous ovarian activity (ovulations) throughout the year. At least 3 of 9 pony mares in a study in Ireland (327) reportedly cycled regularly throughout the winter. Results of evalua- tions of reproductive seasonality in ponies and horses in Poland (1622) and in Arabian mares in Iraq (498) were similar to those of other studies. Seasonal distri— bution of ovulations has been studied with slaughterhouse specimens in Australia (1185), England (94, 376), Denmark (1167), Mexico (1379), and the United States (Figure 4.8, 1759). Historically, the Australian study is espe- cially noteworthy because it served to direct attention to the great disparity between physiologic and operational breeding seasons. Although the slaugh- terhouse data most likely included a greater proportion of cull mares, the data generally agree With those shown in Figure 4.2. 4.2. Annual Distribution of Periods of Estrous Behavior Behavioral estrus occurs during all months of the year, even during the anovulatory season (Figure 4.1). The estrus preceding the first ovulation can be especially prolonged, sometimes exceeding a month or more (pg. 174). As examples, data from two herds are shown in Figure 4.3. The horses (but not the ponies) were mated, and therefore, some selection bias and decrease in numbers occurred as the breeding sea- son progressed. During the winter and early spring, when ovulation rate was low, a large percentage in both herds showed estrous behavior. Later, as the percentage of mares ovulating reached 100%, there was close agreement between number of mares in estrus and number ovulating. Estrous behavior in the presence of seasonally inactive ovaries is common in mares. The biology Ovulations & estrus 100 I’- . 80 ,I "\ Horses 50 0/0 in S Africa estrus (”=75) ‘- 40 % ovuiating 20 E 0 a) 0 3100 n. 80 60 ‘\. Ponies 40 Wisconsin USA 20 0 NHemJ FMAMJJASON D SHemJASONDJ FMAMJ Month FIGURE 4.3. Percentage of mares in estrus or ovu- lating per month in 75 light farm horses and 14 ponies. Considerable estrous activity, unaccompa- nied by ovulation, occurred during winter and early spring, whereas there was closer agreement between these characteristics during late spring, summer, and fall. Adapted from (horses, 1665) and (ponies, 573). of unseasonal estrous periods and the problems they bring to breeding pro- grams are discussed elsewhere (pg. 136). 4.3. Reproductive Seasonality in Other Equids Donkeys. Seasonality was studied in Brazil (1908; 720) in 13 jennies that were 3 to 18 years of age. Only two jennies ovu— lated regularly throughout the 15—month study. A third jenny cycled and ovulated throughout the first winter but apparent- ly did not ovulate in the spring. Four of the 13 jennies did not cycle during the 15—month study, suggesting to this reviewer that this may not have been a representative sample of reproductively sound jennies. In another study, 12 adult jennies were monitored every 1 to 3 days throughout the year in northern United States (43°N; 620). The percentage of jen- nies ovulating at least once each month and the length of the first interovulatory interval and first estrus beginning in each month are shown (Figure 4.4). An apparent anovulatory season occurring in Jennies (n=12) 100 E 8 80 a“, % ovulating n. 60 28 Interovulatory interval 17; ab Cd ab ; 26 3 E. 8’ 24 m _i 22 "3 16 a a. Estrus length 3 E a: C c» _| FIGURE 4.4. Percentage of jennies ovulating and mean lengths of the interovulatory interval and estrus for each month of the year. Differences among months were significant for all three end points. Within each end point, any two means with no common superscripts differ significantly. Adapted from (620). Reproductive Seasonality 109 winter in 4 of the 12 jennies was relative- ly short (39 to 72 days) and was terminat- ed by a long period (17 to 41 days) of estrous behavior in the continued pres- ence of large follicles (>20 mm). Prolonged estrus accounted for much of the lower incidence of ovulations during December and seemed similar to the transition between anovulatory and ovu- latory seasons in mares. There was a significant effect of month on length of the interovulatory interval, due primarily to shorter inter- vals during May to September (means: 23 and 24 days) than during October to April (means: 25 to 27 days). Length of estrus differed significantly among months, due primarily to shorter peri— ods during May to October (means: 6 and 7 days) than during November to April (means: 7 to 15 days). These results indicate that this species is sub— jected to seasonal effects on reproduc- tive function. However, the dramatic partitioning of the year into ovulatory and anovulatory seasons, as occurs in most mares, was absent (8 jennies) or limited (4 jennies). Zebras. The months of foaling in Cape mountain zebras were studied in South Africa (320 to 34°S; 1253). On average, foaling for individuals occurred every second year. Foals were born through- out the year with a peak in spring and early summer, a time when there is apparently ample grazing after a wet winter. Because the gestation period is approximately ‘12 months, the yearly foaling distribution likely reflected a similar distribution of ovulations and conceptions. This group of zebras did not have an absolute seasonality phe- nomenon, since some foaling occurred in every month. Reproductive traits of Hartmann’s zebra were examined in August in Namibia (1765). Based on size of fetuses, most pregnant zebras appeared to have conceived in November to April (equivalent to May to October in the Northern Hemisphere). 110 Chapter 4 4.4 Factors Affecting Reproductive Seasonality 4.4A. Length of Photoperiod and Type of Mare A graph depicting relationships among latitude, month, and number of hours of daylength is shown (Figure 4.5). Seasonal changes in length of twilight are not con- sidered. At all latitudes, the shortest day (winter solstice) and the longest day (summer solstice) occur on approximately December 22 and June 22, respectively, in the Northern Hemisphere and June 22 and December 22, respectively, in the Southern Hemisphere. Day length at the summer solstice and rate of change in the light2dark ratio are progressively greater as the distance from the equator increases. Daylength is 12 hours through- out the year at the equator. The association between natural changes in daylength and reproductive seasonality has been summarized graphi- cally (Figure 4.2), and several observa— tions may be made. Incidence of ovula- tions was lowest in the winter when daylength was shortest. The increasing incidence of ovulations in the spring approximately paralleled the increasing daylengths. The ovulation rate seemed to increase simultaneously with increasing daylengths in the herds of horse mares, whereas there was a two- to three-month lag between increasing daylength and increasing ovulation rate in the herds of pony mares. Maximum incidence of ovu— lation in both horses and ponies generally coincided with time of maximum daylength. Decreasing daylength preced- ed the drop in incidence of ovulation, resulting in a lag of several months for the herds of horses. However, the lag period between decreased daylength and decreased percentage ovulating was not as great for the herds of ponies. 4.43. Latitude Because the stimulus for equine sea- sonality is primarily daylength, several questions arise during study of Figure 4.5. Is the peak of the ovulatory season roughly synchronized throughout each hemisphere, using winter or summer sol- stice as a reference? Is there a range of latitudes where there is a relative absence of seasonality? Do the lengths of the ovulatory and anovulatory seasons or Latitude & daylength 60° 18 16 73‘ 3 14 5 53 12 3 6‘ *5 1° FIGURE 4.5. Relationship '5. between latitude and number g 8 of hours of daylength through- 3 out the year. Adapted from 6 Winter Vernal Summer Autumnal tabulated data (400) based on solstiCe equinox solstice equinox daylengths at the memdlan NHem Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec SHem Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun of Greenwich using the local mean time of sunrise and beginning of astronomical twilight. the proportions of a population that show seasonality change progressively as lati- tude changes? Few documented answers to these questions are available. There are no obvious differences in reproductive seasonality due to latitude in Figure 4.2. Herds E and F were located near the northern (43°N) and southern (33°N) extremes of the United States (Wisconsin and Florida). The animals were ponies of similar breeding. The curves for percentage of mares ovulating seem similar, although the duration of the 0% interval may have been longer for the Florida herd. One survey was done in southern Mexico (15°N to 22°N) within the Torrid Zone, yet a seasonal reproduc- tive pattern was obtained (1379). The dif- ferent methods of obtaining data and defining reproductive activity preclude direct comparisons of this study with other slaughterhouse studies. Based on inspection of the reports, however, no obvious differences can be attributed to latitude. In a recent abstract (854), it was stated that mares at 60°N latitude started to cycle a month later than what has been reported for latitudes closer to the equator; direct latitude comparisons were not possible in this experiment or in others. Although convincing evidence for dif- ferences attributable to latitude appar- ently is not available, it has been noted (1188) that the peak incidence of foaling for Standardbred mares occurs approxi- mately one month earlier in the Australian mainland (28° to 3808) than in Tasmania (4208) and New Zealand (36° to 46°S ). Foaling data for Standardbreds in Pennsylvania (867) tend to bolster the interpretation (1188) that peak foaling occurs approximately one month earlier at latitudes of <400 than at latitudes >400. Of course, it is a tenuous conclusion that such a difference, if real, is attributable to latitude differences in daylength. If the length of the ovulatory season is reduced at greater latitudes, it Reproductive Seasonality 1 1 1 may be attributable to shorter days in winter and accompanying greater depth of ovarian inactivity. If so, more time may be required for the mares at higher lati- tudes to restore ovarian activity in the spring. In this regard, in experiments on the stimulatory effects of artificial light, mares in which the ovaries were more inactive at the start of light exposure took longer to achieve ovarian activity (905). This section is sprinkled with hedge- words, reflecting the present inconclusive state of our knowledge on effects of lati— tude. A good controlled experiment is needed in which the seasonal light cycle at different latitudes is simulated for dif- ferent groups of mares. Also, the effects of twilight length should not continue to be ignored. 4.40. Age Reproductive biology from birth to puberty is discussed elsewhere (pg. 488). The effects of age (after puberty) on repro- ductive function is a surprisingly neglected research area in equine reproductive biolo— gy. A major research thrust in reproduc- tive gerontology could do more for the eco- nomics of the equine industry than any other research endeavor (opinion). The influence of age and season (month) on reproductive activity in pony mares was examined in a slaughterhouse survey (1759). It was noted that many very old mares (>20 years) were reproductively active. However, the ovaries of a few mares, 17 to 25 years old, had apparently reached senescence. The ovaries were small with no evidence of follicular or luteal activity during the time of year when such activity would be expected. N 0 other information on the incidence and nature of reproductive senescence was found, reflecting the informational void in this important area. Late entry. Seven of 19 mares (37%) greater than 24 years old did not ovulate during the ovulatory sea- son (1873). 112 Chapter 4 Ovulations & age 07 0 Percent ovulating .1: O N O N Hem Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec S Hem Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun In the slaughterhouse survey (1759), reproductive tracts from 1,003 nonpreg- nant ponies were examined over a three- year period. A mare was classified as ovu- latory if she had a corpus luteum or a corpus albicans plus a large follicle. Only sexually mature mares with indications of past or current luteal activity and in good body condition were included. The number and percentages of mares accord- ing to age groups that were reproductive- ly active (ovulatory) is shown (Figure 4.6). Totaled over all months, the percent- age of ovulatory mares was significantly lower for $2, 2.5 to 5, and >15 year age groups than for the 6 to 10 and 11 to 15 year groups. The lower percentage for the S2 and 2.5 to 5 year groups was attributable to a significantly earlier decline in the percentage ovulating dur- ing the fall. This result indicates that younger mares (2 to 5 years) have a shorter ovulatory season due to an earlier cessation of activity. In the old mares (>15 years), the lower total yearly ovula— tory productivity seemed due primarily to the absence of ovulatory mares during February to April; the decline in the fall months seemed similar to that in the middle-aged groups (6 to 15 years). Significant overall age effects also were FIGURE 4.6. Monthly changes in percentage ovulatory in slaughterhouse specimens according to age of mare. Percentage ovulatory in September was significantly lower in the S2 year and 2.5 to 5 year groups than in the other age groups. Adapted from (1759). found for paired ovarian weight and num- ber of follicles 2 to 20 mm but were not found for diameter of largest follicle and number of follicles >20 mm (Table 4.1). These results suggest that age effects involve number of follicles <20 mm and not diameter of largest follicle. Inspection of the table indicates gradually increasing number of follicles between puberty and five years, a plateau over 6 to 15 years, and a decline after 15 years. 4.5. Mechanisms for Control of Reproductive Seasonality Based on, the previous sections, it can be concluded that photoperiod (daylength) is in the forefront of the envi- ronmental factors affecting equine repro- ductive seasonality. During the past decade, workers in Florida have done a series of projects on the mechanisms by which photoperiod exerts such a profound effect on ovarian function in mares. This landmark research has focused on the roles of photoperiod, pineal, and hypo— thalamus and required adapting various research techniques to the horse. Approaches included ganglionectomy (1439), pinealectomy (655), pituitary stalk Reproductive Seasonality 1 13 TABLE 4.1. Effect of Age on Mean Ovarian End Points (n=934) Age (years) End point <2.5 2.5 to 5 6 to 10 11 to 15 >15 Paired ovarian weight, (g) 2881 11.7 38b 11.9 52C 11.7 540 12.2 47be 12.4 Diameter of largest follicle (mm) 16 11.1 18 11.0 18 10.6 19 10.7 19 11.0 No. of follicles 2m 10 (mm) 93 10.7 128113107 160 10.7 16C 10.8 14b 11.0 11 to 20 (mm) 28‘ 10.3 28 10.3 4b 102 4b 10.3 3C 10.3 >20 (mm) 0.4 10.1 0.5 10.1 0.6 10.1 0.7 10.1 0.6 10.1 Means in the same row with no common superscripts are different (P<0.05). Adapted from (1759). sectioning (1432), push-pull hypothalamic sampling (1434), quantitating melatonin (875), and localizing and quantitating GnRH (1570). The recent development of a method for cannulating the pituitary venous effluent by New Zealand workers should become a valuable tool in the 19903 (803); it allows measurement of the hypothalamic and pituitary secretion rates before the hormones are diluted in the general circulation. Transrectal ultra- sonography began to be utilized in the 1980s and is the ultimate tool for nonin- vasive monitoring of the dynamics of the follicular population (590). Large portions of this section will draw upon results obtained by these modern techniques. Results from laboratories utilizing equine and nonequine species will be incorporat- ed. Much of the information from nonequine species will be based on reviews (1291, 845). 4.5A. Experimentation on the Effects of Light It was reported in the 1940s in England that exposure of pony mares to artificial light, beginning on January 1, stimulated estrus during February and March in four mares; estrus did not occur until April in four control mares (260). It should be noted, however, that the mares also were treated with an ovulating hor- monal preparation. The rationale for this small but pioneering experiment was based on previous work in ferrets, voles, and birds and is a good example of utiliz- ing knowledge gained from other species. A similar small project was done in Japan (1153). Both workers appreciated the potential utility of their findings, and dis- cussion of the applied use of artificial light will be found elsewhere (pg. 158). Despite these early reports, it was not until the 1970s that extensive controlled research was initiated on the effects of light on equine reproduction. Simulation of the increasing length of photoperiod. Controlled environmental chambers were used to program tempera- ture and daily photoperiod to outdoor con- ditions in northern United States (43°N). Conditions from October 17 to February 15 (decreasing photoperiod and tempera- ture) were simulated for the controls, while conditions from March 1 to July 1 (increasing photoperiod and temperature) were simulated for the treated group (1431). By 69 days, all studied ovarian end points were significantly greater in treat- ed mares than in controls. The profile for 114 Chapter 4 Environmental factors & follicles (n=7/group) 20 Temperature .1", 5a 7‘, P 3' Degrees centigrade O ,,,,, 14 Daylength , . — — ‘ 12 x' 10 ' Photoperiod (hours) Diameter (mm) Day of treatment FIGURE 4.7. Effect of altering seasons (tempera- ture, length of photoperiod) on diameter of largest follicle. Mares were housed in environmental cham- bers beginning October 17. Adapted from (1431). diameter of largest follicle is shown as an example (Figure 4.7). The estrous period associated with the first ovulation was prolonged, similar to what occurs in the spring under natural light (pg. 108 and pg. 174). The average day of first ovulation was March 11 for the treated mares and June 4 for the control mares. Thus, the ovulatory season was hastened by 2 to 3 months. The project convincingly con- firmed the earlier reports that the onset of ovulatory seasonality is a function of environmental factors. No attempt was made, however, to differentiate between the effects of light and temperature or to analyze their interactions. The ovarian stimulatory effects could not have been caused by nutritional differences since both groups received identical diets and body weights were maintained. Long fixed—length photoperiods. The effect of a constant daily light period on the onset of the ovulatory season in ponies has been studied (905). Fixed pho— toperiods of 24 hours, 16 hours, 9 hours, or natural daylength were used beginning November 13 (Table 4.2). The 16- and 24- hour daily photoperiod significantly has- tened the onset of the ovulatory season with other environmental factors held constant. Results indicated that a gradu- ally increasing daily photoperiod, natural or artificial, is not essential to stimulate the onset of the ovulatory season. Interestingly, mares exposed to 24 hours of light per day entered the ovulatory sea- son later than those exposed to 16 hours. In another study (1211, 1209), a 20—hour fixed photoperiod was less effective than a 16-hour period. However, the 24-hour group (905) had a significantly shorter interval to onset of the ovulatory season than did the control (natural light) mares. Therefore, alternating periods of light and dark may favor, but are not essential for, induction of the ovulatory season in the mare. The reason for the intermediate TABLE 4.2. Effect of Daily Photoperiod on Ovulation and Hair Coat Changes Mean interval (days) from Nov 13: Light/day No. To To beginning ovul. by first smooth Nov 13 Jun 13 ovulation hair coat Control 8/8 1923 (May 24) 205a 24 hours 7/8 15Gb (Apr 18) 17410 16 hours 9/9 107c (Feb 28) 146c 9 hours 4/9 >200a (Jun 1) >ZOZa Within each column, means with different super- scripts are significantly different. Adapted from (905). effectiveness of excessively long light exposure is not known. Some investiga- tors working with other species have hypothesized that both dark and light are important. It was also found that, aver- aged over all groups, the interval to first ovulation was significantly shorter for mares with active ovaries (follicles >15 mm) at the onset of the light treatments (mean: 149 days) than for mares with inactive ovaries (mean: 176 days). Thus the depth of ovarian inactivity at the time the lights were turned on affected the interval to ovulation. There was no signif- icant difference in the time of termination of the ovulatory season between control mares and mares with an induced early onset (Table 4.3). Thus, termination of the ovulatory season apparently is not dependent on the time that the ovulatory season begins but is an independent func- tion of decreasing daylength. Short fixed-length photoperiods. A nine-hour photoperiod was included in the above-described study. Nine hours had‘a retarding effect on the onset of the ovulatory season, as indicated by the number of mares ovulating by the end of the experiment in the controls (8 of 8) and in the nine-hour group (4 of 9; Table 4.2). TABLE 4.3. Effect of Altered Photoperiod on Termination of the Ovulatory Season in the Fall Interval (days) from Jun 22 to N 0. last ovulation Treatment group mares of the season Control photoperiod 7 1343 (Nov 2 ) Early onset of ovulatory season induced by increased photo- period in the spring 6 1443 (Nov 12) Continuation of maxi- mum photoperiod after summer solstice 7 235b (Feb 11) Means with different superscripts are significantly different. Adapted from ( 905). Reproductive Seasonality 1 15 Similar results were obtained in a subse- quent study (575); 5 of 5 ovulated by June 13 in the control group, and 3 of 5 ovulat- ed in the nine-hour group. Thus, over two studies, 13 of 13 mares ovulated by June 13 when under a control photoperiod, and 7 of 14 ovulated when under a fixed nine— hour photoperiod per day. These results indicated that some mares were retarded by dark treatment, whereas others were not. The effect of age on the retardation incidence was not studied. Further study of the association between artificial short- ening of the photoperiod and retarded ovarian function could provide needed insight into the photoperiod control of reproductive seasonality in mares. Extending light after the summer sol- stice. Maintenance of maximum day- length after the summer solstice length- ened the ovulatory season considerably (Table 4.3). Two of the mares in this group continued to cycle throughout the year, a condition that is not usually observed in pony mares kept under natu— ral daylength (pg. 106). However, five mares eventually did enter the anovulato- ry season despite the maintenance of maximum daylength. April 18 was the average date of onset of the subsequent ovulatory season for the five mares which eventually became anovulatory when exposed to continuous light. This com- pares with the average May 12 date of onset observed in pony mares under natu- ral conditions (573). Apparently, exposure to a 16—hour, fixed daily photoperiod throughout the year caused mares to have both an earlier onset and a delayed termination of the ovulatory season. That is, the breeding season was extended on both ends. In a subsequent study (1412), starting a fixed program on July 1 or August 1 produced the following results: 1) Five of 17 mares ovulated throughout the winter (did not have an anovulatory season) compared to O of 19 controls; 2) About 50% of the mares that had an anovulatory season ovulat- ed later in the fall and/or earlier in the 116 Chapter 4 spring than control mares; and 3) The mean interval from January 1 to first ovulation was intermediate between control mares and mares in which stimu- lation began in the winter (Table 5.4, pg.161). It appears that in some mares there are factors that eventually will override the stimulatory effect of artificial light in the fall, as well as the retarding effect of artificial darkness in the spring. It has not been critically determined what effect exposure for more than one year to daily photoperiod regimens of various lengths would have on reproductive activity of the mare; this question demands attention because mares on farms may be treated with light over many consecutive years. Photosensitive period. Investigations in the nonequine species have directed much attention to defining a narrow period in the 24-hour day when an animal is most photosensitive, regardless of the total length of light per day. This research area was extended to horses by workers in France (1211, 1209). These sci- entists concluded that the time of light sensitivity begins 9.5 hours after the beginning of the dark phase and is no more than one hour in duration. The fol- lowing lighting schemes produced similar ovarian stimulation: 16L:8D, 14.5L:9.5D, 8L:9.5D:1L:5.5D, and 4L:9.5D:1L29.5D. All of these programs have in common one or more hours of light beginning 9.5 hours after the beginning of a dark phase. Note that the latter scheme provided for only five total hours of light per day, yet was effective. Other schemes with an hour of light provided at other times of the 24—hour period were ineffective. A similar conclusion was subsequently reached by New Jersey workers who used similar test systems, except that the shortest light phase was two hours (1011). They concluded that the photosensitive phase occurred 8 to 10 hours after dark, confirming the earlier work. The results and conclusions of the above studies on a photosensitive period, however, do not adequately explain the mechanism of photoperiodic time mea- surement under natural conditions. The experimental and natural schemes differ as follows: 1) abrupt versus gradual change between light and dark phases each day, 2) constant versus variable intensity of darkness (e.g., moonlight, cloudiness), and 3) fixed (experimental) versus gradual changes in lengths of phases. An hour of light beginning 9.5 hours after official sunset (provided by National Weather Service) may not be an operational photosensitive period under natural conditions based on the following: 1. Light exposure (sunrise) 9.5 hours after sunset does not occur until May 11 at 43°N when many mares would already have entered the ovulatory season (Figure 4.2); 2. Sunrise 9.5 hours after sunset does not occur at anytime at latitudes nearer to the equator (Figure 4.5), but mares still show reproductive seasonality (pg. 111); and 3. An artificial one—hour light pulse 9.5 hours after the official definition of natural sunset significantly shortened the interval to ovulation compared to controls but was considerably less effec- tive than a 15-hour fixed photoperiod (1412). One problem contributing to the apparent disparity between natural con- ditions (gradual change between light and dark) and experimental situations (abrupt change between light and dark) may be that the horse’s perception of sunrise and sunset deviates consider- ably from the official definitions. The photosensitive period discovered by the above cited workers provides an excel- lent experimental model, but care will be required in projecting the results into the natural scheme. Reproductive Seasonality 1 17 SUMMARY: Effects of Photoperiod Based on the findings summarized in this section, daylength is the environmental signal for regulation of reproductive sea- sonality in mares. Other environmental and internal factors may be Viewed as sec- ondary modulators of the effects of pho- toperiod, and these will be discussed in later sections. The indicators that daylength regulates reproductive seasonality are as follows: 0 Daylength is the most predictive fac— tor and, therefore, the one to which mares most likely key. 0 Increase in daylength parallels the increase in proportion of mares ovulating. Reproductive seasonality can be manipulated in mares by manipulat— ing duration of light with other fac- tors held constant. Some aspects of artificial manipulation of length of photoperiod are as follows: 0 Either a gradually increasing or a fixed photoperiod will advance the ovulatory season. 4.5B. Neuropathway from Eye to Pineal In most mammals studied, based on the above-cited reviews, light is perceived by the retina of the eyes, resulting in messages that are transmitted to the pineal gland. Transmission is believed to involve a multineuronal pathway, includ- ing the superior cervical ganglia and postganglionic fibers that terminate in the pineal parenchyma. During light stimulation, the function of the pineal gland is inhibited, but in darkness the postganglionic fibers from the superior Prolonged fixed photoperiods (>20 hours) produced intermediate results, indicating that alternating light and dark periods of an optimal length are important. A longer period of light treatment is required when the ovaries are inac- tive at the beginning of treatment. ' Fixed short photoperiods (e.g., 9 hours) retard the onset of the ovula- tory season in some mares but not in others. Inducing mares to ovulate early in the year does not affect the day of cessa- tion of the ovulatory season in the fall. Artificially extending daylength beyond the summer solstice causes some mares to have a prolonged ovu- latory season. Under experimental conditions of abrupt changes and fixed lengths of photoperiods, the photosensitive peri- od is short (e.g., 1 hour) and occurs approximately 9.5 hours after the dark phase begins. cervical ganglion release norepinephrine (neurotransmitter substance) to the pineal gland, stimulating many aspects of pineal metabolism. The pathways that transmit light stimuli beyond the retina are apparently distinct from the pathway for normal Vision. Thus, certain parts of the optic tract can be disrupted resulting in normal vision but absence of a pineal response to continuous darkness. Care must be taken in interpreting clinical observations of blind animals since Visual blindness is not necessarily indicative of ability of the pineal to respond to light changes. 118 Chapter 4 4.50. Role of the Pineal Gland The pineal gland carries much of the burden for the mechanisms of reproduc- tive seasonality. The pineal is considered the mediator between the light receptors and the hypothalamic-pituitary axis. Current theories of pineal function hold that the pineal acts as a neuroendocrine transducer, converting a neural input (norepinephrine released from nerve end- ings) to a hormonal output; the rate of hormone synthesis is thought to be inversely controlled by environmental illumination (1099). Pineal morphology and hormones in noneguine species. The morphologic approach is one way of studying the involvement of the pineal in reproductive seasonality. Such work has not been done in the equine species, but morphologic studies in other species are supportive of a pineal role (see reviews cited above). Based on studies in laboratory animals, remarkable variations occur in the biosynthetic and presumably secretory pattern of the pineal, likely reflecting the influence of the photoperiodic environ- ment. The pineal is very rich in indoles, and a number of these have been classi- fied as pineal hormones, primarily because of their ability to experimentally inhibit gonadal function. The indoles include serotonin and related compounds and melatonin (pg. 57). Melatonin is the prime candidate for the role of a pineal hormone because exogenous melatonin inhibits various pituitary-gonadal func- tions in certain species. Implication of the pineal in eguine sea- sonality. The first study to implicate the pineal in seasonality in mares utilized the temporal relationship between pineal hydroxyindole-O-methy1transferase (HIOMT) activity and ovarian activity (1764). The enzyme HIOMT is responsible for the final step in the synthesis of mela- tonin. The melatonin—forming activity and the percentage of pony mares ovulat- ing each month are shown (Figure 4.8). The pineals formed more melatonin in Melatonin-forming activity 5 O 2 ><4 A? .E 3 3 52 E 5. S, o o NH:ASONDJFMAMJJ SH: FMAMJJASONDJ Month FIGURE 4.8. Monthly changes in percentage of pony mares which were ovulating and in mean melatonin-forming activity (MFA) of pineal gland in the Northern Hemisphere (NH). Equivalent months for the Southern Hemisphere are shown (SH). Means with no common superscript letter are signif- icantly different. Adapted from (1764). November to January than in the other months, and percentage of mares ovulat- ing was low during December to April. Decreased ovarian activity was associ- ated with increased pineal activity, and ovarian activity was not restored until well after pineal activity had decreased. Most noteworthy, the first significant decrease in pineal activity occurred in February, a time when the hypothalamic- pituitary axis is being released from inhibitory control (pg. 122). Although these results did not directly demonstrate an antigonadal pineal activity in mares, they did indicate that pineal melatonin—form- ing activity is temporally related to repro- ductive seasonality. Melatonin experiments. An initial attempt to alter ovarian activity in mares by administration of melatonin was not successful (1599). However, melatonin con- centrations in mares are higher at night (327, 655, 875), and denervation or removal of the pineal abolishes the daily rhythm (Figure 4.9, 1435). In addition, the stimu— latory effect of 2.5 hours of light applied in the evening was abolished by treat- Melatonin (n=6/group) 100 ‘0 O Pineal-intact 80 70 60 Concentration (pg/ ml) 50 40 7 11 15 19 23 3 7 Time of day (hours) FIGURE 4.9. Circulating melatonin in pineal- intact and pinealectomized pony mares throughout a 24 hour period. There was a significant hour by group interaction that was due to the lack of time trends in pinealectomized mares. Adapted from Sharp et al. (1435). ment with 25 mg of melatonin at the time of natural sunset (Table 4.4). These data support the hypothesis that artificial light at sunset blocks the secretion of endogenous melatonin. When mares were exposed to continuous darkness, mela- tonin levels increased but still fluctuated, indicating that the species is not under total dominance of photoperiod. However, the fluctuations were not as regular and synchronized as when mares were under a normal light/dark cycle. Recent compar- isons of melatonin treatments in the evening versus morning confirmed that melatonin was effective in mimicking prolonged darkness only when given in the evening (657). Denervation of the pineal. Removal of the superior cervical ganglia denervates the pineal and may result in reproductive changes that are essentially comparable to removal of the pineal gland itself. Sectioning the superior cervical ganglia in mares, as in other species, resulted in altered ovarian function with respect to Reproductive Seasonality 1 19 environmental lighting (Table 4.4; 1439). Ganglionectomy of mares in the winter did not upset the onset of the first ovula- TABLE 4.4. Results of Experiments Involving Melatonin Administration, Removal of Ganglia, and Removal of Pineal N0. days No. from Jan 1 to Experimental groups mares ovulation Melatonin treatmenta Natural daylength No melatonin 5 109 (Apr 19) Melatonin treated 4 100 (Apr 10) 2.5 hr photoperiod extension (PM) N o melatonin 6 63 (Mar 4) Melatonin treated 5 122 (May 2) Removal of superior cervical ganglia in winterb 1st year after removal Control 4 125 (May 5) Sham operated 4 136 (May 16) Ganglia removed 4 129 (May 9) 2nd year after removal Control 4 125 (May 5) Sham operated 4 125 (May 5) Ganglia removed 4 192 (Jul 11) MIME) lst year after removal Control 9 125 (May 5) Removal in summer 3 142 (May 22) Removal in winter 3 128 (May 8) 2nd year after removal Control 9 121 (May 1) Removal in summer 3 121 (May 1) Removal in winter 3 160 (Jun 9) W W0 Control 5 137 (May 17) Light (PM) 5 77 (Mar 28) Pineal removal and light 4 126 (May 6) a Significant interaction between light treatment and melatonin treatment. b 1st year, no difference among groups; 2nd year, removal of ganglia or removal of pineal in winter significantly delayed onset of ovulatory season. C Removal of pineal negated the effect of light. Adapted from Cheves and Sharp (301) and Sharp et al. (1435, 1439). 120 Chapter 4 tory season. However, the mares grew long shaggy hair coats during the sum— mer and shed to a sleek summertime hair coat in the fall. During the second year, all end points studied (follicular develop- ment, estrus, first ovulation, hair coat) were delayed by more than two months. In other species (e.g., ferrets), ganglionec- tomized animals continued to display breeding seasons that are out of phase with normal seasonality. Pinealectomy. Comparative work indi- cates that if the pineal is removed, sea- sonal rhythm is abolished (e.g., in ham- sters), or the rhythm is modified so that the breeding period is out of phase with the appropriate season of the year (e.g., in ferrets; see reviews cited above). Removal of the pineal in hamsters pre- vents light-deprivation atrophy of the reproductive tract. These types of investi- gations have been extended to mares (Table 4.4). Pinealectomized mares showed the following responses: 1) delayed onset of the second ovulatory sea- son after surgery, 2) failure to hasten the ovulatory season when treated with lights, and 3) lack of elevated circulating melatonin concentrations during dark- ness (655). Pineal removal in the summer resulted in a delay (although not signifi- cant) of the ovulatory season of the first postoperative year (Table 4.4); however, the season was not delayed in the second year. Pinealectomy in winter did not delay seasonality until the second year. An enigma. Although it is commonly stated that light stimulates ovarian func- tion in mares, inhibitory mechanisms are involved, at least partly. That is, stimula- tion of the pineal by decreasing daylength (increasing nightlength) results in anti- gonadal activity of the pineal, and ovari- an function is thereby inhibited. A change in photoperiod in the opposite direction (increasing daylength) removes the anti- gonadal activity of the pineal, and ovari- an function is resumed. However, it is enigmatic that the stimulatory effects of extended photoperiod can be negated by either administration of melatonin (the hormone produced by the pineal) or, under certain experimental conditions, removal of the pineal (Table 4.4). If the pineal produced only an inhibitor, abla- tion of the gland would be expected to result in the loss of ovarian seasonality; that is, the anovulatory season would be eliminated, rather than extended. Perhaps the surgery resulted in sec- ondary or side effects or the pineal pro— duces not only an inhibitory substance (melatonin) in response to decreasing photoperiod but also a stimulatory sub- stance in response to increasing photo— period. Clearly there are many unan- swered questions, but much progress has been made. Exciting findings on the effect of the pineal on reproductive seasonality in the mare are emerging. The mare may prove to be a valuable comparative research model in this regard. 4.5D. Role of Hypothalamus Another matter of concern is the direct site of action of the postulated pineal fac- tors. It has been shown in nonequine species that the effect of the pineal on the pituitary-gonadal axis is exerted through the hypothalamus. In mares, results of experiments involving administration of GnRH during the anovulatory season are compatible with the postulate that the hypothalamic area is part of the light- responsive chain between the retina and ovary. A single injection of GnRH during the anovulatory season caused an imme- diate (within 5 minutes) release of LH (490, 622) and FSH (490). Various GnRH treatment regimens effectively stimulate ovulation during the anovulatory season, even in mares in January and February that have little follicular development at the start of treatment (pg. 168). Furthermore, the gonadotropin profiles induced by GnRH treatments can be very similar to the natural profiles that precede the first ovulation of the year (pg. 150). In addition to the pituitary and ovarian stimulatory effects of exogenous GnRH, the following research results have impli- cated the hypothalamus and GnRH in reproductive seasonality and some of these results implicated the pineal gland: 1. Treatment with melatonin depressed the GnRH content of the hypothalamus from 14 ng in controls to 10 ng in mela- tonin—treated mares (1569); 2. GnRH content of the hypothalamus, as well as its distribution within the hypothalamus, was different during the anovulatory and ovulatory seasons, and hypothalamic GnRH levels increased dur- ing the return to the ovulatory season (1570, 690, 1481, 1434); 3. Melatonin implants during the sum- mer resulted in brain tissue distribution and content of GnRH that were similar to those occurring normally in the winter (1435); 4. GnRH content of the hypothalamus was elevated early in the daily dark phase, and was followed by a decline; this pattern was abolished by pinealectomy (1435); 5. Hypothalamic tissue appears to bind melatonin (1435); 6. Light treatment tended to increase (P<0.07) the hypothalamic content of GnRH and significantly increased circula- tory concentrations of LH and FSH (309, 310); 7. Mares treated with light beginning in January responded sooner than did control mares to an LH-inducing chal- lenge of a single injection of GnRH (1140); light treated mares responded in February Whereas controls did not respond until April; and 8. The first ovulation of the year was inhibited by active immunization against GnRH (1378, 557). It also has been demonstrated that the cells lining the third ventricle over the median eminence and other hypothalamic Reproductive Seasonality 121 areas are associated with reproductive changes in horses, including changes involving seasonality (1079, 1076, 1077, 1078). Studies on the concentrations of hor- mones and catecholamines in the cerebro- spinal fluid of horses have begun (1080). Results suggested that humoral sub- stances in the cerebrospinal fluid may be involved in the coordination of reproduc- tive processes. Seasonal effects on the lev- els of dopamine were dependent on the ovaries; seasonal changes were not detected in ovariectomized horses. The above findings are compatible with the hypothesis that the hypothalamus, the site of production of GnRH, is part of the chain that controls equine reproduc- tive seasonality and that photoperiod and the pineal are the initial links in the chain. Discussion of the effects of estro- gen and inhibin feedback in the transi- tion between anovulatory and ovulatory seasons will be found elsewhere (pg. 155). 4.5E. Role of the Pituitary As discussed above, the retina is the main receptor responding to daylength changes in mammals. The anterior pitu- itary is the effector, altering ovarian func- tion through changes in circulating levels of gonadotropins. Involvement of the pitu- itary in the chain of events that lead to reproductive seasonality in mares is well documented. LH and FSH after ovariectomy. Studies of seasonal changes in circulating concen- trations of gona'dotropins in ovariec- tomized mares have been helpful in the development of a concept on the interrela- tionships between season (length of pho- toperiod) and ovarian control of pituitary function. An earlier study (554) demon- strated that LH levels were low to a simi- lar extent in ovarian-intact and ovariec- tomized mares during the anovulatory season, but were high in ovariectomized mares during the ovulatory season. Distinct seasonal profiles were obtained 122 Chapter 4 in ovariectomized mares subjected to photoperiods of normal length (Figure 4.10; 540). The shape of the LH profile seemed similar to the curve for mean monthly LH values in ovarian intact mares (Figure 4.12) and to the profile depicting monthly ovulatory rates for ponies (Figure 4.2). The FSH levels in ovariectomized mares also followed a sea- sonal pattern in mares exposed to the control photoperiod (Figure 4.11). The FSH levels rose above base-line levels earlier than the corresponding rise in LH. Seasonal LH and FSH profiles. Mean monthly circulating concentrations of LH and FSH were determined in ovarian- intact pony mares (Figure 4.12; 1644). Mean concentrations per month per mare were based on samples collected every three days throughout the year. The mean concentrations for each month averaged over all mares were used as indicators of the average output of each hormone per month. The seasonal LH profile produced no surprises; the concen— trations were maximal during the sum- mer, attributable to ovulatory LH surges, Luteinizing hormone (n=5/group) Concentration (ng / ml) N Hem Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun S Hem and minimal during the winter (Figure 4.12). Similarly, concentrations of LH in the pituitary, as well as in the peripheral circulation, were higher during the sum- mer (1601). Concentrations of FSH did not differ significantly among months (1644) in contrast to the results in ovariec- tomized mares. Similarly, FSH concentra- tions in the pituitary, as well as in the peripheral circulation, were not different between summer and winter in ovarian- intact mares (1601, 690). Failure to detect seasonal (monthly) changes in FSH (1644) was disconcerting. However, within mares, associations were found between FSH concentrations and degree of follicu- lar activity during the anovulatory season (Figure 5.14, pg. 151). It appears that monthly FSH output is constant through- out the year, reflecting negative pres- sures of season during the winter and of the ovaries during the summer. Ovariectomy plus light treatment. Treatment of ovariectomized mares with prolonged daily photoperiod resulted in elevation in LH (Figure 4.10) and FSH (Figure 4.11) concentrations approxi- FIGURE 4.10. Seasonal LH pro- file in ovariectomized mares receiving control photoperiod or 16 hours of light/day throughout the year (OVX-16L). Means with— in and across lines without any common letters are significantly different. Adapted from (540). Reproductive Seasonality 123 Follicle stimulating hormone (n=5/group) 40 30 N 0 Concentration (ng / ml) _L D OVX—control light NHem Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec SHem Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun mately two months earlier than for pho— toperiod controls. The first significant rise in FSH preceded the first significant rise in LH by two months. Furthermore, light treatment resulted in a delay in LH and FSH decline in the fall and Winter months. Thus, extension of photoperiod broadened the seasonal LH and FSH curves in ovariectomized mares, as well as the seasonal ovulatory profile in intact mares (pg. 113) in both spring and fall. It is especially noteworthy that both LH and FSH were released in response to pho- toperiod and seasonal changes in the absence of ovarian influences. These results indicate that season exerts an effect through the pituitary, thus clearly demonstrating that the pituitary is part of the light-responsive chain in mares. LH, FSH and GnRH tissue content. Recently, it was demonstrated that LH content of the pituitary began to increase in February at a time when GnRH content of the hypothalamus was also beginning to increase (148]). The num- ber of GnRH receptors and the FSH FIGURE 4.11. Seasonal FSH profile in ovariectomized mares receiving control photoperiod or 16 hours of light/day throughout the year (OVX-16L). A # indi— cates a significant difference between groups for the indicated time, and a star indicates the first time that FSH levels signifi- cantly exceeded the initial level for each group. Adapted from (540). Gonadotropins (n=11) ASO FMA J J D J Month FIGURE 4.12. Average monthly concentrations of LH and FSH in ovarian-intact pony mares. Mean for each month was calculated from determinations made every three days for each mare. The vertical bar (lsd) indicates magnitude of least significant dif- ference for LH concentrations. Mean concentrations of FSH were not significantly different. Adapted from (1644). 124 Chapter 4 concentration of the pituitary, however, did not change during this time. Apparently, the pituitary is ready to respond immediately throughout the year whenever it receives a GnRH message to release FSH. The standby status for pituitary FSH, however, does not apply to LH. Release of an LH ovulatory surge is preceded by a prolonged build-up in pituitary LH content. Treatment-to-response time lag. In ewes, melatonin infusions altered LH secretion with a lag of 30 to 50 days (843). The melatonin-to-LH time lag in sheep may be related to the long time lag in mares between the beginning of photope- riod treatment and ovulation. In this regard, it is noteworthy that the lag between the initial significant FSH increase and the initial significant LH increase in light-treated ovariectomized mares (2 months; g. 122) is also equivalent to the lag between beginning of an effec— tive light program and ovulation (pg. 113). Administration of gonadotropins. Administration of equine pituitary extracts caused ovulation during the anovulatory season, even when the mares had inactive ovaries at the beginning of treatment (pg. 168; 435). The pituitary extracts contained LH, as well as FSH, which accounted for the much more rapid ovarian response to extracts than to light treatment. Along these lines, repeated administration of hCG, eLH, and com- bined eLH and eFSH preparations during the anovulatory season caused follicular growth and ovulation (pg. 154). The results of these experiments support the hypoth- esis that ovarian inactivity during the anovulatory season in mares results from secretory inactivity of the pituitary. Prolactin. Prolactin concentrations fol- low a seasonal pattern in seasonally breeding mammals, although the func- tional importance of the seasonal pro- lactin profile is unknown (1827). A marked rise in plasma prolactin concentrations occurs during the summer in mares (828); this observation has been confirmed (1827, 1611, 1601). Thyroid-releasing hormone stimulates the release of prolactin in many species, including the horse (983, 1611, 828), regardless of season of the year. Relationships between levels of prolactin, FSH, and LH in the serum and pituitary have been reported for horses (1601). Seasonal prolactin profiles are also tem— porally related to temperature, photo— period, and hair coat. An experiment on the interrelationship of these factors with artificially extended photoperiod indi- cated that photoperiod plays an impor- tant role in levels of ePRL, but that other seasonal factors are also involved (e.g., temperature; 830). There is a photosensitive phase (8 to 10 hours after dark) during which ePRL secretion occurs (834); the sensitive phase is similar to the experimentally deter- mined photoperiodic period for induction of the ovulatory season (pg. 116). A rise in PRL preceding the rise in LH that is associated with the first ovulation is a temporal indication that PRL may be part of the hormonal cascade leading to the onset of the ovulatory season. The seasonal profile of PRL had a distinct decrease between August and September at a time when mares were expected to still be in the ovulatory season. It has been suggested (831) that seasonal decline in PRL is not the result of increased sen— sitivity to a proposed prolactin-inhibiting factor. Perhaps the decline in ePRL is involved in a chain of events that culmi- nate in the end of the ovulatory season. The hypothesis that ePRL is involved in the series of events controlling onset and termination of the ovulatory season needs to be tested. Approaches could include administering prolactin or stimu- lating or blocking endogenous prolactin production. Dopamine antagonists and agonists that elevate and suppress pro- lactin levels in mares are metoclopramide (168) and bromocriptine (pg. 471; 799), respectively. These products are candi— dates for use in seasonality studies in mares. Reproductive Seasonality 125 SUMMARY: Pathway from Photoperiod to Ovaries The pineal (melatonin), hypothalamus (GnRH), and pituitary (LH and FSH) are the vital links in the chain extending from changes in, daylength to the ovaries. Some of the indicators for the involve- ment of these organs and hormones are as follows: Pineal ° Melatonin-forming activity of the pineal is temporally related to repro— ductive seasonality. ° The stimulation of 2.5 hours of light in the evening is abolished by mela- tonin administration. ° Exogenous melatonin simulates some of the effects of darkness. 0 Denervation and removal of the pineal alter ovarian function With respect to photoperiod. Hypothalamus 0 Exogenous GnRH stimulates circu— lating LH and FSH concentrations and ovulation during the anovula- tory season. 0 GnRH content of the hypothalamus is different during the anovulatory and ovulatory seasons and is increased by light treatment. 0 The ovulatory season is delayed by immunization against GnRH. Pituitary ' Mean monthly profiles of LH follow a distinct seasonal pattern. 0 In ovariectomized mares, LH and FSH follow a seasonal pattern that can be manipulated with light treat- ments. 0 Exogenous gonadotropins stimulate ovulation during the anovulatory season ' LH content in the pituitary is mini- mal during ovarian inactivity in the Winter and gradually begins to increase in late Winter. Monthly FSH output is constant through— out the year, reflecting negative influence of season during the Winter and negative influence of ovaries during the summer. Major areas needing clarification include: I) the role of PRL, 2) the role and interac- tion of internal rhythms, and 3) the possi- bility that the effect of increasing pho- toperiod may involve production of a pineal reproductive stimulant as well as suppression of a pineal inhibitor (mela— tonin). It is likely that prolactin has a role in the cascade of events leading from ovarian inactivity to ovulation; (e.g., circulating levels of prolactin increase before the increase in LH). NH J F M A M J J A S 0 N D SH J A S O N D J F M A M J \‘9 \1‘ Receptors / 0‘0 in eye 9,) Neuro- l . hw V pat ay Pineal gland Decreasing increasing melatonin V melatonin Hypo- thalamus lncreasing ; Portal Decreasing GHRH ‘ system GHRH V Anterior pituitary increasing General Decreasing gonadotropins t curc gonadotropins Ovaries FIGURE 4.13. Diagrammatic overview of postu- lated chain of events by which changes in dayiength affect ovarian function in the mare. World hemi- spheres are indicated by NH and SH and months by single letters. 126 Chapter 4 4.6. Role of Other Extrinsic Factors Light is the major modulator of sea- sonal reproductive rhythms, but other environmental factors play a modifying role. In support of this View in nonequine species, are the observations that expo- sure of rats to temperature extremes resulted in altered pineal function (1152, 1653). Similarly, stressful situations (immobilization) resulted in hyperplasia and hypertrophy of pinealocytes. Whether or not these mechanisms (temperature, stress) act in the horse is unknown. Exercise also could be an important factor if horses restrained in stalls responded in the same, though attenuated, fashion as immobilized rats. Restricted feed intake was shown to increase pineal activity in rats; the increased activity was apparently mediat— ed through adrenal cortical steroids (1653). Similarly, food supply and body condition have a well documented modifying role in equine reproductive seasonality. That is, increasing daylength cannot readily acti- vate the ovaries in poorly nourished mares, whether the poor nourishment is the result of inadequate quantity or qual- ity of feed or inadequate consumption, digestion, or assimilation (e.g., para- sitism, disease). In an early Swedish study (174), mares fed oats (considered to be inadequate) were compared to mares fed better-balanced pelleted feeds. Before the onset of the ovulatory season, signifi- cantly more mares in the pellet group (32 of 36) had large follicles (>10 mm) than mares in the oat—fed group (6 of 20). In an experiment in South Africa (1676), maiden mares were divided into a group kept on winter pasture and a group that was also given a supplement. The project began 36 days after the winter solstice and contin- ued for 53 days. In the unsupplemented controls, only 2 of 6 mares ovulated by the end of the experiment, compared to 8 of 8 in the supplemented group. During a study on reproductive seasonality (573), some information on the effect of nutri— tion on the onset of the ovulatory season was obtained insofar as changes in weight of mares reflected changes in the level of nutrition (quantity and quality of feed consumed or utilized). The length of the interval from January 1 to the first ovulation of the season was shorter for six mares that gained weight (mean: 114 days) than for eight mares that lost weight (mean: 145 days). Furthermore, a correlation analysis indicated that the greater the loss in weight, the longer the ovulatory season was delayed. In another study (716), barren and maiden Quarter Horses were assigned body scores of one (very thin) to nine (very fat). The interval from January 1 to first ovulation was significantly shorter for mares scoring five or greater (68 days) than for mares scoring less than five (94 days). The effects of body condition in pregnant and postpartum mares found in this and other studies are discussed else- where (pg. 478). In a more recent study, mares were classified as thin, good, and fat and were fed at high and maintenance levels of energy intake (917). The first ovulation was 23 days earlier for the fat category than for the good and thin cate— gories. High—energy intake hastened the onset of the first ovulation at the end of the transitional period in the mares in the thin category but not in the good category or fat category. These studies demonstrate that mares in good or fat body condition will, on the average, begin the ovulatory season earlier than mares that are not. The interaction of nutrition or body condition with pituitary gonadotropins on the initiation of the ovulatory season has received limited attention. It was observed (230) that mares in good body condition were better able to respond to eLH administration (induced ovulation) during March than were mares in poor body condition. This observation could be well utilized in building a hypothesis for experimental testing in regard to nutrition-gonadotropin interactions in the mare during the onset of the ovulatory season. 4.7. Role of Intrinsic Factors It has been suggested that there is an intrinsic annual rhythm of reproductive function that is modified, but not entirely controlled, by seasonal changes. That is, an inherent rhythm may act as a sec- ondary regulator of sexual activity in the absence of adequate photoperiod changes. In mares, the possibility of an inherent rhythm may be related to the state of evo— lutionary change or change due to human influence; that is, some individuals of the species have broken away and the species, as a whole, is beginning to break away from photoperiodic dominance. In this regard, discussions with nonequine seasonality specialists have given the impression that such changes have occurred quite rapidly in several species, such as pigeons, due to human influences. It has been suggested (986) that the management practices associated with domestication in mares may be gradually relieving the pressures for reproductive seasonality and that breeds may differ in reproductive seasonality according to the various environments in which they evolved. Indications that horses are not rigidly controlled by photoperiod. Indicators com- patible with the possibility that mares are beginning to break away from the regulatory dominance of photoperiod are as follows: 1. Many horse mares, but few pony mares, ovulate throughout the year (pg. 107); 2. The more primitive breeds appear to have shorter ovulatory seasons (pg. 107); 3. Young mares have shorter ovulatory seasons (pg. 492); 4. Denial of increasing photoperiod by constant 15 hours of darkness resulted in retardation in the onset of the ovulatory season in some mares (50%) but not in Reproductive Seasonality 127 others; all mares eventually ovulated despite the constantly short photoperiod (Pg- 115); 5. Maintaining a fixed 16-hour pho- toperiod after the summer solstice caused some, but not all, mares to cycle through- out the winter (pg. 115); 6. Removal of the superior cervical gan- glia resulted in eventual loss of seasonal synchrony but did not eliminate seasonal- ity (1439); and 7. Mares exposed to continuous dark- ness continued to have daily, but modi- fied, fluctuations in melatonin levels (pg. 118). Comparative studies. Recent and con- tinuing studies in sheep indicate that there is an interplay between an endoge- nous rhythm and environmental cues (primarily length of photoperiod) that governs seasonality (845). That is, repro- ductive seasonality arises from within the ewe and is modified, but not driven, by daylength. When the endogenous rhythm and the photosensitive phase coincide, the physiologic response occurs. The abili- ty of mares to “break away” from photope- riodic control in the above listed examples indicates that an endogenous rhythm, subject to modification by the environ- ment, also exists in mares. Research in sheep indicates that the development of refractoriness to photoperiod involves postpineal processing of the photoperiodic message, but does not involve changes in the 24-hour secretory pattern of mela- tonin (844). The white-footed mouse and some other mice species may be good compara- tive species for scientists studying equine seasonality. The breeding season of the white-footed mouse is similar to that of horses. Exposure to fixed short photoperi- ods (10 hours) resulted in regression of the gonads in 70% of mice but did not affect the other 30% (838). Furthermore, the mice that underwent gonadal regres- sion escaped from the retarding effects of short photoperiod after 30 weeks. It has been proposed that some mice secrete 128 Chapter 4 insufficient pineal antigonadal factors or are refractory to them and that other mice eventually develop refractoriness when continually challenged by short photoperiods. These research results seem similar to the above discussed find- ings in mares. 4.8. Hair Coat and Reproductive Seasonality It is frequently stated that shedding of the winter coat and development of a sleek, smooth coat mirror the activity of the ovaries. Although development of a short coat and the onset of the ovulatory season both occur in the spring, there are apparently no reports demonstrating that change in coat is useful for selecting those mares within a herd with early onset of the ovulatory season. Length of interval from January 1 to beginning of the ovula- tory season and from January 1 to the appearance of a smooth coat were posi- tively correlated (r=+0.37, n=14), but the correlation did not reach significance (573). Also, the coat changes in the fall were not significantly correlated with the time of termination of the ovulatory season. The effect of lighting regimens on hair coat is illustrated by number of days to shedding, number of days to appearance of a smooth coat (Table 4.2), changes in hair length and coat depth (Figure 4.14), and by photographs of representative mares (Figure 4.15). Shedding of hair in tufts preceded the first ovulation of the season by approxi- mately two months in both control and light-treated mares in the study described for Table 4.2; a smooth coat appeared, on the average, after ovula- tion. It seems, therefore, that shedding of hair can be used as an indicator that the ovulatory season is approaching, at least on a herd basis. The temporal asso- ciation between hair coat change and reproductive activity also has been seen after removal or denervation of the pineal (pg. 119). It appears that mares use daylength rather than temperature as a signal for changes in hair coat. Presumably, this occurs because daylength and temperature changes par— allel one another, but daylength is less variable, day to day, and therefore is a more stable cue. The need for more research in this area is indicated from both the biologic and applied views. An interesting increase in hair length and coat depth occurred in light-treated mares approximately one month after their coats had become smooth and sleek (905). This effect is illustrated in Figures 4.14 and 4.15 (June 13, Group L162D8). The renewed growth of hair was similar to that which occurs normally in the fall, and there was no evidence of shedding of the new coat by the end of the project (June 13). Thus, experimentally altered photoperiods induced these mares into Effect of photoperiod on hair coat Depth (mm) 12-13 1-30 3-25 5-6 Date FIGURE 4.14. Effect of continuous lighting regi- mens on depth of hair coat; 9L, 16L, and 24L refer to number of hours of light per day and C refers to controls. Within each date, means with no common superscripts are significantly different. Adapted from (905). . control ,Jun 13 . " control Reproductive Seasonality 129 FIGURE 4.15. Examples of effect of lighting regimens on hair coat. Light treatments began on November 13, using fixed daily photoperiods of 9 hours light:15 hours dark (L9zD15) and 16 hours light:8 hours dark (L16:D8). Adapted from (905). wearing heavy coats on hot summer days. All of the mares continued to cycle nor— mally, indicating a dissociation between these two end points. In foals, hair coat changes and reproductive stimulation by 16 hours of light were dissociated because of a negative effect of light treatment on the age of puberty but not on hair coat (pg. 494). It appears, therefore, that hair coat and ovarian activity are associated temporally, but experimental manipula— tions can dissociate one from the other. 4.9. Seasonal Infertility Many aspects of equine reproduction are different and in some cases unique among domesticated mammals. But it works and has assured species survival for millions of years. The species evolved according to environmental pressures and therefore reproductive patterns and envi- ronmental changes were in harmony. Through domestication, man added dis- cord, much of which can be attributed to inadequate knowledge or consideration of equine reproductive seasonality. 4.9A. The Physiologic Problem It will be recalled from the previous sections that estrus is not a reliable indi— cator of ovarian function during those months when the ovulation rate in a herd is low. During the anovulatory season, many mares will show all the signs of estrus, sometimes including sexual recep— tivity. The ovaries may contain few palpa— ble follicles or, if large follicles are pre- sent, ovulation does not occur; the estrous period is, therefore, a sterile one. In addi- tion, very long ovulatory estrous periods (>10 days) occur frequently in the spring. Once aware of these normal phenomena, we should not be surprised that pregnan- cy rates are low during the winter and early spring, as many investigators have reported. 130 Chapter 4 Month of foaling was studied for 1,024 Standardbred mares in Pennsylvania (867). The mares were bred every time they came into estrus, beginning December 15. However, 58% did not foal until April and May; pregnancy did not occur until May and June. These data show the result of breeding mares before many in the herd are ready to conceive. It should be noted that the stallion, as well as the mare, may be involved in poor pregnancy rates when breeding is done early in the year (1258). Illustrations of the low pregnancy rates during the winter and early spring are shown in Figure 4.16, based on reports from Virginia (792) and South Africa (1665). During this time, the percentage of mares in estrus was considerably higher than the percentage of mares ovulating and conceiving. Monitoring the ovaries by pal- pation or ultrasonic scanning should increase pregnancy rate at this time by withholding breeding from those mares with poor follicular development. Pregnancy rates (166) 60 Percent pregnant 0.: O N O Thoroughbred 10 NHem J F SHem J A M S A M J O N D J Month FIGURE 4.16. Monthly pregnancy rates for Thoroughbred horses in Virginia (792) and non— Thoroughbred horses (light-farm type) in South Africa ( 1665). Numbers in parentheses are number of mares. The operational breeding season will be different under various management schemes. It is influenced not only by sea- son, but also by the proportion of mares that are barren (did not become pregnant or failed to maintain pregnancy during the previous year), maiden (to be mated for the first time), or foaling (postpartum mares). For example, the mating of mares that foal late will necessarily be delayed. Conversely, those that foal early may have opportunity to foal even earlier the next year because of the return to cyclic- ity shortly after parturition and a gesta- tion length of less than 12 months. The month in which mares of the three types (barren, maiden, and foaling) were first submitted for service is shown in Figure 4.17. The mares were Thoroughbreds and Quarter Horses primarily from Florida and Oklahoma. Most barren mares were first bred in March and April, compared to April and May for foaling mares. Month of first mating Maiden (n=201) 40 30 20 Percent mated 10 Feb Mar Aug Sep Oct Nov N Hem Jan S Hem Jul Apr May Jun Jul Dec Jan FIGURE 4.17. Month in which maiden, barren, and foaling horses were submitted for first service of the year. Numbers in parentheses are numbers of mares. Adapted from research data of American Breeders Service, courtesy of J. Sullivan. 4.93. The Official Birth Date Problem It may not be apparent why so much effort is expended in attempting to mate mares before they are in prime mating condition. Much of the problem centers around the tradition of birth date recogni- tion among various registries. The author surveyed many of the breed registries in the United States in 1990 about their policies in the recording of birth dates. Responses were received from 21 registries, and of these, 43% use January 1 of the year in which the foal was born as the official birth date, and one additional registry uses March 1 (Peruvian Paso; Table 4.5). The three major racing breeds (Quarter Horse, Standardbreds, and Thoroughbreds) use the January 1 birth date. Some of the reg- istries record the actual birth date, but for show purposes, January 1 is used. The official birth date problem, therefore, is not limited to the racing breeds. Many TABLE 4.5. Birth Date Policies of Various Registries in the United States in 1990 Actual birth date American Connemara Pony Society American Hanoverian Society American Morgan Horse Association American Saddlebred Horse Association American Suffolk Horse Association Arabian Horse Registry of America Belgium Draft Horse Corporation of America Clydesdale Breeders of the United States Friesian Horse Association of North America Paso Fino Horse Association Welsh Pony and Cob Society of American January 1 official birth date American Hackney Horse Society American Paint Horse American Quarter Horse Association Appaloosa Horse Club Missouri Fox Trotting Horse Breed Association Pony of America Club Tennessee Walking Horse Breeders and Exhibitors Association The Jockey Club (Thoroughbreds) U.S. Trotting Association (Standardbred) March 1 oficicial birth date Peruvian Paso Horse Registry of North America Reproductive Seasonality 131 countries in the Northern Hemisphere use January 1 and in the Southern Hemisphere use August 1 as the official birth date for many breeds, especially the racing breeds. Thus, a foal born on February 1 and a foal born on August 1 would both be considered one year old on the following January 1 in the Northern Hemisphere, even though there is a half- year difference in actual age. These two foals would compete on the racing and show circuits with an advantage for the foal that was born earlier in the year. The promotion of yearling sales in the sum- mer also contributes to early breeding pressure since big yearlings bring higher prices. Thus, farm operators are under pressure to mate mares so that foals will be born as soon as possible after January 1 (Northern Hemisphere) or August 1 (Southern Hemisphere; Figure 4.18). The importance of this situation to the reproduction specialist lies in the imposi- tion of a breeding season that has been set by man without consulting the horse. There is approximately a three-month discrepancy between the onset of the imposed breeding season (early February Imposed breeding seasons Breeds with Jan 1 K— official birth date ——>_ 3 (N Hem) § : Breeds with Aug 1 ; 100 E ;<— official birth date +§ E i (S Hem) = m 80 .E E g 60 o '5 g 40 co 0. M O N Hem Dec Jan Feb Mar Apr May Jun Jul SHem Jun Jul Aug Sep Oct Nov Dec Jan FIGURE 4.18. Discrepancy between imposed breed- ing seasons and optimal, natural breeding season. 132 Chapter 4 in the Northern Hemisphere and early September in the Southern Hemisphere) and the onset of the optimal natural breeding season (May and November in the two hemispheres; Figure 4.18). Thus, activity each year among the operators and veterinarians commences long before that of the mares. The competition to breed horses before they are in prime breeding condition has become a well- engrained facet of the equine industry and is a major cause of horse breeding problems. To the reproduction-oriented indivi— dual, the solution to the problem may seem simple—change the official birth date so that it is compatible with the breeding season. Osborne (1185, 1186) in Australia is recognized and cited as the principal veterinary authority in this area. She proposes that the spring equinox be used as the beginning of the official breeding season. Birth dates of foals under this scheme would be March 1 and September 1 in the Northern and Southern Hemispheres, respectively. It was suggested that such a scheme would cause minimal interference with the cur- rent spring and summer racing programs, except for the maturity of juvenile horses. One registry (Peruvian Paso) in the United states has adopted March 1 as the official birth date. The history of the establishment of the official birth date has been reviewed (1186). In 1751 the date of May 1 was pro- claimed to be the official date for first foals in the racing areas of England. The May 1 date was entirely reasonable since breeding would then occur at the ideal time of early June. However, May fell within the racing season, and confusion was caused among racing administrators because of the necessity for an age change in the middle of the racing sea- son. Therefore, the English Jockey Club decreed in 1833 that January 1 would be used. It was apparently believed that a standard birth date would be easier to administer and enforce. The English racing influence, including the tradition for a universal birth date, spread throughout the world. In the Southern Hemisphere, July 1 was initially used since it corresponded to January 1 in the Northern Hemisphere. In 1859, however, the birth date was changed to August 1, and this date is used in Australia and South Africa. From the above historical account it is obvious that the current official birth date arrangement is a long-standing tra- dition. Correspondence with officials of the American Jockey Club (277) and the American Quarter Horse Association (638) indicates that a change in birth date policies would be a tremendous task with vast economic implications. It seems that registries will not use the natural birth date because of policing and administrating problems. Changing to a later official birth date would force more than 100 North American race tracks to modify their racing programs. The racing rules and laws of many states would have to be changed. A birth date change would require industry-wide cooperation with large economic ramifi- cations and therefore would likely be met with vigorous opposition by racing and, perhaps to a lesser extent, show and sales management people. It seems, therefore, that the official birth date problem for the foreseeable future will be a problem borne mainly by breeders and veterinarians. An objective analysis is needed to compare the costs that would be borne by regulators if a biologi- cally compatible birth date was used against the costs that are now borne by owners. Reproductive Seasonality 133 HIGHLIGHTS: Reproductive Seasonality The percentage of ovulating mares is minimal in winter, increasing in the spring, maximal during the summer, and decreasing during the fall. The effects of latitude on reproductive seasonality have not been studied; the study closest to the equator was at 15°N to 22°N and the mares were seasonal. The ovulatory season begins about one month later in ponies than in horses; young mares stop ovulating sooner in the fall. Light is the only environmental factor that has been demonstrated to affect repro— ductive seasonality, except that factors expressed as health and body condition have modulating rolls; mares in poor condition enter the ovulatory season later. Either a gradually increasing photoperiod or a fixed period of 15 hours begun in December causes the ovulatory season to begin about two months earlier. Prolonged fixed photoperiods (>20 hours) produce an effect intermediate between the effects of natural daylength and a 15-hour photoperiod. Artificially shortening the photoperiod (e.g., 9 hours) delays the ovulatory season in some mares but not in others. Light treatment requires more time when the ovaries are less active at the begin- ning of treatment. Artificially extending photoperiod beyond the summer solstice causes some mares to have a long ovulatory season. Under experimental conditions (abrupt changes between light and dark), a photo- sensitive period occurs 9.5 hours after the beginning of darkness. Light exerts its effect through pineal production of melatonin during darkness, which in turn blocks production of GnRH by the hypothalamus. Denervation or removal of the pineal upsets reproductive seasonality. In ovariectomized mares, LH and FSH follow a seasonal pattern, and the months of maximum production can be extended on either end (spring or fall) with light treatment. Monthly LH output reflects the influence of season, whereas monthly FSH output is constant, reflecting the influence of season in the winter and the influence of the ovaries in the summer. Seasonal ovarian activity and hair coat are associated temporally, but experimen- tal manipulations can dissociate one from the other. For example, altered pho- toperiod can induce mares to carry heavy hair coats on hot summer days. The greatest obstacle to operational and physiologic harmony in reproductive management is the use of biologically incompatible official birth dates. 134 Reproductive Seasonality MILESTONES: Reproductive Seasonality Discovery that artificial lengthening of the photoperiod hastens the ovula- tory season (260). First study on the influence of nutrition on reproductive seasonality (174). Characterization of reproductive seasonality by study of ovaries from a slaughterhouse (1185). 1974-75 Demonstrations that GnRH stimulates LH (622) and FSH (490) release. 1975 Initiation of studies on the effects of various light regimens (905, 1431}. 1976-79 Characterization of seasonal control of circulating levels of LH and FSH throughout the year based on ovarian—intact and ovariectomized mares. (554, 540). Demonstration of the temporal association between pineal melatonin forma- tion and reproductive seasonality (1764). 1979-82 Initiation of a series of studies on the role of the pineal in reproductive sea- sonality based on pineal denervation, pineal removal, and melatonin assay ( 1439, 655). Initial studies on the morphology of the lining of the third ventricle and its role in reproductive seasonality (1076, 1077). -—Cfiapter 5— ANOVULATORY SEASON As defined and discussed in Chapter 4, the anovulatory season extends from the last ovulation of the ovulatory season to the first ovulation of the subsequent ovu- latory season. This chapter will examine the phases of the anovulatory season, including the behavioral, anatomical, and hormonal aspects and will review the methods for artificial induction of ovula- tion in seasonally anovulatory mares. The terminology to be used here is shown in Figure 5.1. The anovulatory season will be divided into three phases: receding, inactive, and resurging. The receding phase or recession is defined as a gradual retrenchment to an inactive condition, beginning after the last ovula- tion of the year. The phase is character— ized by failure of a large follicle to ovulate at the expected time following luteoly— sis.The resurging phase or resurgence is defined as a gradual return to the ovula- tory season. At the beginning of the Ovulation Anovulatory season resurging phase, the ovaries of the inac- tive phase are subjected to follicular stimulation. Eventually a large follicle grows and ovulates, or the ovulatory- sized follicle may regress and be replaced by another. Terminology for the phases is intended to divide the anovulatory season into mentally manageable and meaning- ful parts. The phases apply to individual mares that are under profound influence of photoperiod; in other mares, one or all phases may be indistinct or absent. There has not been a standardization of terminology for the anovulatory sea- son. The term recrudescence is sometimes used by seasonality specialists and is a synonym of resurgence. The term transi- tional period is used frequently and is usually in reference to spring transition (resurging phase) as opposed to fall tran- sition (receding phase). Sometimes the transitional terminology is subdivided into early and late transition. Most Ovulation GOO GOO 000 5X— Receding phase ——> <— Inactive phase —> 4—— Resurging phase —> V Oct Nov Dec Jan (Apr) (May) (JUN) (Jul) v Feb Mar ’ Apr May (Aug) (Sep) (Oct) (Nov) FIGURE 5.1. Terminology used herein for various portions of the anovulatory season. Months in parentheses are for the Southern Hemisphere. 136 Chapter 5 authors appear to use the term late tran- sition for mares that have developed large follicles (e.g., >25 mm) but have not yet entered the ovulatory season. Other common terms include deep anestrus and shallow anestrus. Presumably, deep anestrus is similar to the inactive phase and shallow anestrus is similar to the early portion of the resurging phase. 5.1. Overview 5.1A. Sexual Behavior Frank estrous behavior unaccompanied by major follicular development is com- mon in mares that are in the anovula- tory season. Such periods have been called unseasonal estrus, pseudoestrus, false estrus, and paradoxical estrus. Unseasonal estrus occurs even in mares with very small ovarian follicles (inactive phase). There is a paucity of information about these anovulatory estrous periods even though they could be a rich source of information about the relationships among ovarian function, hormonal changes, and expression of estrus. Lack of awareness of the occurrence of unseason- al estrus seems to have caused some con- fusion, particularly in attempts to charac— terize equine reproductive seasonality. An account of the incidence of unseasonal estrus has been reported (573) and is sum- marized below. Other aspects of the phenomenon are given in Chapter 3 (pg. 95). In the pony herd described in Figure 4.1 (pg. 106), unseasonal estrus occurred in all months of the anovulatory season. All of the 14 mares except one (Mare 4, Figure 4.1) had one or more periods of unseasonal estrus, with an average of 7 such periods per mare. Many (71%) of the periods were only 1 or 2 days long and the average length was 2 days. The unseason— al estrous behavior occurred very irregu- larly and occurred most often at the beginning and end of the anovulatory sea- son. The number of such estrous periods was not significantly different between the 30 days after the last ovulatory estrus and the 30 days preceding the first ovula- tory estrus. The cause of unseasonal estrus during the inactive phase is discussed elsewhere (pg. 96). It is noteworthy that similar estrous activity occurs even in ovariec— tomized mares. During the inactive phase, therefore, the estrous behavior is not caused by the ovaries. Presumably, during the receding and resurging phases, estrous behavior can be caused by the synthesis of estrogen by the growing follicles. 5.13. Follicular Dynamics Changes in diameters of follicles, as determined by transrectal palpation or ultrasonic scanning, provide a convenient gross indicator of the status of the repro- ductive system during the anovulatory season. The size of the largest follicle seems to be a convenient indicator of the mare’s position within the three phases and can be taken as a representation of seasonal pressure on ovarian activity. This guideline can be especially useful in judging the expected response to a given treatment to end the anovulatory season (pg. 168). Follicular dynamics. Follicular mea- surements were recorded during a 12-month study in 14 pony mares (575). The follicular aspects were based on ovar- ian transrectal palpation every three days, except that daily palpation was used when a large follicle (>30 mm) devel- oped. Overall relationships between month of the anovulatory season and the percentage of mares with various levels of follicular activity are shown in Figure 5.2. Note that during January and February no mare developed a follicle >30 mm, and the majority of mares did not develop a follicle that reached 20 mm. Using these criteria, follicular activity was minimal Anovulatory Season 137 Diameter largest follicle (n=14) 100 oooooooooocoooooooooooooo. ..O.............. 0 °-. . .. °. .' 30 0. ,°<-— >30 mm °-. o ....... . 0. : O o C (I) .. a“. O. m 60 0 .° ' : a O. O .0 O E '. , -' '.o° E °. .' -' 3 0 . o. . 0. 0... e 40 3... '. o O. .0 lg.) .0. 9.. '. o. 1 : :6 20-30 mm .0 :0. .ooocooo. 2 : 1 o. ' .0 o... .'. 20 .0. .‘ 0. .‘t .0. a .' 0 .. .- .0 .' ,0 ;<— <20 mm -. ... . -_ .0 Q0? ... Q... . ' ..'o 0 o-oco-o-o-o-o-o-o-o-o‘o‘o-u-o-o.o-o-o‘o-o-o-o~o.o .00.... ------------ 'w-ooo-o-o-ooo-o-o-o-o-o- N Hem Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug 3 Hem Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb I‘— Summer _|l__ Fall ——ll'— Winter _‘ll— Spring ——ll‘— Summer —| FIGURE 5.2. Percentage of 14 mares in which the largest diameter attained by a follicle for each month was >30 mm, 20 to 30 mm, or <20 mm. From (575). during the two winter months of January and February (equivalent to July and August in the Southern Hemisphere). At the other extreme, almost all mares developed at least one follicle >30 mm during each month of the ovulatory sea- son (May to October). The number of mares that developed a large follicle decreased during November and December and increased during March to May. Atresia. Follicular atresia (Figure 5.3) involves degeneration of the follicle and may begin at any stage of development. The following account of the histologic aspects of follicular atresia during the anovulatory season in mares is based on the report of van N iekerk and co-workers (1671). In small follicles (<5 mm), the internal and external layers of the theca are indistinguishable. The first sign of apparent atresia in such follicles is the absence of mitotic figures in the stratum granulosum. The nuclei of the granulosa cells subsequently become pycnotic (show shrinkage and loss of vesicular appear- ance), and the cells lose contact with the basement membrane. The inner portions of the stratum granulosum slough into the antrum as large, round, degenerative bodies. Fibroblasts of undifferentiated theca proliferate and grow in among the remaining granulosa cells, obliterating the antrum and forming a nodule of scar tissue. In larger follicles With differentiat- ed thecal layers, the first Sign of atresia is the appearance of lipoid droplets in the granulosa cells with pycnosis of nuclei. The cells slough into the antrum, and the basement membrane of the stratum gran— ulosum thickens to form a distinct hya- line membrane. Contraction of the hya- line membrane causes collapse of the follicle with obliteration of the antrum. Ultimately, the follicle is replaced by scar tissue (corpus fibrosum atreticum). “ m A -\ WALL B. EARLY ATRESIA VA NORMAL FOLLICLE C. OOCYTE, EARLY ATRESIA FIGURE 5.3. Histologic examples of follicular atresia in seasonally anovulatory pony mares. A. Wall of a normal follicle showing mitotic figures B. Early atresia. Note the disorganization of the (arrow) in the stratum granulosum. The granulo- granulosa with loss of arrangement of basal layer of sum is well organized with a row of cells with tight- cells, dark-staining cytoplasm, pycnotic nuclei and 1y packed nuclei at its base. degenerative bodies in the follicular fluid. C. Close-up of oocyte and cumulus in early atre- sia. Section is from an 8 mm follicle. Remaining portion of follicle was in a much more advanced stage of atresia. Anovulatory Season 139 D. ADVANCED ATRESIA F. REGRESSED FOLLICLE D. Advanced atresia with the development of a dis- E. Regressed follicle consisting of a distinct, con- tinct hyaline band (solid arrow) between the theca tracted hyaline band (arrow). The antrum is gone interna and the sloughed granulosa. The granulosa and has been replaced by fibroblasts within the has been replaced by a network of fibroblasts. Note collapsed hyaline band. Note the wall of a normal degenerative bodies (open arrow) or remnants of the follicle at the top of the section. granulosa in the follicular fluid. F. Remains of a regressed small follicle. The antrum is gone and has been replaced by a network of fibroblasts. The fibroblasts of the thecal layers have proliferated to form a compact mass or r‘ nodule around the regressed follicle. 140 Chapter 5 5.2. Receding Phase 5.2A. Follicular Dynamics Apparently only one study on the first portion of the anovulatory season has been done (1498). This is regrettable because both ends of the anovulatory sea— son are of interest from a scientific View- point. The heavy research emphasis on resurgence with little emphasis on reces- sion is understandable and is due to prac— tical pressures. The diameter of largest follicle and number of follicles >20 mm are shown for the receding phase (Figure 5.4; 1498). On the average, follicular activity for the two weeks following the last ovulation of the year was similar to that during pre- vious diestrous periods. At the time when the next ovulation would have occurred, follicular activity increased. That is, the mares did not, on the aver- age, enter immediately into a state of fol- licular inactivity. Apparent attempts were made to bring up a crop of follicles in cyclic fashion, but final growth of a preovulatory-sized follicle failed. Inspection of raw data indicated that 11 of 14 mares manifested increased follicu- lar activity approximately 24 days (equivalent to an estrous cycle length) after the last ovulation of the ovulatory season. It may be concluded that most of the mares initially attempted to develop an ovulatory follicle but gradually reced- ed into the anovulatory season. 5.2B. Endocrinology The association between follicular and gonadotropic changes during the fall fol- lowing the last ovulation of the year was considered in the above described study of follicular changes (1498). Emphasis was given to the deficiencies that were associ- ated with failure of the mare to ovulate at the expected time. The experimental design involved a comparison for follicu- lar and gonadotropic end points between End of ovulatory season (n=11) After last OV M. J l \ \ Concentration Concentration (ng/ml) (ng/ml) Diameter (mm) Number of follicles Coded value 0 6 12 18 24 30 Number of days after ovulation FIGURE 5.4. Gonadotropin, follicular, and behav- ioral changes during the transition from ovulatory to anovulatory seasons for 30—day periods after the second-to—last and last ovulations of the year. Adapted from (1498). the 30 days following the second-to—last ovulation (control period) and the 30 days following the last ovulation. There were no significant differences between the two experimental periods in number of folli- cles >20 mm, expression of estrus, and circulating levels of FSH (Figure 5.4). However, on the average, the diameter of the largest follicle and the level of LH at the time of the expected ovulation were less than during the last periovulatory period. These data were reinforced by recent studies that demonstrated that the pituitary content of LH decreased pro- gressively from the middle of the ovulato— ry season to the middle of the anovulato- ry season (1481). Within the limits of the end points studied (1498), the failure to ovulate was associated with a deficiency of both LH and the final preovulatory growth spurt of a large follicle. Failure to ovulate was not associated with a defi— ciency of FSH or number of large follicles available for a final growth spurt. The FSH profile preceding the failed ovulation was not different in magnitude or relative location from the FSH profile that preced- ed the last ovulation of the year. Based on limited data involving inspection of FSH profiles for a few indi- vidual ponies (Figure 5.5), FSH surges may continue sequentially during at least a portion of the receding phase. Perhaps developing follicles produce FSH-inhibitory substances (inhibin and estrogen) that periodically inhibits FSH, thereby resulting in FSH surges. Such surges may partially account for failure to show a significant decrease in the monthly FSH productivity during the receding phase (Figure 4.12, pg. 123). The GnRH output of the hypothalamus presumably decreases during the reced- ing phase. The melatonin-forming activi- ty of the pineal gland increases in tem— poral association with decreasing daylength after the fall equinox (pg. 118). Much study is needed to further charac— terize the mechanisms associated With the receding phase. Anovulatory Season 141 25 20 -1tin‘-- _ _ _ 15 1O ‘}...__-_-__ -m— FIGURE 5.5. Fluctuations throughout the year in plasma concentrations of LH and FSH in four indi— vidual pony mares. Plasma samples were collected every three days. Arrows indicate days of ovulation. From ( 5 75). 5.3. Inactive Phase 5.3A. Follicles and Tubular Genitalia The dynamics of the follicular popula- tion (e.g., turnover rate of individual folli- cles) during the inactive phase have not been elucidated. During this time, ovari- an follicles usually do not develop above 10 or 15 mm. Seasonal effects on follicular develop- ment have been studied by serial section- ing of ovaries and by counting and cate— gorizing the follicles (447). Three stages were considered: 1) anestrus (February; corresponds to inactive phase), 2) early ovulatory season (May 15), and 3) late ovulatory season (August 15). This investigation, unlike others, considered 142 Chapter 5 follicles that were <2 mm. There was a difference among stages in the movement of follicles out of the primordial pool. In the preovulatory stages early in the ovu- latory season, the mitotic index (a mea- sure of the extent of cell division) was higher for the preantral follicles, leading to an accumulation of antral follicles (Figure 5.6). The anestrus and late ovula- tory seasons did not differ from one another, except for the absence of large (>15 mm) follicles during anestrus. The only stage of folliculogenesis lacking dur- ing the inactive phase was growth of large follicles. Therefore, the term “inac- tive ovaries” may be appropriate only in that follicles do not attain large size (e.g., >10 mm). Perhaps small follicles during the inactive phase develop and regress without hormonal stimulus. The authors commented that such a situation has been shown in ewes. The similarities in follicular histology between anestrus and the late ovulatory season occurred despite considerable expected differences in hor— monal milieu. Further studies of this type are needed in mares, with emphasis on the various phases of the anovulatory season, as well as the ovulatory season. Ovaries taken from two mares during the anovulatory season are shown in Figure 5.7 . Certain aspects of the macro- scopic and microscopic anatomy of the Activity of small follicles .75 Early breeding i season \ . in o Anestrus _.-'——>,' Mitotic index [0 01 Late breeding season .00 50-80 120-190 300-1100 80-120 190-300 Diameter (pm) of follicle FIGURE 5.6. Mean (iSD) values for mitotic index for various follicle-size categories. Note that activity is reduced during the inactive phase (anestrus) and late in the breeding season. Adapted from Driancourt et al. (447). reproductive tract of mares during the inactive and resurging phases have been described by van Niekerk and co-workers (1671) in South Africa. The study was made during July to October which corre- sponds to January to April in the Northern Hemisphere. The salient fea- tures of the organs of the reproductive tract for three classifications of season- ally anovulatory mares are summarized in Table 5.1. The overall picture during nummnigiiriginqnninigungmsgnugr 1 3 5 2 4 FIGURE 5.7. Ovaries taken from mares during the anovulatory season (February). Note the difference in fol- licular status between the two sets of ovaries. Anovulatory Season 143 TABLE 5.1. Characteristics of Uterus and Vagina for Three Reported Classifications of Seasonally Anovulatory Mares Deep Shallow Prolonged Organ anestrus anestrus estrus UTERUS Characteristics Thin, atrophic, toneless, Thin, flabby, walls can Flaccid, easily per rectum difficult to discern be rubbed together distinguished Characteristics Pale, dry; only a few thin Pale, but glistening; folds Moist, glistening; folds at slaughter longitudinal folds; thickness, 2 mm thicker with small 20 folds; thickness, 3 mm more voluminous; thickness, 5 mm Luminal Cuboidal (6 um); small Low columnar (10 um); Tall columnar (20 um); epithelium nuclei and scant scant cytoplasm, but much cytoplasm and cytoplasm larger nuclei large nuclei Uterine Small diameter (32 um), Greater diameter (52 um) Further increases in glands few in number (15/19 mm2) and numbers (25/19 diameter (65 um) and and confined to a narrow mm2) and involving a number (38/19 mm2), zone (0.2 mm); cells short wider zone (0.5 mm); width of zone (0.9 mm), (5 pm) with scant cytoplasm cells taller (10 um) and height of cells and dark nuclei (inactive) with more cytoplasm (13 um) VAGINA Characteristics Pale, dry walls stick Similar, but not as Moist, glistening, pale at slaughter together sticky pink (increased vascularity) Epithelium <— Nonkeratinized; uniform thickness —> Slightly keratinized; (32 to 39 um) and consisting of about 5 layers of polyhedral cells; round nuclei irregular thickness (65 pm) with papillae jutting into stroma; oval; more crowded nuclei; more leucocytes in stroma Deep anestrus: small, bean-shaped ovaries with follicles <5 mm; Shallow anestrus: follicles 5 to 30 mm, but no estrus; Prolonged estrus: follicles 5 to 30 mm and in prolonged estrus (28 to 63 days). Adapted from textual data (1671). the inactive stage suggested minimal estrogen production. The ovaries were small (2.4 x 1.6 X 1.6 cm; 17.5 g/ovary), firm, and kidney shaped. The follicles were <5 mm, by definition, and most were undergoing atresia. The ovarian surface was smooth or contained small, firm stro- mal nodules. Such nodules also have been noted by other workers (190). The small palpable nodules apparently are caused by proliferation and dense packing of fibroblasts in the thecal stroma surround- ing small atretic follicles. Inactivity of the tubular organs is indi- cated by dryness (minimal secretion) and paleness (minimal vascularity). Histologic indications of inactivity of the epithelial lining of uterus, uterine glands, and cervix are the lOW cuboidal cells with scanty cytoplasm and dark-staining nuclei (Figures 5.8 and 6.18). The stro- ma is dense, indicating minimal edema. The endometrial glands may appear long and straight due to minimal branching and atrophy. On endoscopic inspection, the folds of the cervix and endometrium are nearly indistinguishable or thin (Figure 6.19, pg. 205). Ultrasonically, cross-sections of uterine horns may be flat or irregular because the flaccid uterus is easily distorted by other abdominal viscera (590). Minimal stratifi- cation of the vaginal epithelium also indicates minimal activity (Figure 5.8). 144 Chapter 5 ENDOMETRIUM ""\ VAGIA FIGURE 5.8. Photomicrographs of endometrium, cervix, and vagina from pony mares during the anovula- tory season. Specimens were taken during March (Northern Hemisphere), and mares were in shallow anestrus, as defined in Table 5.1. Minimal activity is indicated by the low cuboidal cells with scant cytoplasm and dark-staining nuclei in the epithelium of uterus, uterine glands, and cervix, by the small size and num- bers of uterine glands, and by the minimal keratinization of the surface cells of the vaginal epithelium. 5.33. Endocrinology of the Inactive Phase Steroids. Concentrations of circulating estrogens and progestins during the anovulatory season have received only limited attention. Michigan workers (1194) found that peripheral estradiol and pro- gesterone were minimal during the anovulatory season. Concentrations of estrogens in the urine have been studied in seasonally anovulatory mares (730). Seven mares were described as being in deep anestrus characterized by firm inactive ovaries, pale dry vagina, relaxed dry cervix, and a flaccid uterus (equiva- lent to inactive phase). Urinary estrone and estradiol were minimal. The preg- nane-3,20-diols have been measured col- orimetrically in the urine of mares during January and February (418). Concentra- tions were consistently high. Similar high levels also were found after ovariectomy of anestrous mares. It was concluded that the urinary pregnanediols were derived from a precursor whose source was extra- ovarian, probably the adrenal cortex. LH concentrations. Mean circulating LH levels are minimal during the inactive phase, comparable to the low levels of mid-diestrus (554). As described in Chapter 4 (pg. 120), exogenous GnRH will cause LH and FSH release during the anovulatory season. Exogenous estradiol caused an increase in LH plasma concen- trations in ovariectomized mares during the anovulatory season, whereas proges- terone and a combination of estradiol and progesterone had no effect (555). The LH stimulatory effects of exogenous estro- gens during the Winter, however, were less profound than during the summer. FSH concentrations. In a study in 11 ponies, the mean output of FSH (monthly mean concentration in plasma samples taken every 3 days) was not dif- ferent throughout the year (Figure 4.12, pg. 123); however, mares with a greater level of follicular activity during the inac- tive phase had higher levels of FSH Anovulatory Season 145 (pg. 150). Apparently, the circulating levels of FSH during the inactive phase repre- sent the suppressive effects of season even though pituitary output, as repre- sented by monthly means, does not increase during the ovulatory season. This enigma is discussed elsewhere (pg. 122 and pg. 150). Briefly, it is postulated that the negative effects of the ovaries override the positive effects of season dur— ing the summer, thereby resulting in a constant monthly mean output. Within this balance between seasonal and ovari- an influences, marked fluctuation in FSH levels occur reflecting the varying extent of negative ovarian influences on a given day. Also, based on inspection of daily FSH profiles for a few ponies, consider— able fluctuation occurs during all months (575), and this also may contribute to fail- ure to find differences among months. As shown in Figure 4.11 (pg. 123), FSH is at minimal level during January and February in ovariectomized mares under natural daylength. In this regard, ovariec- tomized mares exhibited fewer peaks of FSH concentrations in winter than in the summer (1602). The peaks in winter were, however, larger and longer in duration. Considerable study Will be needed to unravel the complexities of seasonal- FSH relationships during the inactive phase, as well as for the other anovu- latory phases. Pulsatility of FSH, follicu- lar dynamics, and FSH microheterogene- ity are some of the aspects that need to be incorporated into a research thrust. As a further complication, FSH control mecha- nisms may exist that do not involve GnRH (pg. 154). Melatonin. Melatonin-forming activity reaches its zenith during the inactive phase but drops off rapidly beginning in February (Figure 4.8, pg. 118). This high pineal activity is temporally associated with quiescence of the hypothalamic- pituitary-ovarian axis. Further discussion of photoperiod (pg. 113) and pineal involve- ment (pg. 118) in seasonality is given on the indicated pages. 146 Chapter 5 5.4. Resurging Phase 5.4A. Follicular Dynamics Overview. During early resurgence (e.g., March), the number of follicles >20 mm increases. Early resurgence seems equivalent to the frequently used terms shallow anestrus and early transitional period. During this time, the ovaries are breaking out of the inactive phase in preparation for transition to the ovulato- ry season. The ovaries during the early resurging phase have been described as much larger (3.5 x 2.9 x 2.6 cm; 37.8 g/ovary) than those during the inactive period (1671). The ovaries felt knobbly and usually contained several developing and atretic follicles as large as 3 cm diameter. The gross and histologic picture of the tubular genitalia indicated an increase in activity (Table 5.1), suggesting an increase in estrogen production. A transrectal palpation study (575) indi- cated that, on the average, the number of smaller (<20 mm) follicles began to recede in the last half of March, and a rapid increase began in the number of large fol- licles and in the diameter of the largest follicle. During the late resurging phase (spring transitional period), follicles reached preovulatory diameter (>35 mm). However, the follicular pool is dynamic; individual follicles are growing or regress- ing. In many mares, large dominant folli- cles continue to emerge and regress until one is ultimately favored to be the ovula- tory follicle. Early versus late onset of the ovulatory season. Data on follicular changes preced- ing the first ovulation of the year were obtained by transrectal palpation and normalized according to day of first ovula- tion (1644). Comparisons were made between the seven mares that ovulated first (mean date of ovulation: May 2) and the seven that ovulated last (mean date: May 25). A prolonged period (approxi- mately 30 days) of considerable follicular activity preceded the first ovulation. During this time, the early ovulators had more large follicles than the late ovula- tors, whereas the late ovulators had more small follicles. A preovulatory reduction in number of follicles occurred after Day -9 and involved primarily large follicles in the early ovulators and small follicles in the late ovulators. Length of estrus associated with the first ovulation was longer for the early ovulators (mean: 14 days) than for the late ovulators (mean: 4 days). These data indicate that growth of large follicles during the resurging phase is most prominent for the earliest ovulators. Monitoring individual follicles. In a recent study, individual follicles were monitored by ultrasonography in 13 horse mares during the resurging phase (598). Seven mares developed 1 to 3 anovulatory follicular waves, each characterized by a dominant follicle (maximum diameter: 238 mm); a few individually identified subordinate follicles were apparent in some waves. The distinction between sub- ordinate and dominant follicles was made retrospectively after the growth and regression profile of an individual follicle was known. The day of emergence of a wave refers to the first day of ultrasonic detection of the follicles of a wave and was also determined retrospectively. The dominant follicle had growing, static, and regressing phases. The subordinate folli- cles seemed to regress a few days after retrospective identification of the emer- gence of a wave (Figure 5.9). The emer- gence of a subsequent wave (anovulatory or ovulatory) did not occur until the domi- nant follicle of the previous wave was in the static phase. After the emergence of the subsequent wave, the previous domi— nant follicle regressed. Length of the interval between emergence of successive waves was 10.8 i2.2 days (mean iSD). Before the emergence of waves (identified by a dominant follicle), follicular activity seemed erratic and follicles did not reach >35 mm. In the remaining six mares, fol- licular activity remained erratic (no obvi- ous pattern) throughout the resurging Anovulatory Season 147 Individual follicles during resurgence 50 40 30 20 10 50 40 30 20 Diameter (mm) 10 U W—§_ W-s- m-a—s- s IQ - 3% 50 4o x 30 20 10 a— _s— Bis ma s Wlmmw U Im_§l §\\V\§_§§ -m W m § -10 Number of days from ovulation -30 -20 0 -50 FIGURE 5.9. Daily diameters of individual follicles during resurgence in six mares. In Mares A, C, E, the ovulatory follicle was preceded by growth and regression of large anovulatory follicles, whereas in Mares B, D, F the first large follicle (>38 mm) became the ovulatory follicle. Bar U = uterine ultrasonic echotexture. Bar B = estrous behavior scores (black bars, estrus-like; white bars, nonestrus-like; and shaded bars, inter- mediate). Adapted from (598). phase, with the absence of distinct anovu- latory waves (large dominant follicles; Figure 5.9); the first dominant follicle to develop was the ovulatory follicle. Thus, about 50% of the mares had prominent transitional periods with distinct anovu- latory follicular waves, whereas the remaining mares did not. COmpared to the end of the first interovulatory inter- val, the ovulatory follicle at the end of the resurging phase reached 20 mm earlier (Day -10 versus Day -15), grew more slowly (2.6 versus 3.6 mm/day), but reached a greater diameter at Day -1 (44 versus 51 mm; Figure 5.10). The interval from cessation of growth of the largest subordinate follicle to the occur- rence of ovulation was longer for the end of the resurging phase (mean: 9.5 days) than for the end of the first interovulato- ry interval (mean: 6.8 days). The ultrasonically obtained growth and regression profiles of individual follicles for the resurging phase seem consistent with 148 Chapter 5 Comparison of 1st & 2nd ovulatory follicles 50 .b O ist ovulation (n=16) i l 2nd ovulation (n=9) Diameter (mm) 00 O N O 10 -20 -15 -10 -5 0 Number of days from ovulation FIGURE 5.10. Sequential diameters of follicle that resulted in the first ovulation of the year (end of transitional period) and the follicle that resulted in the second ovulation (end of first interovulatory interval). The follicle involved in the first ovulation was identified sooner before ovulation, grew at a slower rate, and reached a greater diameter on Day —1 than the follicle involved in the second ovulation (P<0.05 for each end point). Adapted from (598). individual profiles obtained previously by transrectal palpation (575); the studies dif— fer primarily in the greater detail obtained by ultrasonography. On the basis of these findings and comparative studies during the interovulatory interval in cattle (612), the following overall work— ing hypothesis is suggested: During resurgence, follicles grow and regress until one reaches a critically large size (e.g., 40 mm). The large follicle either ovulates or becomes part of an anovulato- ry follicular wave. Such anovulatory waves develop sequentially until condi- tions are right for ovulation. The domi- nant follicle of each wave contributes to the regression of its subordinates and perhaps is involved in preventing the emergence of another wave until the dom— inant follicle reaches a static phase (anovulatory wave) or approaches ovula- tion (ovulatory wave). 5.4B. Endocrinology of Resurgence Much was learned during the 19805 about the mechanisms involved in the profound increase in follicular activity during resurgence. Steroids. In the study of urinary estro- gens, mares emerging from the inactive phase began excreting increasing amounts of estrogen in the urine for 16 to 73 days before showing estrus (730). Two mares were described as being in shallow anestrus (follicles of 15 to 30 mm) for 80 and 157 days, respectively, and excreted an average of 29 and 32 pig/liter of estrone and estradiol, respectively. These values were approximately 10—fold higher than during the inactive phase. Circulating concentrations of estradiol also are mini— mal during the inactive phase and early resurgence (1194). An early, but less pronounced, increase in estradiol probably occurs before the first ovulation, as indicated by the follow- ing: 1) prolonged estrus-like ultrasonic uterine echotexture (Figure 5.11), 2) pro- longed estrus, and 3) the presence of large Uterine echotexture 2.5 (“=23) 2nd Ovulation Score -15 -12 -9 -6 -3 0 3 Number of days from ovulation FIGURE 5.11. Mean ultrasound uterine scores associated with the first and second ovulations of the season. The presence of a star on the horizontal axis indicates a significant difference between groups for that day. Adapted from (6.99). follicles for a prolonged period. Scores for estrus uterine echotexture before the first ovulation of the year rose about five days earlier but did not rise as high as before the second ovulation (Figure 5.11). Assuming that estrus-like uterine echo- texture mirrors circulating estrogen con- centrations (590, 699), a longer but lower estrogen surge occurs in association with the first ovulation. Circulating proges- terone has not been detected in mares during resurgence (1194). LH concentrations. Mean circulating concentrations of LH remain minimal during most of resurgence until the occur- rence of the ovulatory surge beginning a few days before ovulation (540, 509, 1430). In one study (540), follicular diameters Gonadotropins & follicles at end of resurgence (n=10) 12 .l E E 8 C .2 E E 8 g 4 0 I w LI. 0 T 40 7% Follicles15-25 mm | I ....+-~+-v+ E 20 I ..... ”,]|:: “Ind/"l o lfl/l ..... l l'\ -60 -52 -44 -36 -28 -20 -12 Number of days from ovulation Anovulatory Season 149 and LH and FSH concentrations were quantitated every 4 days for 60 days before ovulation. Mean LH values remained consistently low before Day -8 and increased rapidly beginning on Day -4. Diameter of largest follicle and number of large follicles increased pro- gressively. However, during the last 8 days before ovulation, the number of large follicles decreased and diameter of the largest follicle increased rapidly and ovulated. The growth spurt of the preovulatory follicle was temporally asso— ciated with the rise in LH. In this study, the gonadotropin profiles before the first ovulation were not different between con- trol and light-treated mares. In contrast, results of a recent study indicated a more 6 r. I O 4 S O ('D 5’. E. a. 3 2 E i o ‘ ["4 6 1' 5 2 C i 4 3 U 9; FIGURE 5.12. Mean concentra- tions of FSH and LH and follicu- lar changes in pony mares during the transition between anovula— tory and ovulatory seasons. The vertical bars (lsd’s) represent the magnitude of the least significant difference. Adapted from (540). -4 -1 150 Chapter 5 abrupt LH surge in light-treated mares than in controls (509). The gonadotropin changes at the end of resurgence were similar to changes asso- ciated with subsequent ovulations. However, the magnitude of the LH surge was less for the first ovulation than for subsequent ovulations (Figure 5.13). This observation has been confirmed (509). The lower LH surge at the first ovulation of the year suggests that at the time of the Gonadotropins at onset versus middle of season (n=10/group) 14 FSH 10 Concentration (ng/ml) -28 -20 -12 -4 -1 1 4 Number of days from ovulation FIGURE 5.13. Comparison of LH and FSH curves between onset (first ovulation) and middle of the breeding season in ponies. Means with no common superscript letters are significantly different. Adapted from (540). first ovulation maximum photoperiod effects have not yet been reached. That is, the hypothalamic-pituitary axis is not yet completely free of pineal inhibition. In addition, the lower LH surge is compati- ble with lower circulating estrogen in association with the first ovulation (pg. 148). A recent study (1480) found no indi- cation that the attenuated LH surge associated with the first ovulation reflects the absence of a preceding diestrus period (absence of progesterone priming). Furthermore, the profile of the LH surge associated with the first ovula- tion was associated with time of year rather than ovulation sequence; postpar- tum LH surges for mares foaling in April versus July reflected the time of year that the surge occurred. FSH concentrations. Despite increased follicular development, a corresponding change in concentrations of circulating FSH during early resurgence was not detected when FSH was measured every three days in 11 mares (1644). However, the mean level of FSH was higher dur- ing January to March in mares classi- fied as having a higher level of follicular activity (Figure 5.14; 1644). These results and others (731) indicate that FSH plays a role in follicular develop- ment, but factors other than a simple increase in circulating concentrations of FSH are involved in follicular develop— ment during the resurging phase. Preliminary trials in two mares (600) suggested that each dominant follicle, whether anovulatory or ovulatory, was associated with a surge of FSH (Figure 5.15); the FSH surge peaked at the time the dominant follicle was only about 20 mm, and the FSH surge declined soon thereafter. These preliminary findings indicate that the association between follicular waves and FSH surges in mares needs intensive study. Mean FSH values fluctuated prior to Day -20 and then steadily declined to the low levels characteristic of estrus (540). The decline in FSH began an average of 16 days Anovulatory Season 151 FSH &fo||icles before ovulation and appeared to pre— cede the decline in number of large folli- cles. Higher FSH levels in early resur- 10 E ngh follicular gence compared to late resurgence have ‘2, /aCiiVity (”=7) been confirmed (731, 24). 7; 8 The selection of the ovulatory follicle .2 seems associated with the decline in g 6 L f ”_ I FSH. Decline in FSH is followed by a g agilivvit‘; 231:2; decline in number of large follicles and a 8 __/ rapid increase in LH during the final 4 ----------- V“ growth of the selected follicle. The inter- val from the preovulatory decrease in FSH to the occurrence of ovulation is F°"i°'es more prolonged for the first ovulation than for the ovulation at the end of the first interovulatory interval (Figure 5.13); by the same token, it takes longer for the first ovulatory follicle to grow, and it ulti- mately reaches a greater size than does the next ovulatory follicle (pg. 147; 598). Pulsatility of the gonadotropins. The increasing mean levels of LH in ovariec- tomized mares during the spring (March N Hem Jan Feb Mar to May) was accomplished by increasing pulse frequencies (512); pulses were char- FIGURE 5.14. FSH and follicular changes during acterized by sampling eVery 15 minutes January-March in mares classified as having a high for 8 hours each day. In general, pulse or low level of follicular activity in January. Number Adapted from (1644). Dominant follicles & FSH in individual mares 10 Dominant follicle 50 Anovulatory Ovulatory h o co 0 (LULU) Jelawela Concentration (ng/ml) 20 10 -24 -20 -16 -12 -8 -4 0 -24 -20 -16 -12 -8 -4 0 Number of days from ovulation FIGURE 5.15. Associations in two mares between apparent FSH surges and the first ultrasonic detection of the emergence of a dominant follicle (anovulatory and ovulatory) prior to the first ovulation of the year (OV). Follicular data from (598); FSH levels determined by D. R. Bergfelt. 152 Chapter 5 frequency was lowest when mean LH con- centrations were low and gradually increased as the daily mean levels increased (Figure 5.16). The first indica- tions of LH pulsation did not occur until late March or early April when the mean daily levels were beginning to increase. The pulses increased from about 1 per 8 hours in early April to about 4 per 8 hours in May. Pulses continued to increase in frequency and became so rapid that accu— rate characterization could not be done, even with samples collected as often as every five minutes. The phenomenon of pulsatility also has been demonstrated in ovarian-intact mares during resurgence (509). The frequency of pulsatile LH secretion increased during resurgence. Inspection of the tabulated data indicates that pulse frequency was equivalent to approximate- ly one per day during the inactive phase and approximately six per day during the transitional period. Pulse amplitude decreased as pulse frequency increased, apparently accounting for a failure to detect increases in mean daily composite samples. During the 60 days before ovulation, blood samples collected daily LH pulses (n=5) _L O OD Concentration 0') A '.—--.‘ I], \ ~ ~.’ Pulse flequency Number of pulses/8 hr or concentration (ng/ ml) N ’L. Feb Mar Apr May FIGURE 5.16. Changes in mean serum LH concen- trations and mean LH pulse frequency in ovariec— tomized mares between February and May (38°N). Adapted from Fitzgerald et al. ( 512). appeared to provide a reliable estimate of circulating LH concentrations. That is, values in daily samples were similar to the mean values of frequent-sample composites. However, pulse frequency increased fivefold from 60 to 11 days before ovulation even though daily means did not. Similarly, pulse frequen- cy in a recent study (24) ranged from one pulse per day when ovaries were inac- tive to one pulse per hour when periovu- latory follicles were present. Another recent study on LH levels and pulses during resurgence in intact and ovariec— tomized mares is currently available only in abstract form (8). Pulses of LH have been reported to occur simultaneously with those of FSH during the seasonal transition (731). In the preliminary data shown in Figure 5.15, apparent pulses of FSH sometimes occurred in synchrony with LH pulses. Apparently such synchrony diminishes as ovulation approaches, resulting in high mean levels of LH but minimal levels of FSH. In a recent study (731), FSH pulse amplitude decreased with the onset of the ovulatory season in association with a decrease in the mean daily concentrations. . Administration of xylazine, an ocz-adrener- gic agonist, during the anovulatory season increased the pulse frequency of LH and FSH (513). The results were interpreted as an indication that the seasonal suppres- sion of gonadotropin pulses involves inhibitory neural mechanisms. Inciden- tally, a recent study (351) failed to find an effect of a single injection of xylazine on length of the interovulatory interval or concentrations of circulating progesterone. Pulsatile release of GnRH. Many lines of research have implicated GnRH in the return to the ovulatory season and are reviewed elsewhere (pg. 120). Release of GnRH from the hypothalamus occurs in pulses (pg. 53) and the patterns have been characterized in the venous system of the equine pituitary (22). Pulses of GnRH and LH occurred in harmony. During the inac- tive phase, pulses of LH were not measur- able; the pulse frequency during the luteal phase was about 1 pulse/10 hours and increased to about 1 pulse/hour during estrus. The exogenous delivery of 5-second GnRH pulses at a rate of 1 pulse/hour (832) apparently mimicked the pattern of endogenous pulses. The pulse delivery experiments of Johnson seemed to have produced more consistent results than the non-pulse delivery systems used by others, especially for mares with small fol— licles at the start of treatment (for further discussion of pulsatile versus constant delivery see pg. 169). Very low doses (e.g., Effect of GnRH on gonadotropins 90 l FSH .. 5‘ """ ‘ } 20 pg GnRH/ hr l .. 1 / 11:7) _. 2ngnRH/hr / ---- I so 1 ' ‘1'] 11 1;" "l. [IJL 30 I E Tm Control 5 (n=4) : 10 .9 g 12 C 8 S 10 O 0 2 4 6 8 10 12 Day of treatment Anovulatory Season 153 3.3 pig/hour; 22) induced an LH response similar to endogenous LH pulses. Gonadotropin profiles from GnRH treatment. An exogenous pulse delivery system involving 2 or 20 ug/hour resulted in normal-appearing gonado- tropin profiles (829). The LH, FSH, and estradiol profiles were similar to what occurs naturally before the first ovula- tion of the year (Figure 5.17); subsequent progesterone profiles seemed normal (829, 69, 797). It has been noted that LH con- centrations during GnRH treatment often followed a bimodal profile with peak concentrations occur- ring approximately 15 and 5 days before ovulation (686, 797). Mares that failed to ovulate did not show the reciprocal relationship be- tween FSH and LH that char- acterizes the first ovulation of the year (decrease in FSH and increase in LH before ovulation). In recent studies (pg. 168), administration of a GnRH analogue every 12 hours dur- ing the inactive phase result- ed in ovulation but with inadequate LH levels and diminished follicular and luteal development during the ensuing pregnancy. The inadequate LH output may have been related to time of year at the start of treat- ment or to differences in the treatment protocol and prod— uct. Clearly, further studies are needed, especially for GnRH stimulation when the ovaries are inactive. FIGURE 5.17. Effect of doses of GnRH in hourly pulses on concen- trations of LH and FSH in season- ally anovulatory mares. Adapted from Johnson (826). 154 Chapter 5 Differential effects of GnRH on LH and FSH. The release of LH and FSH some- times appears to be coupled, and at other times, release of the two gonadotropins appears to be independent. These associa- tions and disassociations occur even in the absence of ovaries. For example, both gonadotropins followed a seasonal pat- tern in ovariectomized mares, but signifi- cant increases in FSH in the spring pre- ceded significant increases in LH (Figures 4.14 and 4.15). Administration of GnRH during the anovulatory season caused an increase in both LH and FSH, indicating association, but after approximately a week they became dissociated; FSH decreased and LH did not (Figure 5.13). As another example, treatment of ovariectomized mares with testosterone propionate resulted in an increase in FSH without an effect on LH (1324), and treatment with estradiol resulted in an increase in LH but not FSH (552). Circulating concentrations of LH, but not FSH, decreased drastically during the anovulatory season in ovarian—intact mares (1644); in ovariectomized mares both gonadotropins decreased (540). Furthermore, the ratio of concentrations of the two gonadotropins changed consid- erably during resurgence. Concentra- tions of FSH reach high levels during resurgence but may fluctuate during the transitional period. On the other hand, LH does not increase until ovulation approaches and a decrease in FSH has occurred. An additional indication of dichotomy of response occurs when GnRH is administered during the anovulatory season; FSH is more responsive than LH (3.7 -fold versus 24—fold increases; 492). Colorado workers reported that the amount of LH in pituitaries in winter was only 15% of the amount found in summer (690). In a recent experiment (1140), mares were challenged with a single injection of GnRH every three weeks beginning in January. The mares did not release LH in response to the GnRH challenge until April, indicating continuing low pituitary content of LH. In contrast, pituitary con— tent of FSH did not differ due to season (690). This result is compatible with the greater response of FSH than LH to exogenous GnRH in the winter (557, 492). The low pituitary LH content in winter was temporally related to low hypothala- mic GnRH content (690, 1482). These work- ers postulated that the LH response to exogenous GnRH during resurgence is limited by pituitary LH content. The release of FSH is, however, more com- plex. During the winter, FSH secretion is low, probably due in part to decreased GnRH. An injection of GnRH releases much FSH because the pituitary content of FSH and the number of GnRH recep— tors remain high, even in winter. During the resurging phase, GnRH increases and therefore FSH surges and the ovarian fol- licular diameters also increase. An alternate control mechanism also may be involved in the differential LH and FSH responses to GnRH. Thompson and associates used GnRH immunization studies to test the hypothesis that some FSH dynamics do not depend on GnRH (557). Mares immunized against GnRH failed to return to a cyclic condition. Concentrations of circulating LH were suppressed to undetectable levels, but FSH concentrations were only partly sup- pressed. The LH secretion and production were entirely dependent on GnRH, whereas GnRH affected only some of the FSH secretion. Furthermore, FSH stor- age and perhaps production were rela— tively independent of GnRH. These results and interpretations could at least partly account for the greater depression of LH than FSH circulating concentra- tions during the inactive period (1644, 1596}. Gonadotropin administration. Daily administration of small doses of hCG (200 in) or eLH induced ovulation when given after follicular development had begun (230). When such treatments were given to ponies in March (early in resurging phase; largest follicles, 20 mm), more mares ovulated (7 of 8 in a mean of 9 days) than when treatment was initiated in February (inactive phase; largest folli- cle <15 mm; 1 of 7 ovulated). In another experiment, a combination of eFSH and eLH induced ovulation in February when follicles were small (9 of 10 ovulated in a mean of 9 days). These important studies showed the dependence of initial follicu- lar growth on FSH and showed that LH, alone, was adequate to induce ovulation when large follicles (>20 mm) were pre- sent. Incidentally, mares unsuccessfully treated with hCG in February developed antibodies and subsequently showed higher LH levels than control mares in association with the first ovulation; apparently, part of the LH response was tied up by the antibodies, thus requiring more LH for induction of ovulation. 5.4C. Late Resurging Phase Associations between follicular and hormonal activity. During successive waves of follicles in the spring transi— tional period, each wave, characterized by a large dominant follicle (>35 mm), devel- ops in some mares at a mean interval of approximately 10 days between emer- gence of waves (pg. 146). Perhaps large fol- licles produce substances that have a reg- ulatory role in follicular periodicity; smaller follicles do not produce adequate quantities of such substances, and wave— like patterns are not obvious. The sup- pressing substances may be inhibin and estrogen. Results of a recent in vitro study (379, 378) suggest that the large folli- cles (>30 mm) that initially develop are less competent in the production of estra- diol. A proteinaceous fraction of follicular fluid (free of steroids and likely contain- ing inhibin) suppressed circulating FSH and follicular development in mares (181), and the proteinaceous fraction combined with estradiol had a greater FSH-sup- pressing action in ovariectomized mares than did the proteinaceous fraction alone (1095). Production of estrogen by the large Anovulatory Season 155 follicles was consistent with the estrus- like uterine echotexture that seemed approximately related to the growing phase of large follicles (Figure 5.9) and to the occurrence of behavioral estrus, some- times for greatly prolonged periods. Perhaps during the transitional period the suppressing substances reduce circu— lating FSH while the large follicle is in its growing phase. This assumption is consis- tent with periodic FSH waves seen in individual mares during the resurging phase (575). The decrease in FSH before ovulation apparently does not involve an inhibitory effect exerted at the hypothala— mus since FSH decreases in the face of continued pulsatile administration of GnRH (829). Apparently, the dissociation in the LH-FSH response to GnRH is exerted at the pituitary level and results in diminished secretion of FSH and enhanced secretion of LH. The dissocia- tion is likely a function of substances pro- duced by the preovulatory follicle. Presumably, the reduction in FSH is involved in cessation of growth of a domi- nant follicle (anovulatory wave) or, if ade- quate LH is available, the dominant folli— cle ovulates. Direct comparisons are needed between follicular patterns and circulating concentrations of FSH and LH during resurgence. It is likely that the initial gradual increase in LH toward the end of resur- gence is a function of the photoperiodic- pineal-hypothalamic mechanism. This has been demonstrated by an initial increase in LH in both ovariectomized and ovarian intact mares at a similar time of the year (pg. 121 and pg. 254). However, the final portion of the LH ovu- latory surge is a function of the ovaries, indicated by the elevation above the lev- els that occur during this time in ovariec- tomized mares. The ovarian substance responsible for the final LH surge is likely estrogen from the growing preovu- latory follicle; the positive effect of estrogen on circulating LH has been well documented (pg. 248). 156 Chapter 5 SUMMARY: Endocrinology of the Anovulatory Season Melatonin-forming activity of the pineal increases in temporal association with decreasing daylength after the fall equinox (Figure 5.18). For this reason, at least in part, the pituitary LH content and magnitude of LH ovulatory surges decrease during the latter portion of the ovulatory season. Melatonin exerts its effects through the suppression of GnRH release. The failure of ovulation at the beginning of the anovulatory season occurs because pituitary and circulating levels of LH no longer meet the require- ments for an ovulatory LH surge. Failure of ovulation is not due to a deficiency in FSH or number of follicles available. Pineal melatonin—forming activity reaches its zenith in association with quiescence of the hypothalamic—pituitary- ovarian axis (inactive phase). Concentra- tions of LH are minimal comparable to the low levels during diestrus. These low levels are entirely attributable to season- al pressures, since similar levels occur at the corresponding time in ovariectomized mares. Both LH and FSH follow a distinct sea- sonal profile in ovariectomized mares, with FSH decreasing later than LH in the fall and increasing earlier than LH in the spring. Yet, in the presence of the ovaries, an index of monthly FSH output does not change, whereas the monthly production index of LH follows a profile similar to that of ovariectomized mares. Pituitary stores of LH, but not FSH, are also low during the inactive phase. The contradiction of an overall seasonality profile of FSH in ovariectomized mares, but not in ovarian-intact mares, is explained on the basis of a continuing negative ovarian effect during the ovula~ tory season. The extent of negative ovari~ an pressures apparently varies during the estrous cycle, resulting in FSH surges and pulses. Circulating melatonin concentrations drop rapidly in response to increasing daylength in the spring. This action is , , responsible, at least in part, for the initi- ation of follicular reSurgence. As a result of the seasonal influencecexerted through the pineal and hypothalamus, the LB stores in the pituitary increase slowly and gradually during resurgence and may account for its long length (e.g., 75 days). There are weak indications‘that the pineal also produces a hypothalamic stimulator when daylength increases. ‘ ‘ Prolactin may also play a role in season:- ality, but the nature of its role has not been determined. During late resurgence, follicles become large and competent in the pro- \ duction of FSH inhibitory factors (estro9 gens and inhibin); FSH drops, but an LH surge does not occur because of made quate stores. As a result, the dominant follicle regresses and a new one develops (e.g., every 10 days; a similar phe- nomenon occurs at the beginning of the, anovulatory season). If an estrogen— competent dominant follicle (>35 mm) develops when LH pituitary stores are adequate, the follicle will also enhance circulating levels of LH through estro— gens. The resulting LH surge induces ovulation and thereby ends the anovula- tory season. The magnitude of the LH surge is less for the first ovulation than for subse- quent ovulations, and is a reflection of continuing, although diminishing, sea- sonally controlled suppression and lOW estrogen levels. The regulatory mechanisms controlling the ovarian activity during the anovula- tory season are far «more complex than this simplistic summary indicates. For example, FSH Control mechanisms may exist that do not involve GnRH. It is believed, however, that this working hypothesis capsulizes the current status of knowledge in this important area. Anovulatory Season 157 5< ANOVULATORY SEASON; Stimulant from { pineal? ‘ Prolactin? Melatonin Luteinizing hormone - ~ - — Seasonal influence --- Positive effect of Estrogen —— Negative effect of Progesterone ...............‘.~ \ Follicle stimulating hormone /’ x - — - ~ Monthly output, ovariectomized (I x - - - - ~ - Monthly output, ovarian intact ,’ \ —-——- Daily output, ovarian intact , Anovulatory, major Ovulation : toliicutar waves 2 ~ 10vutatlon 000 000 000 <-— Receding phase -—-> *- inactive phase —> .<-— Resurging phase -—>§ Oct Nov Dec Jan Feb Mar Apr May (Apr) (May) (Jun) (Jul) (Aug) (Sep) (Oct) (NOV) FIGURE 5.18. Follicle and hormonal interrelationships during the anovulatory season. 158 Chapter 5 Late entry. Steroid hormones were given to ovariectomized ponies during the resurgence phase of the anovulatory sea- son, and the ponies were then given an injection of GnRH (1871). Circulating LH levels increased and FSH tended to decrease during estrogen treatment. Estrogen treatment for 10 days reduced the pituitary LH response to GnRH and by 16 days increased the pituitary con- tent of LH, but not FSH. Results con- firmed that estrogen has a positive effect on LH secretion. Results further support- ed the hypothesis that the initial rise in circulating LH is due to photoperiodic stimulation of GnRH secretion, but that estrogen exposure is required for the final ovulatory LH surge. It was suggested that estrogen stimulates LH synthesis rather than enhances the pituitary response to GnRH. 5.5. Applied Techniques for Induction of Ovulation Steadfast adherence to official birth dates (pg. 131) has forced the development of techniques for inducing ovulation dur- ing the anovulatory season or for stimu- lating early onset of the breeding season. Older literature claimed that estrogens and gonadotropic preparations can be used to terminate anestrus. However, these experiments were poorly designed and too unconvincing to warrant recom- mending use of such regimens. Several practical methods have been reported more recently and are summarized below. 5.5A. Use of Artificial Lights A workable and reliable procedure for stimulating early onset of the ovulatory season involves the artificial extension of daylength with lights. As noted earlier, the stimulatory effect of light in mares has been known for decades (pg. 113). Progress in adapting the technique to practical field use has been slow. Approxi- mately 20 years elapsed between the orig— inal demonstration of the stimulatory effects of light in ponies and large-scale field testing in horses. Incremental changes in photoperiod. Gradual increases or incremental changes in photoperiod (natural plus artificial) have been used to approximate the changing natural photoperiod that occurs after the winter solstice. This approach was used in the first document— ed field trials on the practical use of light regimens in horses. As far as the author is aware, the original trials were con- ducted on Thoroughbred and Standard- bred farms in Kentucky by Loy and asso- ciates in 1968 (987). German workers reported on similar field trials the follow— ing year (1083). In the Kentucky trials, artificial light exposure was started on November 27 at 12 hours/day and was increased in increments of 15 to 30 min- utes/week until a maximum of 19 hours was reached on May 6. When compared to pregnancy rates obtained under nor- mal conditions, the lighting regimen seemed to have applicability under field conditions. The results of a lighting regimen that was used for six years in a band of 100 Standardbred mares in the United States have been reported (339). Light treatment began on October 1 at the natural length on that date and was increased at the rate of one-half hour/week. A length of 16 hours was reached on January 16 and was maintained at that level through the remainder of winter. After five years on the program, 50% of the foalings occurred during November to January, as com— pared with 4% at the onset. Kenney and co—workers (867) have made several recommendations for lighting pro— grams involving gradually increasing photoperiods. A ZOO-watt incandescent bulb is recommended for each stall. Timers may be used and set to turn on the lights one-half hour before sunset. The lights are turned off one—half hour after sunset during the first week and progressively one—half hour later during succeeding weeks. The length of photope- riods is increased weekly in increments so that the mares are exposed to 15 to 16 hours of light (artificial plus natural) by the time of the desired beginning of the breeding program. Fixed-length photoperiods. Fixed— length photoperiods (constant number of hours of light per day; natural plus artifi- cial) seem to be as effective as the incre- mental approach (905). Over the years, a number of experiments have been done on ponies in northern United States (43°N). The studies included groups with fixed photoperiods and groups of controls (photoperiod equivalent to natural daylength). Groups for five experiments have been combined into one data set as shown (Table 5.2). The following conclusions seem evident based on inspection: 1. Fifteen or 16 hours were equally effective; 2. The best responses were obtained when the light programs began by December 5, but no advantage was gained by beginning the program earlier; 3. The most effective programs resulted in an overall mean day of first ovulation of March 2 compared to May 16 in controls (75-day advantage); 4. Beginning the program after December 5 (December 16 to January 6) resulted in a progressively decreasing advantage—mares first treated on January 1 did not ovulate until a mean of April 8; and 5. Starting the program in mid-sum- mer (July 1 and August 1) resulted in an interval from January 1 to mean first day of ovulation that was intermediate between the controls and those of the most effective starting dates. Other studies have shown that the most effective length of the fixed photope- riod is 14.5 to 16 hours (1211, 1011). A 13- hour fixed period was less effective (1211). To allow for clock-setting errors, it is rec- ommended that 15 hours of light be used for fixed programs. Anovulatory Season 159 TABLE 5.2. Effect of Beginning a Fixed Photoperiod at Various Times Light treatment No. days No. No. from Jan 1 to Start hrs. mares first ovulation Jul 1 15.5 8 94b (Apr 4) Aug 1 15.5 9 94b (Apr 4) Nov 1 15.5 8 66c (Mar 7) Nov 13 16.0 9 59 (Feb 28) Dec 1 15.5 4 65c (Mar 6) Dec 5 15.0 7 54 (Feb 23) Dec 16 15.0 7 70 (Mar 11) Dec 21 15.0 5 75 (Mar 16) Jan 1 15.5 7 98b (Apr 8) Jan 6 16.0 5 87 (Mar 28) Groups are from five experiments in ponies at 43°N. Groups with superscripts were in a single experi- ment, and the control averaged 132a days; means with a different letter are significantly different. Means for controls in various experiments ranged from 126 to 144 days (May 5 to 24). Adapted from (1412, 905, 540). Light intensity. There has been con— siderable range in the type and intensity of artificial lighting schemes used by various investigators. Specific informa- tion on types of light bulbs, foot candles, and size of rooms in the various studies was tabulated in the first edition (575). One 200-watt incandescent bulb per stall was used in several studies. Outdoor light- ing with quartz lamps also has been used successfully (1412). Light intensity used in various studies ranged from 7 to 45 foot candles. According to Florida workers (1429), a 35 mm camera with light reading capacity can be used to estimate light ‘ intensity. The ASA is set at 400 and the shutter speed at 1/4 second. The camera is held at horse—eye level with a Styro- foam cup over the lens to diffuse the light. If the camera’s meter indicates an aperture (f—stop) of 5.6 or higher, the light intensity approximates 12 foot-candles or greater. Untested observations indicate that shadowing can interfere with the ovarian stimulatory response by decreas- 160 Chapter 5 ing the light intensity reaching the horse’s eyes (600). Examples of interference with light intensity by shadowing include bar— riers (e.g., feed bunks), inadequate light distribution (some mares not facing light source), and too many mares in a corral for light treatment (individuals may stand in the shadow of others). Duration of photoperiod. A preliminary experiment has been done (1412) in ponies on the number of weeks required for ovar- ian stimulation, using a 15.5-hour fixed program beginning December 5 (optimal time). Mares received light treatment for 2, 4, 6, 8, 10, 12, or 14 weeks (n=8 or 9 per group). At the end of treatment, mares were returned to natural daylength. The end point was number of mares developing a follicle >30 mm or an apparent ovulation by April 10. None of the mares in the control group or in the groups treated with light for 2 or 4 weeks developed a 30 mm follicle or ovulated by April 10, compared with 56% to 88% in the groups treated for 6 to 14 weeks. Thus, six weeks was the required mini- mal length of lighting program in this ini- tial study. Alternate light programs. An experi- ment in Florida (1435) involved giving 2.5 hours of light before sunrise, after sunset, or both. Light exposure in the evening advanced the ovulatory season, whereas light exposure in the morning did not (Table 5.3). However, the evening treatment alone seemed to produce a result that was intermediate between the results for the controls and a group treated for 2.5 hours in both morning and evening; the difference in response between controls and the evening group and the difference between the evening group and the evening/morning group were both 23 days. In this regard, the interval to ovulation for the evening/morning group (63 days) was equivalent to the maximal response obtained with fixed treatments by other workers (Table 5.4). The use of a fixed light period imposed at sunrise is equiva- lent to an incremental program since natural daylength is increasing. For field use, the operator should consider that the 2.5—hour evening program is effective, but it has not been demonstrated, convincing- ly, that it is as effective as a 15-hour fixed photoperiod. Many types of light programs have been tested and most have produced intermediate responses—better than the controls, but not as effective (often signifi- cantly different) as 15 fixed hours of light (Table 5.4). Included are programs where- in an hour of light was given during natu- ral dark and resulted in an intermediate response. It appears that mares are toler— ant in response to light manipulations and will partly respond even when an otherwise highly efficient program (15 hours of light) is used only every other day. Postpartum mares. Pregnant and post— partum mares should be considered in lighting programs. Mares foaling during the winter (anovulatory season for non— pregnant mares) may have a postpartum ovulation and then come under seasonal influence (1210). Foaling apparently has a stimulating effect, which sometimes results in a postpartum ovulation during the anovulatory season, but because of seasonal pressures, cyclicity does not ensue (pg. 477). A similar phenomenon occurs when mares in the inactive phase are induced to ovulate with GnRH or pituitary extracts (pg. 168). A surge of FSH TABLE 5.3. Effect of 2.5 Hours of Light Before Sunrise (AM) or After Sunset (PM) on Onset of the Ovulatory Season in Florida No. days Light No. from Jan 1 to treatment mares first ovulation Natural 7 109 $8 (Apr 19) 2.5 hr AM 7 109 :12 (Apr 19) 2.5 hr PM 7 86 i7 (Mar 27) 2.5 hr AM/PM 7 63 i8 (Mar 4) Adapted from Sharp et al. (1435). TABLE 5.4. Effect of Various Light Regimens on Mean Onset of Ovulatory Season in Ponies in Wisconsin Light:Dark regimen N o. N 0. days from Jan 1 Anovulatory Season 161 (N 0. hours of each) mares to first ovulation Experiment 1 Control 7 142a i6 (May 22) 15L:9D every day 7 54C i5 (Feb 23) 15L:9D every other day 8 85Cd i7 (Mar 26) 7.5L:4.5D:7.5L:4.5D 8 760d i7 (Mar 17) 15L23D23L23D 9 71Cd ill (Mar 12) Experiments were done in 6L:3D:6L:9D 7 67Cd :10 (Mar 8) ponies at 43ON. Light treat- 6L:3D:6L:3D:3L:3D 7 98bd 4:13 (Apr 8) mentébegan 0“ December 5 Control plus one hr light 15 hrs (2E11X(p]§r1ment D1; 23d A1218 cember after lights were turned on 8 141a i6 (May 21) Xperimen ' . . groups 1 . were kept under art1fi01al light (Jontrol. plus one hr hght 9'5 hrs b with abrupt transitions between after lights were turned off 9 117 $6 (Apr 27) light and dark. Control photope- E _ riods were adjusted weekly to W a simulate changing daylength. Control 7 12610 i9 (May 5) Within experiments, means 15L:9D 5 75 i7 (Mar 15) with no common superscripts Control plus one hr of light are different (P<0.05). Adapted fixed at 4 AM 5 85b i8 (Mar 25) from (1412). in association with parturition has been demonstrated in mares (1645). Other mares may not have a postpartum ovula- tion and may immediately respond to daylength similar to barren mares. The use of a lighting program has been recom- mended for mares foaling before April 15 (1210), as well as for barren and maiden mares. The interactions of daylength with the length of pregnancy (pg. 329), and the postpartum period (pg. 477) are discussed on the indicated pages. 5.5B. Stabling Apparently the ovulatory season com- mences sooner when mares are stabled especially at higher latitudes where win- ters may be harsh. Michigan workers (1194) found that mares housed indoors began ovulatory estrous cycles about 21 days earlier than those housed outdoors. However, the mares housed indoors were judged to be in better body condition. The stimulatory effects of stabling could be related to better care of the mares (improved body condition and nutritive state), increased temperature and there- fore less energy demand, or associated increases in artificial light. 5.5C. Nutrition It is safe to conclude that mares in good nutritive balance and body condition will enter the ovulatory season sooner (pg. 126). This is a factor under managerial control. The cost factors in good nutritive care should take into account, therefore, the economics of earlier foaling. Some believe that turning mares onto a growth of green grass in the spring has, in itself, a stimulating effect. According to Allen (45), mares maintained in yards on adequate but dried feed during winter and spring remain anovulatory consider- ably longer than do mares moved to grass pasture in the spring. The response of yarded mares when suddenly turned out on fresh spring grass was said to be dramatic, with 80% of the mares ovulat- ing within 14 days. The hypothesis that 162 Chapter 5 Practical Considerations for Lighting Programs- Based on the references cited in this chapter and in Chapter 4, several gener— alizations and recommendations can be made about the use of lighting regimens as a practical method of shortening the anovulatory season: ' The breeding season can be extended on either or both ends with artificial light. 0 Either an incremental or a constant daily lighting interval is effective. ' A 15-hour fixed program has been adequately researched and can be recom— mended; continuous (24 hours) or pro~ longed (20 hours) light is less effective. 0 A program that has been recommend- ed is to constantly provide 2.5 hours of light at the beginning of sunset. This pro— gram does induce early onset of the ovula- tory season, but it has not been docu— mented that it is as effective as a fixed 15-hour program. 0 An example of an incremental pro— gram involves increasing the length of photoperiod at sunset by increments of one—half hour per week to reach a length of 15 to 16 hours at the desired beginning of the breeding season. 0 The optimal starting time for a fixed program is early December in ponies and presumably (not determined) may be ear- lier in horses, since the natural ovulatory onset occurs earlier in horses. 0 It has been recommended that a lighting program should be used for mares foaling before April 15, as well as for barren and maiden mares. ' There is a lag period of approximate- ly two months from onset of treatment to ovarian follicular stimulation and approximately three months to ovulation; however, mares with greater folliCular activity at the start of treatment respond sooner. 0 There seems to be considerable flexi- bility in the nature of light intensity and light source (incandescent, fluorescent, quartz). 0 Light appears to have no effect other than early induction of the ovulatory sea- son (e.g., level of fertility and length of estrous cycle are not changed). 0 Lighting programs can be expected to result in earlier foalings, better distribue tion of the stallion’s workload, and a longer breeding season for working on problem mares. ° No controlled experimentation has been done on the long-term effects of lighting programs, but at least one clini- cal report indicated continued effective— ness for six consecutive years. ° Onset of puberty can be delayed by lighting programs that are used to stimu~ late adults (pg. 494). 0 Lighting may help in overcoming the effects of season on stallion fertility (339, 305, 306, 1608, 684), and therefore stallions should be considered in the implementa— tion of lighting programs. pasture grazing has a stimulating effect on the first ovulation of the year has been tested (281). On May 2, mares were ran- domized into a lush-pasture group (n=14) or into a corral group (n=14) with no exposure to grass. The interval from May 2 to first ovulation of the year was signifi- cantly shorter in the pasture group (13.7 days) than in the corral group (21.5 days). This initial study supported the green—grass hypothesis; however, more tests are needed especially to minimize other confounding variables (e.g., exer- cise). Chemical signals in plants have been shown to have a stimulating effect on the reproductive system of certain rodents (cited in 605). For example, feed- ing fresh green wheat to voles for two weeks during the nonbreeding season resulted in reproductive stimulation. The active plant chemical is believed to be 6-methoxybenzoxyxolinone (6-MBOA). The value of 6-MBOA for hastening the onset of the ovulatory season in mares has been studied (605). Injections during the transitional period did not produce a significant effect. In a study in the fall (1822), 45 saddle- type horse mares were fed a maintenance diet of grass hay without supplementa- tion for 6 weeks. The mares were then randomly divided into a group that con- tinued on the grass hay and a group that was fed high-quality alfalfa hay and pasture (alfalfa and grass) free choice, plus cob corn. The ovulatory mares in the group fed grass hay tended (P<0.1) to have a higher incidence of silent estrus, and fewer (P<0.01) of the anovulatory mares showed estrous behavior. The results of some recent studies on the effects of diet or body condition on the onset of ovulatory season are described in Chapter 4 (pg. 126). In the common practice of flushing, breeding- farm mares are fed to a thin condition in the winter and are then fed at a high nutritive level as the breeding season approaches. The research described in Chapter 4 does not support the flushing Anovulatory Season 163 practice. The studies do, however, indi- cate that mares in good or fat body condi- tion will enter the ovulatory season earli- er than mares that are thin. 5.5D. Progestin Treatment In the late 19708 and early 1980s, there was a burst of research activity on the use of progestins for reproductive management in mares. The use of pro- gestins has become popular in the past decade, and many reviews have been pub- lished for progestin-based reproductive management during the anovulatory sea- son, ovulatory season, and pregnancy (e.g., 1526, 1519, 1518, 51, 976). This section will briefly review the use of progestins during the anovulatory season. Detailed information is available in the above cited reviews. Use of progestins, as well as other preparations, for other purposes will be discussed elsewhere (ovulatory season, pg. 282; pregnancy, pg. 525; postpar- tum, pg. 487). The original investigation into the use of progestins during the second portion of the anovulatory season utilized daily injections of 100 mg of progesterone for approximately seven days (1669). The regi- men was reported to block estrus and result in a return to estrus within a few days after treatment was discontinued; ovulation occurred toward the end of estrus. However, another study did not confirm that a seven-day regimen of pro- gesterone injections advanced the time of first ovulation (1203). Subsequent studies have used synthetic progestins, especially altrenogest (formerly called allyl tren- bolone; commercial product: Regumate). This progestin is given orally by dose syringe or mixed with grain and is espe- cially useful in research studies because it does not confound progesterone assays. Many studies have shown that it is highly effective for suppressing estrus and syn- chronizing the first ovulation of the year. For these reasons, it is an effective man- agement tool for the transitional period; 164 Chapter 5 it reduces the need for frequent breeding associated with protracted periods of estrus and large preovulatory-sized folli— cles without reducing pregnancy rate. It has been frequently and emphati- cally stated that altrenogest hastens the first ovulation of the year when given during the late transitional period (976, 72, 1747). Although its ability to synchronize the first ovulation has been well docu- mented, its ability to stimulate earlier growth of a preovulatory-sized follicle has not. Most research has centered on the post-treatment response; ovulations before or during treatment could have gone undetected or were not considered. In one of the earlier experiments (1520), altrenogest treatments were initiated on February 4, and follicular activity was not affected. Another group was treated after March 6 before mares had ovulated. The post—treatment interval to ovulation was shorter in the treated group (means: 14 days versus 23 days), but the mean interval from January 1 to ovulation was not different. A subsequent altrenogest study (1508) did not list determining the effect of treatment on time of first ovulation as an objective and included no comment in this regard. In a recent study (24), progesterone given daily for 12 days in the spring did not hasten the onset of the ovulatory season. According to an abstract of a study in Hungary (790), a 14—day altrenogest regimen beginning on February 28 apparently did not hasten onset of the first luteal phase. In an experiment designed to test the ability of altrenogest to stimulate early onset of the ovulatory season, a 15-day treatment schedule was begun when the largest follicle was 5 to 15 mm (mean: March 4) or first reached 20 to 25 mm (mean: April 8) or 30 mm (mean: April 30; 1646). The progestin did not hasten onset of the ovulatory season, regardless of the diameter of largest follicle at the onset of treatment. None of seven control mares or seven treated mares in each of the two small-follicle groups ovulated before the end of treatment. In the large—follicle group (30 mm at start of treatment), 0 of 6 treated mares and 3 of 6 controls ovu- lated during the treatment period. Thus, the mean day to first ovulation of the year was 10 days earlier in the control group. In a program consisting of light treatment followed by a 10-day progestin regimen, the first ovulation was hastened compared to controls, but the progestin had no additional effect over light alone (1203). Number and diameter of follicles were not altered during or after treat- ment when treatment began on the day the largest follicle was 5 to 15 mm. Other workers have found a similar lack of effect on mares in the inactive phase (1508). Altrenogest neither suppressed nor stimulated development of small follicles but did suppress development of large follicles (220 mm) during treatment (1646, 1508). Concentrations of FSH were elevat- ed during treatment, perhaps reflecting the loss of an FSH inhibitor from the suppressed follicles. Combination of altrenogest with estradiol did not have an advantage over altrenogest alone in syn- chronization of the first ovulation of the year (1782). The combination had greater follicular suppressive activity than altrenogest alone. Hastening and synchronizing the first ovulation of the year can be accomplished with a combination of light and progestin treatments. French workers applied 16 hours of light per day beginning November 25 (1203). An oral progestin was given for 10 days, followed by hCG 10 days after progestin withdrawal. It was concluded that two months of light treat- ment were needed prior to administration of the progestin. Programs can be devel- oped that incorporate other treatments into the progestin regimen (e.g., PGona to regress any mature corpora lutea that might be present at the start of pro- gestin treatment or hCG to induce ovula- tion of the preovulatory—sized follicles; 314). A program combining light treat- ment with a regimen of a progestin plus estradiol-17B also can be used (1590). Thompson and associates have observed and confirmed that placement of an intravaginal sponge during the anovu- latory season produced an immediate surge of FSH and follicular growth in 50% of mares (1605). The authors suggest— ed that neural impulses may have been involved because the FSH surges occurred immediately after sponge inser- tion. Perhaps this interesting phe- nomenon is related to the FSH surge that accompanies parturition, and sponge insertion activates the same mechanisms that are stimulated presumably during birth of a foal (pg. 481). Sponge insertion each week for six weeks, followed by a 14-day altrenogest regimen, did not indi- cate that there was an additional benefi- cial effect of the sponges, per se, on preparing mares for breeding. Other progestins that have received attention, although limited, as manage- ment tools during the anovulatory season include proligestone (1662) and norgestomet (1781, 1404, 1782). Proligestone is used to suppress estrus in dogs. It was given as a single intramuscular injection when mares developed large follicles after May 1 in Belgium (1662). The authors con- cluded that proligestone may be useful and recommended further study, especial- ly since the product can be given as a sin- gle injection. Norgestomet is given to ruminants to control estrus. However, at the doses used in a trial in horses, the product did not suppress estrus effective— ly (1781, 1782). Limited study indicates that norgestomet is not recognized at the pro- gesterone-binding sites in the mare (1782). 5.5E. Gonadotropic Preparations It was formerly assumed that mares were refractory to stimulation of ovula- tion with exogenous hormones during the anovulatory season. However, hormonal inductions were accomplished in 1974 using equine pituitary extracts (435) and were subsequently confirmed in ponies Anovulatory Season 165 and horses (933, 1814). Gradually increas- ing doses were used initially, but this has been found to be unnecessary. Ovulations were induced with a 14-day regimen even when the largest follicles at the initiation of treatment were small (10 to 15 mm; Figure 5.18). However, when a large folli- cle was present, treatment was more effective in terms of number of days to ovulation, number of mares ovulating, and number of mares with multiple ovulations (1814). In the horse project, 9 of 14 treated mares and 0 of 14 control mares ovulated within 14 days when the largest follicle at the start of treatment was >20 mm. In treated mares with one ovulation, the pregnancy rate, based on transrectal pal— pation, was lower (33%) than in controls (60%). About 50% of the treated mares that ovulated but did not become preg- nant returned to an anovulatory condi— tion. These findings indicated that the endocrinologic sequence that followed induced ovulation was defective in approximately 50% of the mares. Most (95%) of the extract-treated mares ovulat- ed when the largest follicle was 225 mm at the start of treatment, and 64% of the ovulating mares had multiple ovulations. These projects demonstrated that mares, even when in the inactive phase, can be hormonally induced to ovulate. FIGURE 5.19. Uterus and enlarged ovaries of a mare treated with equine pituitary extract during the anovulatory season. 166 Chapter 5 However, the pituitary extract approach for routine use to hasten onset of the ovu- latory season is not practical at this time because of high cost and unavailability, high incidence of multiple ovulations, and the more recent demonstration of the effectiveness of GnRH. The use of pitu- itary extracts to induce ovulation during the estrous cycle (pg. 261) and to induce multiple ovulations for research and embryo transfer purposes (pg. 339) is dis- cussed elsewhere. The pituitary prepara- tions also induced ovulation in some mares that previously did not cycle dur- ing the summer (600). 5. 5F. GnRH Treatment Early studies. In the original experi- ment involving GnRH administration, a single injection to seasonally anovulatory mares, as well as to stallions and ovulato- ry mares, caused a transient increase in LH (622); constant infusion resulted in continuing LH release, and the concentra- tions were enhanced by estradiol treat- ment (555). Enhanced LH release also occurred when GnRH was given after the end of estradiol treatment (1709). Similarly, a single injection of GnRH caused an FSH increase comparable to peak levels during the estrous cycle (494). In 197 6-7 7, it was first reported by Evans and Irvine (491, 493) that a GnRH and pro— gesterone regimen caused ovulation toward the end of the anovulatory season. Although the purpose was to induce ovu— lation by simulating the gonadotropin and progesterone patterns that occur dur- ing an estrous cycle, the project was not designed to determine whether such an elaborate approach was necessary. Indeed, as recognized by these workers, the extent to which exogenous proges- terone contributed to the result was not determined. Other workers (127) in 1977 obtained ovulation by GnRH treatment in 2 of 3 mares during the anovulatory sea- son (month not stated) using 0.4 mg per 8 hours for 14 days. Recent studies. Many workers have shown that many kinds of regimens of repeated or continuous administration of GnRH or GnRH agonists effectively induce ovulation in mares during the anovulatory season. The results of recent investigations are given in Table 5.5; results of more recent studies currently are available only in abstract form (13, 715). Several conclusions can be drawn from the table: 1. Many different treatment regimens and GnRH products are effective; 2. The percentage of mares responding (stimulation of ovulation) increases as the time of year or diameter of the largest fol- licle at initiation of treatment increases; 3. Not all mares with small follicles and treated with nonpulsatile systems respond to treatment, and some of those that do respond revert to seasonal pres- sures after ovulating (603, 793, 1218); and 4. A pulsatile delivery system may be most effective; although currently not a practical method for field work, it could be useful experimentally or for inducing multiple ovulations for embryo transfer. Multiple ovulations. The contributions of Johnson (826, 829, 832) not only demon- strated that GnRH delivered in pulses can stimulate the follicles and induce ovulation in averages of 9 to 12 days, even in mares initially in the inactive phase, but also that high doses can induce multiple ovulations (e.g., means of 2.9, 3.0, and 3.5 ovulations/mare). The occurrence of multiple ovulations has also resulted from injection of GnRH analogue twice daily (603). Anovulatory Season 167 TABLE 5.5 Examples of Results of Experiments on GnRH Regimens for Inducing Ovulation during the Anovulatory Season Product and Year (ref) delivery system Month Results 1986 ( 826) GnRH every hr Feb-Mar 4/4 ovulated; mean, 9 days 1987 (829) for 8 to 18 days Jan 0/4 controls and 13/13 treated mares ovulated; 1988 (832) 2 to 100 pg/dose means, 10 and 12 days J an-Feb 0/ 12 controls and 22/23 treated mares ovulated; mean, 11 days Multiple ovulations common and dose-dependent 1987 {69) ICI 118630; Feb-May 88% of 136 mares ovulated in <18 days 28-day implant 30 to 60 pg/day 1987 (510) Lutrelin Anestrus 2/10 controls and 8/8 treated mares ovulated in i 100 pg, 2X/daya 8 to 14 days GnRH Anestrus 200 pg, 2X/day 2/8 treated mares ovulated 200 pg, 4X/day 3/4 treated mares ovulated 1987 (1100) Fertagyl, 167 pg, 3X/day Jan-Mar Estrus in mean of 12 days in 49/49 mares 1987 (7.97) GnRH by constant Dec-J an 0/10 controls and 10/20 treated mares ovulated; infusion for 28 days mean, 20 days 50 or 100 ng/kg/hr Mar-Apr Mean days to ovulation: 13 control mares, 42 days; 14 treated mares, 19 days 1988 ( 793, 7.95) GnRH by constant Feb 0/ 10 controls and 13/20 treated mares ovulated infusion for 28 days within 34 days 100 or 200 ng/kg/hr 1988 (1218) GnRH for 21 days Anestrus Ineffective 200 pg, 1X/day GnRH for 12 days Jan-Mar 0/ 16 controls and 15/16 treated mares ovulated 10 to 250 pg/hr in 6 to 15 days Continuous infusion or Apparently less effective than hourly pulses even an injection every 3hrs when total dose was greater 1990 ( 686) Buserelin for 28 days Mar 0/ 15 controls, 7/15 treated, and 9/15 implanted 40 pg, 2X/day mares ovulated within 30 days Implant (100 pg/day) 1990 (603) Agonist Dec 17/30 (57%) ovulated within 12 days 100 to 400 pg, Jan Proportion ovulating increased according to 2X/day initial diameter of largest follicle (Figure 5.19) Increased multiple ovulation rate a This regimen also was used to treat 108 acyclic mares during the breeding season, and 80% ovulated. There were no controls. 168 Chapter 5 Beginning treatment during the inactive phase. In a study involving administration of an analogue twice daily, the proportion of mares ovulating within 21 days decreased significantly and the response was less in mares with smaller follicles at the beginning of treat- ment (Figure 5.19; 603). Furthermore, mares that ovulated, despite small folli- cles at the initiation of treatment, had small follicles during the ensuing preg- nancy (Figure 5.20). Some mares that were treated when the largest follicle was small and did not become pregnant at the induced ovulation, subsequently returned to anovulatory status; these results agree with an earlier study (826). In a series of recently completed experiments (184), using the same GnRI-I product and proto- col, the effects on luteal development and embryo loss were considered. Mares in the inactive phase (largest follicle £15 mm; n=24) at the start of treatment were compared to controls (n=20) and to mares that were in apparent early resur- gence (largest follicle 220 mm; n=29). A Effect of GnRH on follicles during ensuing pregnancy Effect of largest follicle on GnRH response A O (6) Percent mares ovulating within 21 days N 0 20-24 <15 15-19 Diameter (mm) of largest follicle at start of treatment 225 FIGURE 5.19. Influence of diameter of largest folli- cle at start of treatment on percentage of mares ovulating Within 21 days. Number in parentheses is total number of mares (e.g., in the GnRH—treatment group in which the largest follicle on first day of treatment was <15 mm, 36% of 25 mares ovulated). Difference between treated and untreated mares for each diameter category: <15 mm (P<0.1), 11-19 mm (P<0.005), 20—24 mm (P<0.005), 225 mm (NS). Adapted from (603). No treatment (n=1?) 30 / 25 I ....... . ‘ I ......... ,0 V! l LF <15 mm (n=11) at start of treatment Diameter (mm) of largest follicle 2 8 14 20 26 Number of days from ovulation LF 20—24 mm (n=19) at start of treatment .4 -~ .4‘ "\ LF15—19 mm (n=7) at start of treatment FIGURE 5.20. Mean diameter of largest follicle (LF) during early pregnancy in control mares and in GnRH-treated mares in which the largest follicle on first day of treatment was <15, 15-19, or 20- 24 mm. Day by group interaction (P<0.0001). The mean in the control group was greater (P<0.05) than in each of the three treated groups on each of Days 17-38. The mean for the <15 mm group was smaller (P<0.05) than that for the 15—19 mm group on Day 17 and smaller than for the 20—24 mm group on Days 11-26. Adapted from (603). 32 38 high rate of embryo loss occurred in treat- ed mares, especially when the mares were stimulated while in the inactive phase (follicle £15 mm, 58% loss; follicle 220 mm, 24% loss; controls, 11% loss). Most of the embryo losses occurred before Day 25, and progesterone concentrations were significantly reduced beginning six days before loss. The mean concentra- tions of LH on Day 3 in mares with embryo loss was lower (0.8 i0.2 ng/ml) than in controls (3.1 i1.0 ng/ml). Perhaps induction of ovulation during the inactive phase led to reduced progesterone produc- tivity due to lower LH levels. In earlier trials with the same regimen (600), embryo loss was prevented by adminis- tration of progesterone. It appears that when the follicles are small at the start of treatment (inactive phase; e.g., >2 month before natural ovu- lation), a mare is less likely to respond, the interval to response is longer, the mare is more likely to return to an anovu- latory status after the induced ovulation, and follicle and corpus luteum develop- ment during the ensuing pregnancy are diminished. In conclusion, such a mare appears to revert to seasonal pressures after an induced ovulation, whether or not pregnancy occurs. Reversion to sea- sonal pressures also occurs when pitu- itary extract is given to induce ovulation (pg. 165). However, when the follicles are large at the beginning of treatment (spring transitional period; e.g., one month before natural ovulation), mares With induced ovulations subsequently continue to cycle (nonpregnant) or, if pregnancy occurs, the follicles and corpora lutea during the ensuing preg- nancy develop normally. Delivery systems. A continuous system (minipumps) and pulsatile delivery sys- tem have been compared (1218). Constant delivery was done at a daily rate of 300 ug, 900 ug, and 2,700 ug, and pulsatile delivery was 10 ug/hr (equivalent to 240 jig/day). All systems induced ovulation (3/7, 4/10, 8/9, 4/5, respectively) compared Anovulatory Season 169 to 0/10 in controls. Treatment by either route was effective (19/31 ovulated com— pared to 0/ 10 controls). However, number of mares per group was too small for definitive conclusions regarding the effi- ciency of continuous versus pulsatile delivery. The pulsatile system appeared to be more effective (not significant) when calculated on the basis of total dose/day (3/7 responded at 300 ug/day with contin- uous delivery versus 4/5 at 240 ug/day with pulsatile delivery). Considerably more study will be needed on delivery systems for field use and especially to determine the importance of pulsatile mechanisms from a basic research point of View. Continuous administration by implant or an injection every 12 hours were equally effective in terms of number of mares induced to ovulate (686), but the LH response was greater in the mares with continuous administration. The implant delivered 100 ug/day, whereas the injection scheme involved 80 ug/day. Recent studies, currently available in abstract form, reached the following con- clusions: 1) pulsatile subcutaneous deliv- ery (10 mg/hour) of GnRH and twice daily injections of GnRH analogue produced similar ovulating response in February and March, but pulsatile delivery seemed more effective in January (4 of 5 versus 2 of 8 mares ovulated; 1042); 2) twice daily injections of GnRH analogue were superi— or to continuous delivery (966); and 3) either hourly pulses of GnRH or continu- ous delivery were effective (1647). La_te entry. A continubus-release depot of a GnRH agonist was an effective delivery system (1866). The effectiveness of various delivery systems will likely be influenced by the reproductive status (diameter of largest follicle) at the onset of treatment. This aspect must be carefully considered in judging the effectiveness of various protocols. 170 Chapter 5 Conclusions on GnRH treatment. There is no question that many types of GnRH programs can be highly effective for bringing an end to the anovulatory season. It is anticipated that long-lasting preparations for ease of administration will become commercially available. In several studies, pregnancy rate did not seem different from the expected or did not differ significantly from controls (e.g., 793, 797, 603). Additional study is needed on the hormone balances of the ensuing pregnancy, especially when mares are forced to ovulate well before the end of anovulatory season. 5.5G. Other Treatments Based on a limited clinical trial, it has been suggested that clomiphene citrate can be used to stimulate follicular devel— opment in anovulatory mares (1336). However, a single intramuscular injection of various doses (10 to 500 mg) failed to alter gonadotropin concentrations and fol- licular growth (1092). An antiestrogen has been tried but Without effect at the doses used (1205). Testosterone immunization also has been tried for hastening the first ovulation, but the results were equivocal (631). Analogues of prostaglandin Fm show promise for stimulating ovulation in mares with low progesterone levels—that is, an effect independent of the well- known luteolytic effect of such products (review and citations of clinical trials: 825). In an experiment involving collection of blood from the intercavernous sinus, prolonged (1 to 2 hours) elevations of LH and FSH occurred after an intramuscular injection of such an analogue (luprostinol; 825). An injection of hCG will induce ovula- tion of a fully developed follicle and if given at the proper time will shorten the transitional period accordingly (314). Transitional mares were given 3,300 iu of hCG when the largest follicle reached 40 mm and the mare had displayed estrus for three days (285). Treated mares ovulated sooner than control mares (means: 3 and 10 days). Progesterone pro- duction was not adversely affected. Research on the minimal diameter (or other criteria) of a follicle that will respond to hCG apparently has not been done. This is a crucial question because of growth and regression of large dominant follicles (>35 mm) that can occur during the transitional period (pg. 146). Most likely an injection of hCG would not be successful if given while a dominant folli- cle was in the regressing phase and the next dominant follicle had not yet reached a receptive stage. It has been reported that administration of hCG induced ovulation when given daily in small doses (e.g., 200 in) when the largest follicle reached >20 mm (pg. 154); however, these workers concluded that this was an unusable practical technique because of the development of antibodies (229). Research is needed to develop recom- mendations on the optimal use of a single injection of hCG. Criteria need to be established for practical selection of a fol— licle with a high probability of response to an ovulating dose during the transi- tional period. Effective selection criteria would negate the necessity for frequent, indiscriminate administration of hCG and would minimize the formation of hCG antibodies. For further discussion of the ovulating-inducing properties of hCG and the effects of anti—hCG antibodies, refer to Section 7.6A (pg. 279). Admin- istration of eCG is not a practical alter- native because of the refractoriness of the mare ovary to its own placental gonadotropin (pg. 50 and pg. 434). Anovulatory Season 171 HIGHLIGHTS: Anovulatory Season The anovulatory season can be divided into recessive, inactive, and resurgent phases, reflecting the activity of the hypothalamic-pituitary-ovarian axis. In some mares, one or all of the phases may be indistinct or absent. Diameter of largest follicle is a convenient indicator of phase. The fall transitional period is associated with a deficiency of LH and final growth spurt of a preovulatory follicle and not with a deficiency in FSH or number of available large follicles. Unseasonable estrus occurs commonly, even in the absence of palpable follicles. . ‘ Circulating LH concentrations, but not FSH concentrations, are at minimal levels ’ during the anovulatory season, except for the high levels immediately following and preceding the last and first ovulations, respectively. Based on mitotic index, follicular activity is lower during the inactive phase than early in the ovulatory season. During resurgence , follicles grow and regress until one reaches a large size (e.g., 40 mm). In some mares, large follicles regress periodically (e.g., every 10 days) until one ovulates. Discharge of LH and FSH occurs in pulses during resurgence . Pulse frequency of LH increases as ovulation approaches. Before the first ovulation, FSH decreases sooner, estrogen increases sooner, LH and estrogen do not rise as high, and the ovulatory follicle grows slower, but to a larger size, than later in the ovulatory season. In addition to melatonin, GnRH, FSH, LH, and estrogen, it may be that prolactin is part of the cascade of hormonal events leading to termination of the anovulato— ry season, but the nature of prolactin’s role has not been determined. Ovulation can be stimulated during the anovulatory season by administration of gonadotropins or GnRH, as well as by artificial extension of the photoperiod. In trials in ponies, light treatments started before December did not have an advantage, and treatments started after December were not as effective as treat- ments begun in December. A recommended light treatment is a fixed photoperiod of 15 hours. Progestins, such as altrenogest, will synchronize the first ovulation but may not be effective for major shortening of the anovulatory season. 172 Chapter 5 MILESTONES: Anovulatory Season First field trials on use of artificial photoperiod for ending the anovulatory sea- 8011 (987). Documentation and characterization of unseasonal estrus (573). Induction of ovulations during the anovulatory season with pituitary prepara- tions (435). Demonstration that GnRH can be used to stimulate ovulation during the anovulatory season (493). Characterization of follicular and hormonal aspects of the recessive phase (1498). Characterization by transrectal palpation of folliculogenesis during resurgence (575). Characterization of circulating FSH and LH levels during resurgence (540). Histologic characterization of seasonal folliculogenesis of small follicles (447). Discovery of changing LH pulsatility during resurgence (512). Extensive trials on the use of GnRH for inducing an end to the anovulatory sea- son (797, 510, 69, 1100). Characterization of follicular dynamics during spring transition by ultrasonic monitoring of individual follicles (598). —Cfiapter 6— CHARACTERISTICS OF THE OVULATORY SEASON This chapter will describe the nonhor— monal characteristics of the ovulatory season, as delineated by the first and last ovulations of the year. For continuity, the estrus associated with the first and last ovulations will be included. Thus, there will be overlap with the previous chapter. 6.1. Estrous Cycles 6.1A. Length Most authors conclude that the estrous cycles of mares are very irregu- lar. A portion of the irregularity, however, can be attributed to the nature of the investigations. Teasing techniques differ considerably, and the criteria used to identify estrus are far from standard- ized (pg. 75). Perhaps more serious are the liberties taken in the definitions of estrus, diestrus, and estrous cycles. It is misleading to conclude, as has been done, that the length of the estrous cycles in a herd ranged from 9 to 124 days. When normally occurring deviations, such as those associated with early embryo loss and unseasonal estrus, are excluded, the variability is considerably reduced. These complicating factors cannot be excluded, however, unless the reproductive tract is examined adequately as part of the data- gathering process. A composite mean, standard deviation, and coefficient of variability have been calculated from many references to obtain overall estimates of the reported lengths of the estrous cycle and its com- ponents (Table 6.1). The means for the lengths of estrus, diestrus, and the estrous cycle were 6.5, 14.9, and 21.7 days, respectively. The estrous cycle, on the average, was two days longer in ponies than in horses (pg. 175; 575) and in TABLE 6.1. Means and Variations for Length (Days) of Components of Estrous Cycle as Averaged Over Many Reports Coefficients of Means Standard deviations variation (%) No. Composite Range of No. Composite Range of Item references mean means references SD SD’s Composite Range Estrus 26 6.5 4.5 to 8.9 8 2.6 2.1 to 4.2 40.4 31.4 to 50.0 Diestrus 10 14.9 12.1 to 16.3 4 2.8 2.1 to 3.9 18.0 14.0 to 24.5 Estrous cycle 18 21.7 19.1 to 23.7 6 3.5 2.5 to 4.5 15.8 11.6 to 21.7 From (575). 174 Chapter 6 one study (1679) was about three days longer in donkeys than in horses. Convenient fig- ures for the lengths of estrus, diestrus, and the estrous cycle, respectively, are 7, 15, and 22 days in horses and 8, 16, and 24 days in ponies. The coefficient of variation (Table 6.1) was more than two times greater for estrus than for diestrus, indicating that estrus was more variable. In a study of pony mares (623), the lengths of 226 estrous periods were significantly more variable than the lengths of 192 diestrous periods. To provide an overall concept of the variation in lengths of estrus, diestrus, and the estrous cycle, distribu- tion curves were prepared (Figure 6.1; 119). The study involved horses of mixed breeding and extended from approximate- ly May to October or November over a two—year period. Mares were teased indi— vidually each day. Length of estrus tends toward repeata- bility within mares (575). In other words, throughout the ovulatory season, certain mares tend to have most of the short periods and others tend to have most of Estrous cycle components Diestrus Estrus 15.0 $2.1 50 6.8 $2.3 ¢ Estrous cycle N O Number of estrous cycles _L O 0 5 10 15 20 25 30 Length (days) FIGURE 6.1. Frequency distributions for length of estrus, diestrus, and estrous cycle. Adapted from (119). the long periods (Table 6.2). In one study (623), the length of estrus within mares was more repeatable than the length of diestrus (intraclass correlation for estrus, 0.58 versus diestrus, 0.39). 6.13. Seasonal Changes Data from eight herds were tabulated in the first edition (575). On average, ovu- latory estrus was quite long in early spring, decreased progressively during the remaining spring months, became shorter and stabilized during the sum- mer, and increased progressively during the fall. Such changes have been statisti- cally documented in ponies (Figure 6.2). Perhaps the length of estrus is a reflec- TABLE 6.2. Repeatability of Length (Days) of Estrus within Mares Wisconsin data Japanese data Length of Length of No. estrus No. estrus Mare estrous estrous identity periods Mean SE periods Mean SE 1 6 2.0 $1.1 5 5.6 $0.8 2 7 3.3 $1.1 4 6.0 $0.4 3 4 3.5 $0.6 6 6.7 $0.6 4 9 3.8 $0.6 4 7.8 $0.6 5 5 4.4 $1.6 2 8.5 $1.5 6 7 5.0 $0.8 2 9.5 $0.5 7 4 5.5 $2.2 4 9.8 $1.2 8 8 7.2 $0.6 6 9.8 $0.7 9 8 7.4 $1.1 6 10.0 $0.5 10 10 7.5 $0.5 5 12.0 $0.7 11 5 8.0 $0.5 6 12.0 $0.7 12 5 9.0 $1.1 6 12.0 $0.7 13 7 9.1 $1.2 14 9 9.4 $0.7 For both sets of data, the mean length of estrus is significantly different among mares indicating a significant amount of repeatability. Intraclass cor- relations are 0.45 and 0.64 for the Wisconsin and Japanese data, respectively. The analysis for the Japanese study was done on published individual observations ( 1396). Wisconsin data from (575). Characteristics of the Ovulatory Season 175 Effect of month on length of estrus & diestrus 3° (n=14) Diestrus Length (clays) NHem M A SHem S 0 FIGURE 6.2. Changes in length of estrus and diestrus during the ovulatory season in ponies. Lines are calculated regression curves which best characterize the monthly changes in length. Adapted from (573). tion of the prominence of the LH surge (i.e., prominent surge = earlier ovulation = shorter estrus). The first ovulatory estrus of the ovulatory season was longer when it began before April 15 than when it began after April 15 (Figure 4.1). This finding indicates that prolonged first estrus of the ovulatory season is more closely related to influences associated with month (daylength) than to its posi- tion as first estrus. A similar conclusion has been reached for the prominence of the LH surge; the first surge of the sea- son is less pronounced than the second (pg. 254). In one study (575), the length of diestrus increased for the first months of the ovulatory season (April to July), remained fairly constant for three months (July to September), and then decreased (October to November; Figure 6.2). The gradual lengthening of diestrus during the first part of the ovulatory season is consistent with previous observations (1666). In another study (623), the length of diestrus, beginning in May (mean: 13.7 days; n=27), was significantly shorter than those beginning in June (mean: 15.8; n=98), July (mean: 16.3; n=45), or August (mean: 16.7; n=23). A study of Thoroughbreds and Quarter Horses in California, however, found no significant changes or tendencies among months in the length of diestrus (780). Changing length of diestrus could also be related to changing LH output since there are indi- cations that LH affects the level of luteal progesterone production (pg. 265). 6.1 C. Interovulatory Intervals It seems reasonable, especially for research purposes, to deal with the num- ber of days between ovulations (interovu- latory interval) rather than with the length of the estrous cycle. This approach eliminates ambiguity associated with variations in methods of detection and in definitions of estrus and minimizes error due, for example, to silent estrus. Reliable ovulation-detection techniques are required, of course, when the length of the interovulatory interval or number of days after ovulation is an integral part of an experiment. The criterion to be used for length of the interovulatory interval in mares with double asyn- chronous ovulations must be stipulated. For example, it must be clear whether the beginning and ending ovulations for the interovulatory interval are based on the first or second ovulation of the asyn- chronous set. , It is convenient to use the day of ovula- tion as a reference point and to define it as Day 0. When this is done, the number of elapsed days from ovulation is equiva- lent to the defined day (e.g., Day 7 is the seventh day after ovulation). It should be noted, however, that under these criteria, Day -1 is as close to the time of ovulation, on average, as Day 0 since Day 0 refers to the day on which the ovulatory follicle is gone. Ovulation occurred sometime between the Day -1 examination and the Day 0 examination. 176 Chapter 6 A frequency distribution for lengths of interovulatory intervals is shown for horse mares (Figure 6.3; 617). Two of 69 intervals (2.8%) were excluded because of short intervals and six (8.5%) because of prolonged intervals (pg. 224). In another study (1092), the mean length of the interovulatory interval was 22.7 i0.7 days for 6 horses and 25.0 i0.6 days for 12 ponies (P < 0.05). 6.2. Follicular Dynamics The field of equine follicular changes or dynamics has in recent years assumed its rightful place as an important research area. Research in the mare, however, has not progressed to the point where it is routinely considered by scientists work- ing on other species. For example, an extensive comparative review in 1988 (645) considered all the usual species of laboratory and farm animals but without mention of the mare. This situation will likely change during the 1990s. Early work in mares has been reviewed (85, 575). In the 1980s follicular dynamics of the interovulatory interval came under closer scrutiny, due primarily to the availability of transrectal ultrasound scanners. These instruments permit fre- quent noninvasive visualization of the dynamic follicular population, the ovula- tory process, and the development, main- tenance, and regression of the corpus luteum (reviews: 590, 615, 1263, 616, 592, 1514). Ultrasonic studies have confirmed earlier sequential folliculogenesis studies involving transrectal palpation (575) and have extended the research to smaller fol- licles (2 to 10 mm). More recently, ultra- sonic imaging has been adapted to the monitoring of individual follicles by selecting ovaries with only one large folli- cle (1206) or by attempting to monitor indi— vidual follicles >15 mm (598, 1484). In addi- tion, several histologic evaluations of follicular populations were made in the past decade by Driancourt and co-workers (446, 442, 444). Photomicrographs of normal Interovulatory intervals —L O 0') O) h Number of interovulatory intervals N O 10 15 Length (days) of interovulatory interval 20 25 30 35 40 45 FIGURE 6.3. Frequency distribution for number of interovulatory intervals of various lengths. Intervals outside of the bracketed area were consid— ered to be abnormal in length. The two short inter- vals were associated with endometritis, and the six long intervals were associated with hemorrhagic fol- licles (n=2), secondary ovulations (n=2), and unknown causes (n=2). From (617). (nonatretic) follicles are shown in Figure 6.4 and of atretic follicles in Figure 5.3 (pg. 138). 6.2A. Underlying Follicular Activity Noneguine species. Based on a review (645), oogonia in most mammals are trans- formed before or soon after birth into pri- mary oocytes. These oocytes enter a pro- longed meiotic or resting stage and are surrounded by a simple layer of squa- mous cells. The entire structure, oocyte and surrounding simple cell layer, is called a primordial follicle. The primor- dial follicles represent the built-in stock- pile that is depleted progressively throughout the reproductive life span. Primordial follicles, through unknown control mechanisms, continuously (pre- sumably) leave the reserve pool, and the surrounding squamous cells are convert- ed into cuboidal cells. This structure is known as the primary follicle; the single layer of cuboidal cells will eventually become the all-important granulosa layer. Characteristics of the Ovulatory Season 177 FIGURE 6.4. Histology of wall of a small (4 mm) developing follicle and a large (40 mm) preovulatory follicle. Note the mitotic figures (arrows) in the granulosa of the small follicle and the row of nuclei at the base of the granulosa. The theca interna is beneath the granulosa. In the large preovulatory follicle, luteal-like cells are present in the theca interna. It takes months for an individual primor- dial follicle to develop into a mature folli- cle, according to studies in sheep (cited in 203); 4 to 5 months are spent moving from a primordial follicle to the first appear- ance of an antrum. Eguids. The concepts reviewed in the above paragraph seem to apply to many mammalian species and can be assumed to be operational also in equids. Antral formation in the growing population of underlying follicles in both horses and ponies occurs when the follicles reach 0.2 to 0.4 mm (446). Atresia (regression) of fol- licles is rare until they reach 1 mm (440). In a histologic study (446), approximately 36,000 primordial follicles and 100 grow— ing follicles were counted in mare ovaries. The authors noted that, in comparison, cows have about 120,000 primordial folli- cles and 280 to 435 growing follicles. Apparently, recruitment of primordial fol- licles into a growing pool, as measured at a given time, involves fewer follicles in mares (e.g., 100) than in cows (e.g., 300). Incidentally, this fundamental species difference may be related to the greater sensitivity of cattle to induction of multi— ple ovulations (pg. 339). If a mare originally had 40,000 pri- mordial follicles and cycled continuously for 25 years using 100 follicles/cycle, she would deplete her reserve of primor- dial follicles (100 follicles/cycle X 16 cycles/year X 25 years = 40,000 primordial follicles). Presumably the pool of primor— dial follicles is finite and does not replen- ish itself; mitosis of oogonia has not been reported in fillies or mares. Dynamics of small follicles. An under— standing of dynamics in the small follicle population (up to 10 or 15 mm) is lacking in the mare. For the sake of continuity, it is speculated that small follicles are con- tinuously growing and regressing and thus provide a reservoir for larger folli— cles. It is further speculated that this underlying basal activity occurs during all reproductive states (anestrus, estrus, diestrus, pregnancy, pseudopregnancy, 178 Chapter 6 and postpartum). The major follicular waves emerge from this underlying bed of activity. It is unknown whether the underlying activity involves independent growth and regression of individual folli- cles or whether groups of follicles develop in synchrony. The level of activity is not necessarily constant. This aspect of fol- liculogenesis in mares is in need of study. Data for follicular changes during known days of estrus or early diestrus were obtained during studies on the ovar— ian effects of anti-pituitary preparations (1268, 1269). There was an apparent increase in number of small follicles (2 to 10 mm) between day 7 of estrus and day 5 of diestrus, but this comparison involved separate experiments. On day 5 of diestrus (mean: 21 follicles), there tended to be more small follicles than on day 8 (mean: 12), and there were significantly more than on day 11 (mean: 8). In a sequential ultrasound study (1264), the number of 2 to 5 mm follicles reached maximum on Day 5 after ovulation in early diestrus. Because follicles of this size could not be monitored individually, the number of growing versus regressing follicles was not known. Results of the study of excised ovaries (442), confirmed by the ultrasound study, indicate that increased follicular activity can occur dur- ing early diestrus without the emergence of large follicles. 6.23. The Wave Phenomenon A major follicular wave refers to sever— al follicles (e.g., 5 or 6) that initially grow in synchrony (approximately same diame- ter and growth rate) but eventually disso- ciate. Dissociation is characterized by preferential growth of one, occasionally two, members of the wave. The favored follicle is termed the dominant follicle of the wave; the physiologically selected dominant follicle grows to a large diame- ter (e.g., >30 mm) and then either regress— es (anovulatory wave) or ovulates (ovulato- ry wave). The remaining follicles undergo early atresia (subordinate follicles). Thus, a selection mechanism for follicular disso- ciation is an inherent part of the defini- tion of a major wave. This definition of a major wave is used to conform to the emerg- ing use of the term “wave” in cattle (612). The mare is apparently unique in that a major follicular wave with ovulation of the dominant follicle sometimes occurs during a progestational state (e.g., di- estrous ovulations). Thus, a major follicu- lar wave can occur during early diestrus and the dominant follicle can be ovulatory or anovulatory. Herein, a major wave forming during early diestrus and giving origin to a dominant anovulatory follicle or a dominant ovulatory follicle (diestrous or secondary ovulation) is defined as a secondary wave; a major wave beginning during mid-diestrus and giving origin to the ovulation associated with estrus (pri- mary ovulation) is defined as a primary wave. It is not known whether syn- chronous growth and regression of a group of follicles can occur without the emergence of a dominant follicle. To rein- force an earlier statement, an increase in underlying follicular activity could repre- sent growth and regression of individual follicles independently of one another or could involve synchronous activity of mul- tiple follicles. 6.20. Major Secondary Wave The dominant follicle of the secondary wave that sometimes emerges in late estrus or early diestrus becomes either an anovulatory follicle or results in a diestrous or secondary ovulation. The wave, whether ovulatory or anovulatory, is nonproductive in that the oocyte of the dominant follicle is not afforded an oppor- tunity for fertilization, except under unusual circumstances (double ovulation with one follicle originating from the sec- ondary wave, pg. 222; planned breeding to a diestrous ovulation, pg. 223). Research indicators for secondary waves are as follows: Characteristics of the Ovulatory Season 179 1. Diestrous ovulations. Large early diestrous follicles (>30 mm) have been described for some cycles, especially in certain breeds, and these follicles some- times ovulate (diestrous or secondary ovulations; pg. 223). 2. Ultrasonic monitoring of individual follicles. Combined for two recent studies (1484, 598), two major waves (secondary and primary) were detected in 11 of 25 interovulatory intervals in which the dominant follicles in each wave reached at least 25 to 30 mm. The remaining intervals had only one major wave (pri- mary wave) which emerged in late diestrus and gave origin to the follicular- phase ovulatory follicle; that is, the pri- mary ovulation. In the two studies, as many individual follicles as possible were monitored during interovulatory intervals in Standardbreds and Thoroughbreds (1484) and in Quarter Horses and Appaloosas (598). Both studies were early in the ovu— latory season. In the study in Standard- breds and Thoroughbreds, 5 of 17 cycles hadtwo major waves involving follicles 225 mm. Three of the five mares with two waves ovulated from the first wave (die- strous ovulations), and in the remaining two mares, the dominant follicle reached 25 to 30 mm but then regressed. The Individual follicles 50 Mare A \ .Ov J) 0 Diameter (mm) 00 O N O 0 5 10 15 20 -5 0 Number of days from ovulation mares with two identified major waves had a significantly longer interovulatory interval (mean: 22 days) than those with one wave (18 days). Inspection of raw data presented by these workers, howev- er, suggested that the occurrence of di- estrous ovulations may have beclouded this result; the three intervals associated with a diestrous ovulation were 22, 22, and 23 days, whereas the two intervals that did not have a diestrous ovulation were both 20 days. In the study in Quarter Horses and Appaloosas, two major waves were detected per interovu- latory interval in six mares (Figure 6.5,B) and one wave per interval in three mares (Figure 6.5,A). The first wave in two- wave intervals was prominent (dominant follicle >25 mm), but diestrous ovulations did not occur. Other studies involving monitoring of individual follicles (600) have indicated, however, that Quarter Horses and ponies usually have only one major fol- licular wave—the wave giving origin to the primary ovulation. This is consistent with the results of earlier work involv- ing transrectal palpation (575) or ultra- sonic imaging (1264); end points were number of follicles in various size cate- gories and diameters of the largest and second largest follicle. The incidence of Mare B ’IOV FIGURE 6.5. Diameters of indi- vidual follicles during the interovulatory interval. U refers to echotexture of uterus (white = nonestrus; stippled = intermedi- ate; black = estrus). Mare A has only a primary wave, whereas Mare B has both secondary (anovulatory in this case) and primary major waves. Adapted from (598). 180 Chapter 6 diestrous ovulations is greater in Thoroughbreds than in Quarter Horses and ponies (pg. 223); this is compatible with a more frequent occurrence of two major waves in Thoroughbreds. 6.2D. Primary Wave The primary wave of late diestrus occurs in all normal estrous cycles. It is the productive wave in that the dominant follicle gives origin to the follicular-phase ovulation (primary ovulation); the oocyte has the potential to be fertilized. The wave emerges at mid-cycle. It is unknown when the follicles of the wave originate from the primordial reserves, but they emerge as an ultrasonically detectable group at mid-cycle. Research indicators for mid-cycle emergence of the wave that gives origin to the primary ovulation are as follows: 1. Transrectal palpation: Follicular diameters were monitored in 24 estrous cycles in ponies by transrectal palpation (575). Diameter of the largest follicle and the number of follicles >20 mm were low before Day 10. By Day 10, however, both these indices of follicular activity began a progressive increase, characterized by the growth of several follicles (Figure 6.6). During the seven days prior to ovu- lation, the diameter of largest follicle continued to increase at an average growth rate of 3.5 mm/day and became the ovulatory follicle. Increasing diame- ter of follicles, beginning at mid-cycle, also has been reported for horse mares (884).Similar follicular dynamics have been described for jennies (1679). 2. Ultrasonic monitoring and categ ; rizing follicles according to size. The ovarian follicles of mares are good sub- jects for ultrasonic imaging (590, 592, 1514). Follicular population dynamics during the estrous cycle have been studied by ultrasonically measuring the largest and second-largest follicle and categorizing all follicles 22 mm into various size classifications (1264, Follicular dynamics (n=24) .h 0 Diameter (mm) on O 20 14 7101316-7-4 -1 Number of days from ovulation FIGURE 6.6. Changes in ovarian end points during the estrous cycle as determined by transrectal pal- pation in ponies. Within each end point and each stage of the cycle (estrus and diestrus), means which do not have at least one common letter are significantly different. Adapted from ( 5 75). 615). In one study (1264), daily ultrasonic examinations were made in 40 horse mares (primarily of Quarter Horse and Appaloosa breeding). The number of large follicles (16 to 20 mm and >20 mm) and diameter of the largest and second largest follicle began to increase after Day 10 (Figure 6.7). The increas- ing diameter of the largest follicle con- tinued until ovulation. 3. Ultrasonic monitoring of individu- al follicles. In both studies, using ultra— sonic monitoring of individual follicles (1484, 598), the ovulatory follicle and its cohorts were first detected (retrospec- tively identified) at mid-cycle. Characteristics of the Ovulatory Season 181 Follicular dynamics 50 Largest follicle .D- 0 Diameter (mm) o: O N O Follicles >20 mm l \J Number -3 024681012141618200 3 Number of days from ovulation 4. Studies of excised ovaries. In one study (1268), there was a significant increase in number of follicles >10 mm between Day 6 and Day 11 (means: 2.8 and 6.8). In a histologic study (442), O of 4 pony mares had a nonatretic follicle >10 mm at Day 6, whereas 4 of 4 mares had a nonatretic follicle >10 mm at Day 14. Several slaughterhouse studies have included measurements of follicles (1759, 719). The results of these studies seem consistent with other more critical work and will not be reviewed. A flaw in all the slaughterhouse studies was that the stage of cycle was estimated by gross appearance of the corpus luteum. The rel- ative size of a central blood clot was a principal criterion; recent ultrasound FIGURE 6.7. Mean diameters of the largest follicle and num— ber of follicles for May to July and August to October during the interovulatory interval. There was a significant effect of season for both end points. Adapted from (1264). studies have demonstrated that many developing corpora lutea do not contain prominent blood clots (pg. 197). 5. Forcing the emergence of a new wave. Growth of small follicles (e.g., 10 to 15 mm) has been experimentally studied in two ways: 1) ‘by removing the ovary with an apparent dominant follicle and studying the remaining ovary, and 2) by suppressing follicles with steroids and then releasing the follicles by termi- nating treatment. In all such approaches, the interval to ovulation has been approx- imately 12 to 14 days. On the basis of the length of an interovulatory interval, the starting point would be equivalent to Day 10 for horses and ponies with mean interovulatory intervals of 22 and 24 days, respectively. 182 Chapter 6 Scientists from France (444) and New York (1484) have used unilateral ovariecto- my to study certain aspects of folliculo- genesis in ponies, Standardbreds, and Thoroughbreds. Removal of the ovary with the preovulatory follicle in ponies delayed the next ovulation for an average of 14.6 days. In the horse study, the ovary with the largest follicle was removed between Day 14 and the fourth day of estrus. The interval to ovulation was 13.7 days, similar to the study in ponies. These results are compatible with the results of the above-cited transrectal pal- pation, ultrasound, and histologic studies. After unilateral ovariectomy, the ovulato- ry follicle grew at a faster rate than in normal mares. The authors noted that this may have been related to the absence of progesterone throughout follicular development in the unilaterally ovariec- tomized mares. In this regard, a temporal relationship between reduced develop— ment of a dominant follicle and high leV— els of progesterone has been reported in heifers (612) and llamas (5). Ovulation synchronization regimens that involve follicle suppression have a treatment-to-ovulation interval of 10 to 14 days (pg. 284 and pg. 186). Notable among the various regimens is daily administra- tion of combined progesterone and estra— diol. In one study (1265), the mean diame- ter of largest follicle at the end of the 10-day treatment was 12.1 mm and the mean interval to attainment of a 35 mm follicle was 12 days. Induction of luteoly- sis by an injection of PGan may result in a similar interval, unless a large domi— nant viable follicle is present at the time of luteolysis (pg. 285). 6.2E. Characteristics of a Major Wave A major wave at time of emergence (day of first detection, retrospectively) comprises several follicles which, by defi- nition, grow in concert until one becomes dominant (continues to grow) and the remaining follicles become subordinates (regress). This dissociation in develop- ment indicates that a selection mecha- nism operates for the designation of a dominant follicle. The mechanism for the primary wave occurs sometime prior to seven days before the primary ovulation. Research indicators for this conclusion are as follows: 1. Transrectal palpation studies. The increase in the number or diameter of large follicles beginning at mid-cycle continued until the beginning of estrus (575). The diameter of the largest follicle then continued to grow until ovulation, whereas the remaining large follicles decreased in diameter, beginning six days before ovulation (Figure 6.6). This was indicated by significant decrease in number of large follicles and in diameter of the second-largest follicle. This palpa- tion study was the first to indicate that follicle diameters begin to increase at mid—cycle and that a dissociation in numbers and diameters occurs by the beginning of estrus. In most mares (62% and 64% in two studies, 575), the follicle that ovulated was the one that was largest on the first day of estrus. In a study of 56 estrous periods in ponies, the mean interval from the day the preovulatory follicle became the largest follicle to the day of ovulation was 5.7 days (575). It should be noted, however, that these characterizations did not include follicular changes associated with the first ovulation of the ovulatory season; this is a special case (pg. 146). In a palpation study in jennies, a significant decrease in number of large follicles was first detected four days before ovulation (1679). 2. Ultrasonic monitoring of follicle pop- ulations. The number of large follicles and the diameter of the second—largest follicle began to decrease siX days before the primary ovulation (Figure 6.7; 1264). This observation confirmed the palpation studies and has been further substanti- ated by additional ultrasound studies (1206, 1208, 615). Characteristics of the Ovulatory Season 183 3. Ultrasonic monitoring of individual follicles. In the Quarter Horse and Appa- loosa study, the mean interval from cessa- tion of growth of the subordinates to the primary ovulation was 6.8 days (598). The corresponding interval seemed to be simi- lar in the Standardbred and Thoroughbred study (1484). This agrees with the conclu— sion from the earlier studies that selection of the dominant follicle becomes manifest 6 or 7 days before ovulation. Follicular waves in both studies appeared to be characterized by the syn- chronous emergence of a dominant follicle (identified retrospectively) and in most cases, subordinate follicles. The subordi- nate follicles grew at a rate corresponding to the dominant follicle but ceased grow- ing after several days. In addition, detectable follicles did not appear to emerge when the dominant follicle was in its active-growing phase. Perhaps (not adequately documented) the dominant follicle is similar in mares and cattle; in cattle, the growing dominant follicle is believed to play a role in regression of its subordinates and also in preventing emergence of a new wave (612). A major anovulatory and ovulatory wave during the first half of diestrus (secondary wave) seemed to follow a sim— ilar pattern in that the regression of sub- ordinates occurred before the dominant follicle reached its maximum diameter. It is emphasized, however, that a relation- ship between dominant and subordinate follicles has not been adequately docu- ment or characterized for major waves occurring during the first half of diestrus. 4. Studies of excised ovaries. In the his— tologic study (442), ovaries were obtained from pony mares on Days 6, 14, and 17 and on Days 17 to 21 (preovulatory mares). At Day 6, none of the mares had nonatretic follicles >10 mm, but two did have large (10 and 21 mm) atretic folli- cles. These probably represented atretic follicles from the ovulatory wave of the previous cycle. At Day 14, all mares had nonatretic follicles >10 mm, but the num- ber of atretic follicles was reduced. This probably represented development of a major wave. At Day 17, the largest follicle per animal had further increased in diameter, and some large follicles were in early atresia. In the preovulatory mares, the largest follicle was 232 mm in diame- ter, and the atretic follicles were smaller than at Day 14. These findings are com- patible with dissociation between a domi- nant follicle and subordinate follicles, beginning on Day 17 (equivalent to 7 days before the primary ovulation in ponies). A study on the nature of the selection mech- anism based on biochemical changes is discussed below. , 5. Forcing the emergence of a new m. When a large follicle was removed by ovariectomy, another follicle devel- oped from a small follicle (5 to 15 mm) rather than from a large follicle (15 to 25 mm; 444). This observation is compatible with the phenomenon wherein a domi- nant follicle reaches large size and the others (subordinates) become atretic. 6.2F. Efl'ects of Month There was significantly greater follicu- lar activity during the first half of the ovulatory season (May to July) than dur- ing the second half (August to October; Figure 6.7). The greater activity was shown by diameter of the largest follicle and number of follicles 2 to 5 mm and >20 mm. There were no interactions with day, indicating that the effects of season were distributed throughout the cycle. In this regard, the diameter of the preovula- tory follicle on Day -1 was greater for the first ovulation than for the second ovula- tion of the year (pg. 148). Significant dif- ferences due to month in the Day -1 diameter of the preovulatory follicle are shown (Figure 6.8). Thus, follicular dynamics during an interovulatory inter- val are profoundly affected by month within the ovulatory season—follicular activity is greater early in the season. 184 Chapter 6 SUMMARY: Folliculogenesis An overall working hypothesis is offered that seems reasonably consistent with the results reviewed in this section. The hypothesis is diagrammed in Figure 6.9. ° Mares have one or two major follicu- lar waves. A major wave is defined by the emergence of several follicles (e.g., 5 to 6). The follicles grow in synchrony with eventual dissociation into dominant folli- cles and subordinate follicles. A physio- logically selected dominant follicle from each major wave grows to large diameter (e.g., >30 mm), whereas the subordinate follicles begin to regress a few days after first detection. Dissociation into domi- nant and subordinate follicles, as indicat- ed by changing diameters, begins about seven days before the dominant follicle ovulates or, if anovulatory, before the dominant follicle reaches maximum diameter. ° In interovulatory intervals with two major waves, the wave that first emerges is called a secondary wave. Emergence (first detectable) occurs in late estrus or early diestrus. The dominant follicle sometimes ovulates during progesterone dominance (secondary or diestrous ovula— tion) and at other times becomes anovu- latory and regresses. The wave that con- tributes the primary ovulation (follicular ovulation) is called the primary wave. It emerges at mid—cycle, whether or not a secondary wave developed in early diestrus, and gives origin to the primary ovulation. 0 It is speculated, for the sake of conti— nuity, that small antral follicles (<10 mm, maximum diameter) are con- tinuously growing and regressing inde— pendently of the hormonal environment or reproductive status of the mare. The nature of the underlying growth and regression of follicles <10 mm is not _ knownwperhaps the follicles grow and regress independently of one another, or , groups of follicles maygrow and regress in synchrony. This continuous activity originates from a pool of many (e‘.g‘., 40,000) primordial follicles, and the pool is probably gradually depleted over the ” reproductive life of the mare. Atresia becomes a factor when the growing follicles begin to exceed 1 mm. When an adequate amount of circulating _ gonadotropins becomes available, follicles _ . are recruited from the dynamic pool“ of smaller follicles (<10 mm) to form the major waves 0 The active underlying small follicles are inhibited from emerging into a major Q _ wave of follicles while the dominant folli—L cle from a prevailing wave remains active. When a follicle reaches a large . size (e.g., 30 mm), it produces factors that directly or indirectly cause regression of other follicles of its wave (subordinates) and prevents the emergence of additional follicles. Thus, a major wave phenomenon is initiated. L ‘ ' Follicular activity is modified by month within the ovulatory season; activity is greater throughout the cycle during the first half of the season. Type or breed of mare also alters follicular ‘ activity. Thoroughbreds and Standard— breds have more follicular activity, as indicated by a greater frequency of di- estrous ovulations (first half of the interovulatory interval) and a higher multiple ovulation rate (second half 0f the interval) than do Quarter Horses and pony breeds. These, and probably other factors, alter activity so that some mares or some interovulatory intervals have two major follicular waves and others have only one. Characteristics of the Ovulatory Season 185 Follicle wave patterns 40 [x ov A 35 ,' 30 primary ovulation .’ subordinate \' follicle 25 20 15 B J .......... _ ,—ov % 40 Dominant -=_-.. 35 follicle, ",v...’ ,2 anovulatory ‘ E so ‘ E, 25 ' a - g 20 Secondary Primary 3 15 ' wave wave 0 _ C 4 . .-~ov ,—0V 0 Dpnlimant x o licle, r 35 secondary ,' ovulation 'x 30 \ :3 I Secondary Primary wave wave Diestrus Estrus -5 0 5 10 15 20 25 Number of days from a primary ovulation FIGURE 6.9. Postulated variations in follicular wave patterns during the equine estrous cycle. Each wave is char» acterized by a dominant follicle and sev- eral subordinate follicles. The subordi- nate follicles regress after a few days, Whereas the dominant follicle continues to grow. A wave that produces the primary ovulation (estrous ovulation) is a primary wave. A preceding wave occur- ring during early diestrus is a secondary wave. Sometimes a secondary wave does not occur (A), and at other times a sec- ondary wave forms and leads to a large anovulatory follicle (B) or an ovulatory follicle (secondary or diestrous ovulation; C). 0V 2 Ovulation. Preovulatory follicle (45) 48 J} 0) Diameter (mm) .5 h h N NHemApr May Jun Jul Aug Sep Oct SHem Oct Nov Dec Jan Feb Mar Apr FIGURE 6.8. Mean (irSEM) diameter of preovula- tory follicle on day before ovulation for April through October. Number of observations is shown at the upper limit of each bar depicting the stan- dard error of the mean. Diameters were significant- ly greater in May than in June, July, August, and September. From (617). 6.3. Selection of the Dominant Follicle The dominant follicle must escape or be protected from the normal degenera- tive process (atresia) which affects the subordinates as well as predecessors in the underlying or basal population. The biologic reason for the waste of many fol— licles during the estrous cycle is unknown. Perhaps the waste reflects a litter-bearing stage in early evolution and serves no real function in today’s mare. A compromise may have been reached wherein the growth of many follicles is retained for an important hormonal con- tribution, and once this necessity is filled in a given estrous cycle, the excess folli- cles are eliminated. That is, many folli- cles may be needed for the hormonal role of the ovaries, but not for the gamete- genic role. The nature of the mechanisms involved in the biologic selection of a dominant fol- licle, whether ovulatory or anovulatory, is 186 Chapter 6 one of the most perplexing research prob- lems in reproduction. A mathematical theory has been proposed wherein an interacting follicle population results in a maturing follicle (or follicles for litter- bearing species) while other members of the population undergo atresia (927). Based on studies in rabbits and monkeys, estradiol is believed to be one of the chemical messengers used by follicles to communicate their maturity to the pitu- itary and therefore to each other. The mare should be used as an experimental model for such studies because of avail- able monitoring techniques, the long interval from beginning of estrus to ovu- lation, the large size of the preovulatory follicle, and the predictability (according to diameter) that a given follicle will ovulate. 6.3A. Atresia Follicular atresia is common to all species (review: 927). It is estimated that >99% of follicles in human ovaries become atretic. In mares, approximately 50% (1175) to 75% (446) of the follicles at a given time, regardless of size classifica- tion, are undergoing atresia. The greater number of atretic follicles is a reflection of slower changes in size during atresia than during growth (e.g., 1.5 mm/day during atresia and 3.5 mm/day during growth; 1206). The biochemical events that determine whether a given follicle will become atretic are unknown. Work has been done in mares on the biochemi- cal and histologic differences between Viable and atretic follicles (334, 296). Follicular fluid of Viable follicles con— tained significantly higher concentra- tions of estrogen and progesterone but lower concentrations of prostaglandin Fm. Estrogen was the best indicator of Viability. Results further indicated that a factor was present in equine follicular fluid from preovulatory follicles that caused degeneration of granulosa cells in tissue culture (296). It was not determined whether the factor was a cause of atresia. It also was shown that the follicular fluid from Viable follicles contained more inhib- in-like activity than the fluid from atretic follicles. Histologic signs of atresia have been developed for bovine follicles (651); the histologic signs preceded loss of steroidogenic function. Histology of atre- sia of equine follicles is discussed and illustrated in Chapter 5 (pg. 137). 6.33. Time of Selection As discussed previously (pg. 182), studies involving follicular monitoring by trans- rectal palpation and ultrasonography and by gross and histologic examination of ovaries all indicate that selection becomes an identifiable phenomenon 6 or 7 days before ovulation or before an anovulatory dominant follicle reaches the end of its growth phase. This is the approximate day when the dominant follicle is identifi- able by its largest size and, when individ- ual follicles are monitored, the approxi- mate day when subordinate follicles cease to grow. The day that selection becomes manifest does not necessarily define the day that selection occurred; this aspect of the phenomenon could have occurred earlier. 6.30. Methods of Study Administration of pituitary extracts. Equine pituitary extracts have been used to study the responsiveness of groups of large follicles (1819). It was assumed that if selection had not been triggered before treatment, most of the large follicles would respond, whereas, if selection had already occurred, only one follicle would respond. This premise is tenuous, however, in that follicles in early atresia (already selected against) still may be able to respond to a stimulant and ovu— late (270). The follicles that were >10 mm on Day 15 represented the number of fol- licles that could be induced to ovulate; there was a positive relationship between Characteristics of the Ovulatory Season 187 number of >10 mm follicles at Day 15 and number of induced ovulations. A substan— tial size difference between the two largest follicles at Day 15 indicated that multiple follicles no longer were respon- sive to stimulation and that selection for a preferred follicle and against all other follicles already had occurred. The differ- ence in diameter between the largest fol- licles was significantly less for mares that responded with multiple ovulations than for mares that had a single ovulation (mean differences: 2.8 versus 7.7 mm). Administration of pituitary extracts after follicle suppression. Follicular devel- opment in Quarter Horse mares was sup— pressed with an estrogen-progesterone regimen to remove the complications that would be associated with a large fol- licle or a corpus luteum (1265). Mares then were treated with pituitary extract after the end of the steroid regimen when the largest follicle first attained 15, 20, 25, or 30 mm (n=6 per group). This experiment, like the above- described experiment, was based on the assumption that mares treated before physiologic selection would have a super- stimulatory response, whereas mares treated after selection would not. On the day the largest follicle reached 35 mm, there were more follicles >30 mm in the groups in which treatment started at 15 mm (2.5 i-O.6 follicles) and 20 mm (2.2 i0.6) than in the group started at 30 mm (0.8 i0.3). The group treated when the largest follicle reached 25 mm had an intermediate response (1.2 i0.4). Results indicated that selection of the ovulatory follicle, to the irreversible detriment of other large follicles, began to occur about the time the largest follicle was 25 mm (intermediate response) and was com- plete in all mares by the time the largest follicle was 30 mm. Administration of porcine FSH. A com- mercial porcine FSH product also has been used to study the nature of selection of the ovulatory follicle (801). The hypothe- sis was tested that the late diestrus decline in FSH is the main signal for initiation of the selection mechanism. A dose was given that was calculated to simulate the circulating FSH levels that precede the FSH decline in late diestrus. The hypothesis was supported since the FSH-treated mares developed an average of 3.8 follicles of preovulatory size. It was suggested that the FSH decline at the end of diestrus occurs when a follicle secretes enough inhibitor to lower plasma FSH concentration. The lowering of FSH then causes the remaining follicles to regress, at a time when the selected folli- cle has become FSH—independent. As indi- cated below, however, it is unknown whether FSH decline precedes or follows triggering of the selection mechanism. The temporal hormonal relationships during the time of selection are further discussed elsewhere (pg. 263). Changes in follicular cells and fluid. Kentucky scientists have studied the receptor and hormonal characteristics of presumptive ovulatory and nonovulatory follicles in order to identify the approxi— mate day of selection of an ovulatory folli- cle (501). Ovaries were removed on Day 14 (late luteal phase) and on the first and fourth day of estrus. One follicle in each of nine mares and two follicles in each of two mares were apparent preovulatory follicles with the following characteris- tics: 1) largest in diameter, 2) greatest amount of protein in the granulosum, probably reflecting increasing number of cells, 3) most vascular, 4) highest concen- tration of estradiol in the follicular fluid (30- to 50-fold greater), and 5) highest content of LH/FSH receptors in the gran— ulosum. These findings agreed with an earlier study that demonstrated that his- tologically viable-appearing follicles from estrous mares bound hCG and were high in estradiol (865). In the late luteal phase, one follicle (presumptive preovulatory fol- licle) had high thecal LH receptor con- tent, but the follicles did not differ in FSH receptor content (501). This LH- receptor characteristic may allow increased 188 Chapter 6 thecal responsiveness to LH and permit an increase in androgen biosynthesis and its aromatization to estrogen (pg. 62). Recently (1651), an immunochemical technique was developed for localization of LH receptors in equine follicles and could be used for further studies of the selection mechanism. Follicular fluid progesterone concen— trations tended to be higher in apparent preovulatory follicles (501). Androgen (testosterone and androstenedione) con— centrations were higher in the follicular fluid of the two estrous groups, and LH was highest in the follicular fluid from follicles on the fourth day of estrus. Size of follicle became indicative of the pre- sumptive ovulatory follicle on the first day of estrus, but biochemical character— istics (especially content of LH receptors in the theca) seemed to identify the ovu- latory follicle before the onset of estrus. These data indicated that selection became manifest by biochemical criteria at least 1 or 2 days earlier than the onset of estrus and preceded the earliest day used in the experiment (Day 14). The authors commented that these findings seemed similar to earlier findings in sheep and cattle. A recent study (1737) found that follicu— lar fluid of preovulatory follicles increased significantly in progesterone and testosterone 28 to 32 hours after reaching 35 mm. An injection of hCG caused an even greater increase in pro— gesterone. Another study (1743) indicated that the preovulatory follicular fluid of mares is increasingly immunosuppres- sive. Phosphatase activity has been mea— sured in equine follicular fluid (714); alka— line phosphatase activity was associated with atretic follicles and acid phos- phatase varied inversely with follicle size. SUMMARY: Selection Mechanisms ¥ Elucidation of the selection mechanism remains one of the great challengesin _ reproductive biology. The‘following reca: . pitulates the principal pOints of know- - ledge on the mechanism in mares: ° The follicle that leads to the primary ovulation originates from a major wave that emerges 12 days before ovulation. About six days before ovulation, the selection mechanism becomes manifest on the basis of follicle diameter, wherein one follicle of the wave becomes the ovu- latory follicle (dominant) and the other , members of the wave undergo atresia . (subordinates). ' ° The size criterion for identifying the ovulatory follicle has been demonstrated by transrectal palpation, ultrasonic imag- ing, and direct measurements of follicles in excised ovaries. 0 Using biochemical or stimulation cri— teria, selection occurred before Day 14 (equivalent to 8 days before ovulation). L The first detected biochemical indication of selection was an increase in LH recep- tor content in the thecal layer. The select- ed follicle soon began to produce estro- gens that exceeded the levels in other follicles of the wave by 30— to 50-fold. 0 Although selection becomes manifest by 7 or 8 days before ovulation, it is not known when selection is triggered and how the favored follicle is determined. 0 Two major waves develop during some cycles and selection, therefore, can occur during the hormonal environments of both early diestrus and late diestrus. ' The selection mechanism can be over- riden by the administration of pituitary preparations, resulting in multiple ovula- tions—that is, failure of atresia of subor- dinate follicles. These findings suggest that a decline in a gonadotropin, presum~ - ably FSH, plays a role in the process. Characteristics of the Ovulatory Season 189 6.4. The Preovulatory Period Behavioral (Chapter 3), hormonal (Chapter 7), morphologic, and ultrasoni- cally detectable events presaging the pri- mary ovulation have been well researched, motivated by a need for crite- ria that can be used to determine the optimum time to breed. Ultrasonically detectable changes in the preovulatory follicle were studied daily in 79 preovula- tory periods in 40 horses (1262). Averaged over all periods, the following significant changes were found in the preovulatory follicle: increasing diameter (mean: 3 mm/day), shape change from spherical to nonspherical (pear-shaped or conical), and increasing thickness of follicular wall Changes in preovulatory follicle (n=79) E 45 Size E. 40 E a) - 35 E .9 0 30 35 cc” Shape '2’ m a, 25 , .c a. 0G ‘55 15 d) 2 ri’ 5 4.5 Wall width 2 4.0 o 0 ‘0 3.5 3.0 -7 -6 -5 -4 -3 -2 -1 Number of days from ovulation FIGURE 6.10. Ultrasonically detectable mean changes in the preovulatory follicle in horse mares. Significant differences among days for each end point. Adapted from (1262). (Figures 6.10 and 6.11). A pronounced change in shape was shown by 85% of the follicles at least once during the pre- ovulatory period. The number of mares in which the follicle changed shape increased as ovulation approached. No significant changes were found for echogenicity (gray-scale value) of the follicular wall or fluid. The combination of diameter, shape changes, and thickening of the wall appeared to be valuable for assessing the status of the preovulatory follicle; however, no reliable ultrasoni- cally visible predictor of impending ovula— tion for individual mares was found. In retrospect, the diameter of the follicle was as useful for predicting impending ovula- tion as any of the other criteria. The growth rate (3 mm/day) and the shape changes in most (89%) of the preovulatory follicles have been confirmed (910). An ultrasonic pixel analysis procedure was developed for research assessment of preovulatory size and shape changes (1630). Cross-sectional area of the antrum of the follicle did not change over the 48 hours preceding ovulation. The folli- cles changed shape considerably (became Growth and shape of preovulatory follicle Growth rate: 3 mm /day Diameter (mm) 27 33 39 45 -7 -5 -3 -1 Number of days from ovulation FIGURE 6.11. Diagrammatic presentation of the growth rate and changes in shape of the preovulato- ry follicle. Note that the average growth rate was 3 mm/day for the seven days preceding ovulation. From (590). 190 Chapter 6 more flattened or irregular) during the three hours before ovulation. Follicles adjacent to the preovulatory follicle changed shape in the opposite direction (nonspherical to spherical) as ovulation approached. This was likely due to a decrease in fluid pressure within the antrum of the preovulatory follicle. Only the larger adjacent follicles that impinged on the preovulatory follicle appeared use— ful as indicators of pressure changes. Decreased pressure within the preovula— tory follicle also appears to lead to irregu— larities or lobules in the follicular outline, as observed ultrasonically and as detect- ed by a softening on digital palpation. In a study involving transrectal palpation at 12-hour intervals, approximately 90% of the follicles were softer at 12 hours than at 72 hours before ovulation (1230). Decreased intrafollicular pressure before ovulation has been demonstrated in rab- bits (1343); the decrease in pressure was attributed to a 6- to 10-fold increase in the distensibility of the follicle. 6.5. Ovulation 6.5A. Inequality in the Side of Ovulation There has been confusion whether the left or right ovary ovulates more fre— quently. The results of several studies were tabulated and examined by chi- square analysis in the first edition (575). The tabulated data indicated that ovula— tion occurred with slightly greater fre— quency from the left ovary. In addition, it has been stated that ovulation occurred 15% to 20% more often in the left ovary, as determined by rectal palpation of 574 mares in 804 estrous cycles (241). The ten— dency for ovulation from the left ovary was also found in a more recent study (54.6% versus 45.5%; 1838). The failure in some of the reports to find differences in side of ovulation could be attributed to the small numbers involved, but it is dis- concerting that several studies failed to find significant differences despite hun- dreds of observations (575, 719, 1759). The inconsistencies among studies suggested that other factors may have nullified the difference between sides for certain mare populations. There was, however, no indi- cation that breed was a complicating fac- tor. Month has been reported to affect side of ovulation (268), but this was not confirmed in other studies (1759, 1838). The puzzle, however, has been at least partly resolved by a study of breeding—farm records that considered reproductive sta— tus (maiden, barren, foaling; 579). The only group with a significant difference was the maiden mares; 62% ovulated from the left ovary and 38% from the right (Table 8.2, pg. 311). The reason for the greater activity of the left ovary in maiden mares, with loss of this asymmetry after pregnancy, is not known. Perhaps vascularization is initially greater for the left ovary. During pregnancy the inequal- ity in vascularization may be permanent- ly eliminated because of hypertrophy of the vessels in both ovaries in association with the massive development of large follicles and luteal structures. In contrast to horses, the right ovary ovulates more often in cattle and sheep. 6.53. Time of Ovulation in Relation to Estrus Many investigators have documented a closer association of ovulation with the end of estrus than with the beginning. The long estrus with the associated delay in ovulation is a challenge for the breeder who would like to breed just before ovula- tion. Cattle breeders have considerable advantage over their equine counterparts because of the short estrus followed by ovulation in cattle. The length of interval from the first day of estrus to ovulation is associated with several interrelated characteristics of the ovulatory follicle. These associa- tions were examined for 56 estrous periods Characteristics of the Ovulatory Season 191 in ponies (575). The relationships may be summarized as follows: 1) The larger the follicle on the first day of estrus, the shorter the interval to ovulation; 2) The greater the maximum diameter reached by the follicle, the longer the interval; 3) The greater the increase in size of the follicle, the longer the interval; and 4) The slower the growth rate of the folli- cle, the longer the interval. It can be con- cluded that an association exists between the duration of estrus prior to ovulation and the size and growth rate of the ovulatory follicle. Palmer (1202) con— cluded that the length of the interval from luteolysis to ovulation (defined as the follicular phase) is dependent on the degree of follicular development, based on circulatory estrogen concentrations, at the time of luteolysis; length of the follicular phase and estrogen levels were negatively correlated (r: -0.47). Furthermore, estrogen levels just before ovulation were positively correlated with the length of the follicular phase. Along these same lines, the diameter of the preovulatory follicle at the time of pro- gesterone decline was negatively corre- lated with the length of the follicular phase (1484). In the first edition (575), the percentage of ovulations that occurred on different days in relation to the end of estrus was tabulated for seven herds. The majority of ovulations (69% over all studies) occurred on the last two days of estrus, and 14% occurred after the end of estrus. When ovulation occurred after the end of estrus in one study (573), the mares fre— quently (55%) showed mild behavioral changes of subestrus before ovulation. Some investigators may have classified a portion of these postovulatory days as the continuation of estrus, pointing out, again, one source of variation. Cessation of estrogen production and onset of pro- gesterone production are associated with the termination of the estrous period and ovulation; more study is needed on the nature of this profound change in hor- monal production. The temporal association between ovu- lation and the end of estrus is good bio- logic economy, since breeding more than 12 hours after ovulation results in reduced pregnancy rates and increased embryo-loss rates (pg. 299 and pg. 537). There were no significant differences in diameter of the preovulatory follicle or in the number of large follicles between mares that were out of estrus the day after ovulation and those that were not (575). Nothing was found in the literature to suggest that the time of ovulation in relation to estrus is affected by age, breed, month, geographic location, nutri— tion, or other intrinsic or extrinsic factors. 6.50. Nature of the Ovulatory Process The biochemical and biophysical aspects of ovulation in the mare are neglected research areas. The massive amount of tissue and fluid in equine pre— ovulatory follicles would be a valuable resource for studies of this type. In one study, for example, the fluid volume of the preovulatory follicle of three pony mares averaged 34.3 i4.0 ml (mean iSD; 1269). The biophysical aspects of follicle rupture must take into account the short- term fragility of the rupture point. Many authors have attested to the great tensile strength of the equine preovulatory follicle and its defiance of digital rupture (e.g., 1230, 1395). Accidental rupture dur- ing ovarian palpation is not common and probably occurs only when the follicle is ready to rupture or is rupturing of its own accord. Presumably, rupture does not occur because of buildup of pressure with- in the follicle; instead, the tensile strength of the rupture point is reduced by proteolytic enzymes so that it can no longer withstand the low hydrostatic pressure from within. For that matter, the consistency of the equine preovu- latory follicle often decreases (softens) rather than increases as ovulation approaches (pg. 189). 192 Chapter 6 The ovulatory fossa. The most distinc- tive feature of ovulation in the mare, com- pared to that in other species, is the occurrence of ovulation from a specialized area, the ovulation fossa (pg. 14). Some older reports (922, 1847) maintain that ovu- lation occurs from practically anywhere on the surface of the ovary, but these statements can be disregarded. The peculiar anatomy of the equine ovary (restricted location of germinal epitheli- um and infundibulum, location of the vas- culature; pg. 16) is not compatible with the occurrence of ovulation beyond the limits of the fossa. In thousands of specimens examined by various investigators (575, 188), the ovulation papilla or the process of the gourd-shaped corpus luteum always, with a single traumatic exception (575), involved the ovulation fossa. The preovulatory follicle may reach the fossa by Virtue of its relatively large size, or special mechanisms may be pre— sent wherein the follicle develops along a line of least resistance so that a portion of it reaches the fossa. Bands of connec- tive tissue radiate from the fossa toward the greater ovarian curvature, and it has been suggested that these bands provide natural boundaries for the follicle’s growth toward the fossa (1292). One worker reported that the leading edge of the follicle consisted of thecal hyperpla- sia and suggested that the leading edge of thecal cells prepares a path of least resistance (687). Another worker indicat- ed that a thecal cone exists in many species, including horses, and provides a pathway to the ovarian surface (1568). It is emphasized, however, that it has not been adequately documented that spe- cial mechanisms other than increasing size are involved in the impingement of the preovulatory follicle upon the ovula- tion fossa. Following ovulation, the pyriform fol- licular cavity becomes luteinized, and a distinct neck-like luteal process marks the path from the fossa to the remainder of the gland, resulting in a gourd-shaped or mushroom-shaped corpus luteum. A dome of the follicle and the ovulation papilla of the newly forming corpus luteum may be seen at the fossa before and after ovulation, respectively. Sur- prisingly, these observations have failed to spark the imagination and interest of those in need of a potentially fruitful basic research area. Here again, the mare offers a biologic model that could con- tribute to basic knowledge. The occur- rence of ovulation from a relatively small, predictable area and the well-marked path of the follicle through a large mass of ovarian tissue should be useful in basic studies of the mechanisms of ovulation. The question of nocturnal ovulation. It has been reported that ovulation tends to occur at night (1803). Seven mares were fol- lowed through three or more estrous cycles and 23 of 25 ovulations occurred at night (11 pm. to 7 a.m.). However, these results were not confirmed in another study (623) involving mares of similar size. Mares were examined at 8 a.m. and 8 pm. Seventeen ovulations occurred during the day (8 a.m. to 8 pm.) and 13 occurred at night. Two other studies (cited in 855) have also failed to find an effect of time of day on the occurrence of ovulation. 6.5D. Clinical Prediction of the Imminence of Ovulation Diameter. An important aspect of intensive breeding—farm reproductive management involves judgment on when ovulation will occur; such judgment is necessary for breeding close to the time of ovulation, yet using a minimal number of breedings. Size of the follicle is the simplest and probably the most exten- sively used clinical criterion. Size can be measured more accurately by ultrasound than by palpation (592). For this and other reasons, an ultrasound scanner is a use- ful breeding aid. In two studies in horses (592), diameter was estimated by calculat- ing the average of two lines of measure- ment; the first line was the greatest Characteristics of the Ovulatory Season 193 distance between opposite walls, and the second line was the greatest distance at right angles to the first line. There were 78 and 103 single ovulations in the two studies. None of the follicles ovulated before reaching 35 mm. It is emphasized that the diameters were made by averag- ing wall-to-wall distances (antral diame- ter) in two directions. Results of other measuring techniques may differ by sev- eral millimeters. Therefore, each operator may want to develop and standardize the measuring system and determine the breeding criterion to be used for that system. In using diameter as a breeding crite- ria, time of year should be considered since diameter of the preovulatory follicle is affected by month (pg. 183). In addition, double preovulatory follicles were smaller on the day before ovulation, compared with single preovulatory follicles (pg. 221). In association with physiologic selection of the ovulatory follicles, the large nonovulatory follicles begin to regress (pg. 182). Sometimes decreased diameter of the nonselected follicles can be used as an indication of impending ovulation. Consistency. Prediction of the time of ovulation by changes in consistency of the large follicle or the ovary has been advo- cated by some and questioned or denied by others. The most extensive study of this possibility was performed by Parker (1230). Follicular consistency was deter- mined every 12 hours during estrus in riding-type mares. Although the ovula- tory follicle was softer on the average at 12 hours than at 72 hours before ovula- tion, it was concluded that the technique was not sufficiently reliable for predicting the time of ovulation in individual mares. Sensitivity. It has been noted that the mare may show varying degrees of sensitivity to palpation as ovulation approaches; the mare may raise a rear limb toward the abdomen, paw or step in place, or turn her head toward the exam- iner (1230, 1638). In this regard, severe signs of colic were temporally associated with ovulation on repeated occasions in two mares (347). Altrenogest (a progestin; pg. 282) prevented ovulation and the asso- ciated recurring bouts of colic. Perhaps such pain is due to stretching of the vis- ceral peritoneum surrounding the ovary. These authors noted that abdominal pain in association with ovulation is common in women (347). Electrical resistance in vagina. In the past few years, intravaginal probes have become available for estimating the imminence of ovulation by measuring the electrical resistance of vaginal mucus (257, 498). As for all techniques discussed here, biologic variation is a challenging obstacle. 6.5E. Follicle Evacuation Evacuation patterns. The extent and pattern of release of follicular fluid at the time of ovulation were studied by trans- rectal ultrasonography, using frequent and sometimes continuous observations (1631, 1628, 1069, 1514, 283). Two distinct evacuation patterns were observed— abrupt and gradual (Figure 6.12; 1631, 1628). Abrupt and gradual evacuation involved a 90% and 50% decrease of the antral area within 60 seconds, respective- ly. In the gradual process, complete evac- uation (as defined) required 6 to 7 min- utes. The reason for the two apparently distinct patterns is not known.- A small residual fluid area persisted in the col- lapsed follicle in 7 of 10 evacuations after the defined end of evacuation (1629). The residual fluid gradually diminished, dis— appearing 0.5 to 20 hours later. Because a large portion of fluid evacua- tion time may involve five minutes or more, one will occasionally observe a folli- cle that is in the evacuation process. Evacuation can be suspected to be under- way when the follicle is reduced in size and is irregular in shape. Examination a few minutes later (and sometimes a few seconds later if early in the process) may indicate further evacuation and confirm 194 Chapter 6 that ovulation is under way. Fate of discharged fluid. A fluid-filled area that appeared to be continuous with the fluid of the evacuating follicle was detected in the approximate location of the ovulation fossa immediately before or during 6 of 11 evacuations (Figure 6.13; 1631). The fluid collection subsequently disappeared at or shortly after the defined end of follicular evacuation. In four mares, the fluid collection. appeared to be transient within the infundibulum or between the inner surface of the infundibulum and the ovarian surface of the fossa; its location could not be deter— mined precisely. In the remaining two mares, there was no detectable decrease in the size of the antrum at the time of the first appearance of the fluid collec— tion. These two nonechogenic areas may have been an extension or bulge of the ovulatory follicle into the ovulation fossa, or some discharge of follicular fluid may have occurred before the onset of evacua— tion was detected. There were no other indications that a detectable quantity of follicular fluid collected within the ipsilat- eral oviduct during or following follicular evacuation. Another group of workers (283) reported that a protrusion or break in the follicular wall, apparently toward 14 —L N _L D co Gradual evacuation Abrupt evacuation Cross-sectional antral area (cm2) 0': 0 20 60 Seconds after the onset of evacuation 100 140 180 220 260 300 340 380 420 the ovulation fossa, was detected 15 to 77 minutes before follicle evacuation. The collections of fluid that appeared to be external to the ovary (i.e., in the infundibular or oviductal area) did not, at any time, approach the quantity of fluid that had been discharged by the evacuat— ing follicle (1631). The largest collection was approximately 15% of the preovula- tory antral area. Unfortunately, the oviducts were not directly identifiable but should have been detectable if the lumen was filled with fluid, especially if the quantity approached that of the preovula- tory follicle. It does not seem likely that the discharged fluid was absorbed imme- diately at the evacuation site or within the oviduct or passed immediately through the oviduct into the uterus. For these reasons, it may be that most of the follicular fluid passed or filtered between the surface of the ovulation fossa and the oviductal fimbria into the peritoneal cav— ity; only a small amount, if any, accom- panied the oocyte into the oviduct. Pre- sumably, the collections of fluid in the area of the ovulation fossa and infun- dibulum during follicle evacuation were temporary accumulations associated with the filtering process. Follicle evacuation time in individuals FIGURE 6.12. Cross-sectional areas of the antrum in five mares in which follicular evacuation was observed continuously. Time 0 refers to the defined onset of evac— uation. Two evacuation patterns were observed: an abrupt decrease to less than 10% of the antral area during the first 60 seconds (solid lines) and a gradual decrease in which approximately 50% of the antral area remained after the first 60 seconds (broken lines). Adapted from (1631). Characteristics of the Ovulatory Season 195 6.6. Luteal Glands A temporary endocrine or luteal gland (corpus luteum) forms at the site of ovula— tion and proceeds through developmental, maintenance, and regressive stages. 6.6A. Detecting Ovulation Transrectal palpation. Transrectal pal- pation is useful for determining that ovu- lation has occurred. As part of the process of ovulation, follicular fluid is discharged along with the ovum through the ovulation fossa, and the wall of the follicle collapses. Immediately after ovulation, the intact surface of the ovary can be digitally depressed into the former follicular cavity, causing a large, soft depression. Some dif- ficulty can occur in detecting ovulation, especially when the interval between pal— pations is more than 24 hours; the follicu- lar cavity may begin to fill with blood and may be confused with an unovulated folli- cle. Daily transrectal palpation of ovula- tion was 91% accurate when based on a depression in the ovarian surface and 60% accurate when based on a firm plumlike structure or the disappearance of a previ- ously identified follicle (1090); 38% of the palpation diagnoses of nonovulation were in error on the basis of a rise in proges- EVACUATING FOLLICLE FIGURE 6.13. Sequential ultrasonograms taken during follicular evacuation showing the development of a fluid collection apparently outside the evacuating follicle in the area of the ovulation fossa. (A) Preovulatory follicle approximately 5 min before the onset of evacuation. (B) Evacuating follicle shortly after the onset of evacuation. The precise time of onset was not determined. (C—F) Evacuating follicle taken 37 min, 60 min, 60 min+15 sec, and 65 min after image B. Note that the fluid collection in the area of the ovulatory fossa is con- tinuous with the antral fluid through a distinct channel. The fluid collection increased (B,C) and then decreased (C—E) in size as evacuation progressed and was not detectable after the fluid from within the antrum was gone (F). From (1631). pof = preovulatory follicle fc ef = evacuating follicle fluid collection outside of evacuating follicle. 196 Chapter 6 terone above 1 ng/ml. These authors sug— gested that in programs based on transrec- tal palpation many second matings may be done unnecessarily because of misdiag- noses that ovulation had not yet occurred. Transrectal ultrasonic imaging. Ovula— tion is readily detected ultrasonically by the disappearance of a large follicle that was present at a recent previous exami- nation (592, 590). In addition, even Without knowledge that a large follicle was pre- sent, an ovulation site usually can be detected ultrasonically on the day of ovu- lation (Figure 6.14,A; 592) and subse- quently can be diagnosed or confirmed by the formation of a corpus luteum (pg. 197). The prolonged period of development of large follicles prior to the first ovulation of the year is a particularly challenging theriogenology problem. During this time, a clinician who is limited to tactile exami— nation may have doubt whether or not an ovulation has occurred. However, visual inspection by ultrasonography will pro- vide clarification. Clearly, ultrasonogra- phy is the method of choice for determin- ing that ovulation has occurred, especially for research purposes. Progesterone values. Monitoring of pro- gesterone values in blood and milk (909) also has been used to estimate the day of ovulation. An enzyme-linked immunosor- bent assay was used for plasma proges- terone determinations in an embryo transfer program (749). Accuracy of esti- mating the day of ovulation was 88% for quantitative progesterone assay and 86% for teasing (first day of diestrus). The first day that the progesterone value increased by at least 0.5 ng/ml tended to be the most useful criterion. Progesterone assay was judged to be sufficiently accurate for embryo transfer programs when transrec- tal palpation or scanning was not avail— able. In this regard, rapid progesterone and LH assay kits are available as a reproductive management aid (pg. 72). Coagulation time. Use of a quick test system for measuring thromboplastin time (an assessment of the blood coagu- lation system) also has been investigated for determining whether ovulation has occurred (662). Values increased signifi- cantly from Day —4 to Day 0. It was judged that the changes in hemocoagula- tion activity were not adequate, howev- er, for predicting day of ovulation. In an abstract (1465), it was concluded that ovulatory hemorrhage produces measur- able alterations in hemocoagulation, but none of several measuring techniques tried by these workers allowed reliable prediction that ovulation was imminent or had occurred. CORPORA LUTEA FIGURE 6.14. Ultrasonic appearance of corpora lutea A. Ovulation site on Day 0. Arrows indicate residual fluid in the evacuated follicle. B. Solid corpus luteum at mid—diestrus. C. Corpus hemorrhagicum on Day 3 resulting from filling of the developing corpus luteum with blood. Characteristics of the Ovulatory Season 197 Other indicators. Laparoscopy is an alternative to palpation for detecting the time of ovulation for research purposes (710). It has been concluded that the quan- titation of urinary and vaginal protein, glucose, and pH (772) and the use of vagi- nal smears (85) are of no value for detect- ing ovulation or estrus. Body tempera- ture and heart rate changes also were not associated with ovulation (485, 79). 6. GB. Transrectal Palpation Characteristics Morphologic organization is gradual and the new corpus luteum progresses through changing consistencies of mushy, spongy, and meaty (1230). Maximum diameter of the corpus luteum has been variously reported as 50% to 75% of the diameter of the pre— ovulatory follicle, based on palpation. In a series of 88 ovulations and subsequent luteinizations, the maximum mean diameter of the new corpus luteum (38 mm) was 79% of the diameter of the preovulatory follicle (48 mm), based on transrectal palpation (575). There is disagreement on the palpabil- ity of the corpus luteum in mares. It has been reported that the luteal structure may be distinguished by transrectal pal— pation for two days (382), three days (722), 10 to 12 days (988), 8.9 i3.7 days (mean iS.D.; n=338; 780), 8.7 days (range, 1 to 19 days; 29), and in some cases, for the entire functional life span (1533). In a series of 88 cycles in pony mares, the percentages of cycles in which the luteal gland was distinguishable on the indi- cated number of days were as follows: 3 days, 94%; 6 days, 79%; 9 days, 50%; 12 days, 22%; and 15 days, 12% (575). It is emphasized that the belief that one has detected the luteal gland in mares by transrectal palpation can be a reflec— tion of prior knowledge of the location of the structure. Palpation of the luteal gland for the first several days is much easier in mares than in cattle. Palpation thereafter becomes progressively more difficult and more prone to error in mares than in cattle. Detection of the time of regression of the corpus luteum by transrectal palpation is certainly a far more useful research or clinical tool in cattle than in mares. 6.60. Ultrasonic Morphology Detectability. One of the major uses of ultrasonography involves the immediate detection and evaluation of the luteal gland. The corpus luteum is detectable throughout its functional life during diestrus and pregnancy using a 5 or 7.5 MHz transducer and high-quality scan— ner. In a study (1261) of 55 interovulatory intervals (mean length: 22 days), using a 5 MHz transducer, the corpus luteum was identified for an average of 17 days. The gland was visible in all mares from the day of ovulation until at least halfway through the interovulatory inter- val. In an earlier study, in 19 mares (615) using the same scanner but with a 3.5 MHz transducer, the corpus luteum was identified for a mean of only six days (range, 4 to 8 days). Development of central clot. Before the availability of ultrasound scanners, it was believed that the equine luteal gland con- sistently went through a developmental stage characterized by a large, fluid-filled central area (corpus hemorrhagicum, 575). In a recent ultrasound study (1261), it was found that many luteal glands did not develop a central area that exceeded 10% of the size of the' gland. The central areas were first seen on Day 0 (day of ovulation; 28%), Day 1 (62%), Day 2 (6%), or Day 3 (4%). In subsequent studies (1629, 1632), the time and incidence of fluid accumula- tion was studied in detail. Combined over the two studies, a detectable non- echogenic central area (fluid) did not develop after loss of the apparent residual follicular fluid in 7 of 22 glands (32%); these glands remained solid throughout their life span (Figure 6.14,B). That is, a 198 Chapter 6 detectable corpus hemorrhagicum (Figure 6.14,C), even of small size, did not devel- op. The time of formation of central fluid areas in the remaining glands is shown (Figure 6.15). In the two studies, detectable fluid began to accumulate at 20 and 30 hours postevacuation, and the first significant increase occurred at 32 and 36 hours, respectively. The central areas developed gradually. After first detection (20 hours), the central areas increased in size for 52 hours, reaching maximum at 72 hours after follicle evacu- ation. The extent of maximum fluid accu- mulation varied in area from 0.5 cm2 to 11.6 cm2 (equivalent to diameters of 8 to 38 mm). Echogenic lines within the cen— tral area were first detected, on the aver- age, 44 hours after follicle evacuation. On the basis of a previous waterbath study (615), the network of echogenic lines with- in the central area was attributable to clotting and fibrinization of the contents. That is, the central areas were likely of vascular origin. Throughout diestrus, the central areas became progressively orga— nized, as indicated by the development of echogenic spots, bands, or networks. The relative proportion of the gland contain- ing a central clot decreased progressively after Day 3, but the clot usually remained visible throughout diestrus. Thus, the progressive fibrinization of the clot and its decreasing proportional size may aid in estimating day of estrous cycle. Functional importance of central clot. The development of a central fluid area did not alter the length of time the luteal structure was ultrasonically Visible and did not alter the length of the interovula- tory interval (1261). Cross-sectional area of the luteal tissue and the intensity of luteal echogenicity did not appear to be influenced by the wide variability in size of the clot. Furthermore, in approximate- ly half of the mares with sequential ovu- lations, a central area formed after one ovulation but not after the other ovula- tion. Similarly, in approximately half of the mares that double ovulated, one ovu- lation was followed by the formation of a central fluid area and the other was not. These observations indicate that the for- mation of a central area is an incidental occurrence that is not functionally impor— tant. A recent study failed to find a rela- tionship between the size of central cavi- ties and circulating progesterone concentrations (1633). Hyperechogenicity. A peculiar ultra- sonic characteristic of the equine corpus luteum is the hyperechogenicity (bright white) of the developing corpus luteum (Figure 6.15). During the 3 or 4 days after ovulation, the gland may be obvious to the ultrasonographer because of the intense echogenicity of the luteal tissue, whether or not it contains a central non- echogenic clot. Increased echogenicity is sometimes seen during regression of the corpus luteum, probably due to increasing density of the gland. The echogenicity cri- terion does not always apply, however. In a series of 55 luteal glands examined daily, hyperechogenicity was seen in 88% of the developing glands and 36% of the regressing glands (1261). Development of central 12 fluid areas in individuals 8 32 56 80 104 128 Hours after onset of luteal development FIGURE 6.15. Cross-sectional areas of the fluid- filled central area of the luteal gland measured in seven mares that developed persistent central areas. Adapted from (1629) Characteristics of the Ovulatory Season 199 6.6D. Microscopic Characteristics In-depth light microscopy studies of the equine corpus luteum throughout the estrous cycle apparently have not been reported. Such studies are needed because the corpus luteum is the struc- ture most involved in the rhythm and control of the estrous cycle. In contrast, the corpus luteum in many other species has been intensively investigated from many angles. More is known about the microscopic anatomy of the corpora lutea in red squirrels (1122), for example, than in mares. An early study of the micro- anatomy of the equine corpus luteum was done at Glasgow University (687). South African workers (1673) extended the earlier descriptions. A fundamental point that needs further investigation concerns the luteinization of the thecal versus granulosa cells. According to both the South African and Glasgow workers, the theca interna does not contribute to luteal tissue in the mare as-it does in other farm species. These workers have concluded that the thecal cells degenerate and are replaced by hypertrophied fibroblasts. They further suggested that the hypertrophied fibroblasts can be confused with the thecal cells, giving the impression that thecal cells contribute to the luteinized tissue. The following descriptive account is based primarily on the South African report supplemented by the Glasgow report and a recent report involving dis- persed luteal cells (1741). To provide con- tinuity and to trace the origin of the cel- lular components of the corpus luteum, the following description begins with the preovulatory follicle: 1. A few days before ovulation. Fibro— blastic cells of the theca interna have pro- liferated and enlarged into oval or round cells with light-staining nuclei (Figure 6.4). These thecal cells are likely secreto- ry. The granulosa cells cease dividing about this time. 2. Just before ovulation. The thecal cells are in various stages of degeneration (condensation of cytoplasm, eosinophilia, pycnosis, fragmentation, phagocytosis). The granulosa cells change from a com- pact mass to a lacelike layer of stellate or spindle-shaped cells. These cells secrete a mucoid substance that lines the antrum. 3. Shortly after ovulation. The granu- losa cells are about 10 um in diameter with a dark-staining nucleus of 5 to 6 mm. The collapsed inner wall of the follicle forms folds consisting of central cores of stromal tissue with accompanying distended blood vessels. 4. 24 hours after ovulation. The granu- losa cells have enlarged to 15 mm. The nuclei are vesiculated, and the cytoplasm contains fine vacuoles, indicating luteinization and secretory activity. By this time, progesterone level in the peripheral circulation is rising (pg. 238). The central cavity may contain consider- able blood, as described in the previous section. Folds or buds of stromal tissue have begun to grow into the granulosa or luteinizing tissue accompanied by prolif- erating capillaries. The capillaries are surrounded by the hypertrophied fibro- blasts that apparently replace the degen- erating thecal cells. These fibroblasts could be confused with luteal cells, but they are more vesicular, have no promi- nent nucleoli, and have small, spindle- shaped cytoplasm; large luteal cells are polyhedral with vacuolated cytoplasm and a single, round or oval eccentric nucleus containing one or more nucleoli. 5. Day 3. Luteinization of granulosa cells is complete. The proportion of large cells in the total population of luteal cells (large plus small) in dispersed cell suspensions was recently reported as 46% on Days 4 and 5 (1741). 6. Day 9. Maximum hypertrophy of granulosa cells has been reached; mean diameters of 38 and 42 um have been reported. The nuclei were 10 um. It has been stated (470) that granulosa luteal cells are the largest endocrine cells in the 200 Chapter 6 body. In addition to the large, light- staining luteal cells, small cells account for about 15% of the luteal cell popula- tion at this time. These small cells (mean diameter: 11 um) have homoge— neous eosinophilia and contain a con- densed, dark—staining, often elongated nucleus of 5 to 6 um. It has been pro- posed that the small dark cells repre- sent a resting stage before conversion into the large, lighter cells (1673). By this stage, the hypertrophied fibroblasts have disappeared without any indica- tion that they were converted into luteal cells; they probably contribute to the development of the stroma of the corpus luteum. 7. Day 12. The large luteal cells begin to decrease in diameter. The proportion of dark cells has increased to about 25%, and the dark cells are probably no longer being converted into the large light cells. A significant increase in proportion of small luteal cells as the corpus luteum ages has been confirmed (1741); the pro- portion of large cells found by these workers for Days 12 to 13 was 24%. Curiously the increased proportion of small cells as the corpus luteum ages in the mare is in contrast to other species in which the proportion of small cells decreases (cited in 1741). 8. Day 16. The diameter of the large light cells has decreased to about 20 pm. 9. Day 20. Next ovulation is approach- ing, and most of the large cells are in an advanced stage of regression (cytoplasm condensed or fragmented, nucleus shrunken and pycnotic). Most of the luteal cells that remain are of the small, dark type. The intercellular spaces are filled with stromal cells that have grown into the luteal tissue from the trabeculae. The blood vessels are under- going sclerotic changes and become obliterated. Photomicrographs of equine corpora lutea are shown in Figures 6.16 and 1.14. Electron microscopic characteristics. Electron microscopic examination of luteal cells has been used in many species to study ultrastructural details or cytolo- gy. The cytologic features of luteal cells in nonequine species have been reviewed (470). An initial study of the changes in ultrastructure of the equine corpus luteum during the estrous cycle has been reported (951). Corpora lutea were exam- ined on Days 3, 9, and 15. By Day 3, full luteinization had occurred, as indicated by a well-developed endoplasmic reticu- lum and numerous lipid droplets. By Day 9, there were few lipid droplets, and there was further hypertrophy of the endoplasmic reticulum. Signs of degener- ation of the luteal cells were evident at Day 15, with indications of cholesterol buildup and accumulations of collagen in the perivascular spaces. The study sup- ported the possibility that luteal regres— sion results from a reduction in blood supply due to sclerotic arterial changes. 6.6E. Gross Characteristics Intact ovary. The intact luteal gland can be identified during surgery by a combination of visual and palpation aids that have sufficient reliability for research purposes (e.g., marking with India ink for later identification), espe- cially for the first several days after ovu— lation. In many instances, a distinct luteal papilla will be seen in the ovulation fossa. In the absence of a papilla, a dark brown spot may mark the site of ovula- tion deep in the fossa. An enlarged por- tion or pole of an ovary, which does not have the translucent appearance of a fol- licle but has the palpable characteristics discussed above, is an additional aid for identification. of the Ovulatory Season 201 g§w ‘ ‘ “i t‘ b‘: ~ - ‘ a. .1 MID-DIESTRUS DAY 7 OF ESTRUS FIGURE 6.16. Photomicrographs of luteal structures showing a low-power View of a fold of developing luteal tissue, two high-power views of mature corpora lutea, and corpora albicantia from Day 2 of estrus and Day 7 of estrus. 202 Chapter 6 Sectioned ovary. Colored photographs of sectioned ovaries, exposing the luteal glands, are shown (Figure 6.17). After midsagittal sectioning, an early corpus luteum that has a large central cavity (corpus hemorrhagicum) has the appear- ance of a blood clot (Figure 6.17,C,D). Because the red cells in mares settle rapidly, a portion of the corpus hemor- rhagicum may occasionally be relatively free of red cells so that one portion is dark red and another whitish or buff- colored (clotted plasma). Presumably, such layering is more likely to occur when the structure fills rapidly with blood, whereas gradual filling leads to a more homogeneous, dark-red structure. In corpora lutea that fill with blood, the cen- tral portion may be organized and dark red or pink, apparently depending upon the amount of red cell pigmentation retained in the fibrinous meshwork. The area of luteinization may be dis- tinguished as a buff— or flesh-colored, peripheral, folded zone that increases in size as the luteal structure ages (Figure 6.17,D,G). The folded or trabec— ulated appearance, when Viewed on cut surface, is very distinctive in the mare and is in contrast to the corpora lutea of other species. The folding results from collapse of the extremely large equine follicle at ovulation so that the follicu- lar wall is thrown into folds that project toward the central cavity. Luteinization may involve the entire structure so that the mature corpus luteum has a solid, although trabeculated, appearance (Figure 6.17,E,F,H). The corpus luteum is irregular, mushroom—shaped or gourd-shaped (Figure 6.17). The lutein- ized process, which leads from the body of the gland to the ovulation fossa, varies considerably in length and promi- nence. Several slices may be needed to expose the process. The process may end beneath the surface of the fossa with closure of the ovulation point, or it may project beyond the surface of the fossa in the form of a papilla. Corpus albicans. As regression begins, the luteal structure takes on a lighter appearance because of decreasing vascular- ization and increasing connective tissue organization. By the time estrus occurs, the luteal structure is straw colored (Figure 6.17,B). Regression of the corpus albicans continues during the subsequent diestrus. As the size decreases, the pigmentation residues are condensed into an increasing- ly smaller mass. The structure consequent- ly becomes darker, with hues of orange, red, or brown (Figure 6.17,F). The corpus albicans eventually becomes a highly pig- mented small streak or oblong structure with the long axis oriented toward the ovu- lation fossa. In 20 pony mares examined at various days of diestrus, a mean of 2.2 cor— pora albicantia (range: 1 to 4) were readily visible during mincing of the ovaries (575). The structures were embedded and not readily removed from the surrounding stroma. Most were oblong, and the largest dimension varied from 2 to 15 mm (mean dimensions: 3.8 x 5.8 mm). In horse mares slaughtered on different days after the beginning of estrus (1732), the mean weight of the luteal structure believed to be from the previous cycle (corpus albi- cans) was as follows: Day 2, 1.83 g (n=8); Day 4, 1.92 g (n=7); Day 7, 0.78 g (n=8); Day 11, 0.50 g (n=8); Day 17, 0.20 g (n=2). The weight of the corpus albicans decreased and color changed from pale yel- low to light brown as the cycle progressed. PHOTOGRAPHIC PLATES The series of plates on the following four pages depicts the gross appearance of whole and sectioned ovaries (Figure 6.17), Videoendoscopic views of cervix (Figure 6.18) and uterus (Figure 6.19), and histology of the endometrium (Figure 6.20). Figure 6.17 was referenced above and Figures 6.18 to 6.20 will be refer- enced in subsequent sections. Figures 6.18 to 6.20 were prepared by G. P. Adams. 203 Characteristics of the Ovulatory Season (B, E-H) d ovaries 10118 . Some midsagittally sect were opened like a book so that the ovulatory fossa is in the center of each specimen. A corpus alb indicated by an arrow (B, D, F). 1ne ovaries FIGURE 6.17. Whole (A) and sectioned (B-H) equ leans is 204 Chapter 6 \::W\\: Egg Characteristics of the Ovulatory Season 205 FIGURE 6.18. Facing page. Videoendoscopic Views of the cervix. A. Estrus cervix showing moderate swelling of cervical folds. B. Estrus cervix with extreme relaxation with ventral cervical folds lying on the vaginal floor. C. External cervical os of estrus showing strand of thin clear mucus between cervix and finger. D. External cervical os of diestrus with strands of thick cloudy mucus between cervix and finger. E. Diestrus cervix showing puckered even-appearance of cervical folds. F. Anestrus cervix. FIGURE 6.19. Videoendoscopic views of the uterus. A. Estrus uterus dilated with saline showing the corpus-cornual junction. Note the swollen appearance of the septum and endometrial folds. B. Estrus uterus dilated with air showing extreme swelling of endometrial folds. C. Diestrus uterus dilated with air giving a flattened appearance to the endometrial folds. D. Anestrus uterus dilated with air. The endometrial folds are indistinct. Chapter 6 206 114~9w€f$lk§§ Characteristics of the Ovulatory Season 207 FIGURE 6.20. Facing page. Histologic appearance of endometrium during estrus (A-D), diestrus (E-F), and anestrus (G-H) using a hematoxylin and eosin stain. A. Magnification, x 40. Swelling due to interstitial edema results in an overall appearance of sparse popu- lation of endometrial glands. B. Magnification, x 400. Note cilia on the tall columnar luminal epithelial cells. C. Margination of polymorphonucleocytes in blood vessels. D. Nesting (normal) of uterine glands during early estrus. E. Magnification, X 40. Diestrus endometrium showing lack of edema (no spreading of tissues), which results in a dense population of uterine glands. Compare with estrus endometrium (A). F. Magnification, x 200. Diestrus endometrium showing density of glands. G. Magnification, x 40. Anestrus endometrium showing shallowness of strata compactum and spongiosum and straightness of endometrial glands. H. Magnification, X 400. Anestrus endometrium showing cuboidal to low columnar epithelium. TABLE 6.3. Histology of Tubular Genitalia during Estrus Organ/tissue Histologic features during estrus compared to diestrus Oviduct Slight increase in height of epithelium, size of capillaries, and number of stromal leucocytes Endometrium Luminal epithelium Height Increase of about 10 um Staining Paler with vacuolation of basal 1/3 Nuclei Larger, lighter, and move from central to basal. Variation of Lamina propria position of nuclei may result in a pseudostratified appearance Leucocyte accumulation in venules toward uterine lumen. Congested Less tortuous, therefore fewer cross sections and loss of the diestrus Occasionally form clusters perhaps due to pressure of edema Staining Paler and more open due to edema Vessels Uterine glands Epithelium Taller, paler, with larger nuclei Shape string-of-pearls arrangement Size Larger, more open, may contain secretions Organization Cervix Epithelium Taller, more swollen, filled with mucus Mucus Layering on surface of epithelium Vaginal epithelium Thicker with more cell layers. Larger cells Basal layer Surface layer Leucocytes Vestibule Increases from 1 layer to 2 or 3 layers. Cells darker, more elongated, and nuclei are crowded May be slightly more cornified More numbers May be cornification and shedding of a few layers of epithelium 6.7. Tubular Genitalia Videoendoscopic Views of the tubular genitalia are shown in Figures 6.18 and 6.19. An impression of the histologic changes occurring when moving from diestrus to estrus can be gained by simul- taneous study of the photomicrographs (Figure 6.20) and Table 6.3. Many of the descriptions in this section are based on clinical observations. With a few exceptions, the rhythmic changes in the organs of the tubular genitalia have not been examined for statistical differ- ences, so the reader may want to reserve judgment on some of the subject matter. The studies of Andrews and McKenzie (85) involved large numbers of observations 208 Chapter 6 with objective measurements, but because of large variations and the absence of critical analyses, conclusions must be guarded. There is need for a crit- ical in-depth study of the rhythmic changes in the tubular genitalia of the mare with concomitant examination of changes in the concentrations of circulat- ing steroidal hormones. Discussion of the hormonal basis and the harmony of form and function for the changes described in this section will be found elsewhere (pg. 276). Certain aspects of the rhythmic changes in the tubular genitalia in mares have been reviewed (85, 190); references will be found to the older literature and to manuscripts written in languages other than English. A systematic method of examining the tubular genitalia, as well as the ovaries, has been reported (133, 644). 6. 7A. Histologic Changes Most histologic studies have used biop— sy techniques and have been limited to the endometrium and the cervical and vaginal mucosa. No report was found of quantitatively and statistically analyzed data for the various tissue layers throughout the estrous cycle. The conclu- sions summarized here are based on the impressions and interpretations of the cited investigators. Andrews and McKenzie (85) reviewed earlier reports and studied vaginal and uterine biopsy specimens from 26 mares during various stages of the estrous cycle and during early and late pregnancy. Hammond and Wodzicki (676) obtained specimens by slaughter and, therefore, the oviducts were included. The study was limited, however, to one mare on each of six selected days of the cycle. More recently (1075), the histologic characteristics of the oviducts were examined using slaughter— house specimens. Kenney (864), and Rossdale and Ricketts (1362) have summa- rized the cyclic histologic changes in the endometrium. Cytologic examinations by the use of uterine swabs or flushes are described in a recent review (1366). Oviducts. The cyclic histologic changes in the oviducts are apparently slight (676). In the recent study of slaughterhouse specimens (1075), the mares were classi- fied according to ovarian structures. The height of the epithelium was not different among groups. The epithelium was ciliat— ed and pseudostratified. It was stated that glands and elastic fibers were in the lamina propria in the infundibulum and ampulla; according to these workers, such structures have not been identified in other domestic species. In some samples, small intraepithelial vesicles were found. Another group of workers (1382) has described the presence of intraepithelial cysts. Endometrium. The cyclic changes in the endometrium are more profound (85, 864, 1362). Even for this organ, however, the changes may be slight for some cycles. The most striking cyclic features involve the configuration of the glands, extent of edema in the lamina propria, and height of mucosal epithelium. During estrus (864) and following estrogen administration (673), the epithelium is high columnar with vacuoles in the cytoplasm. The histo- logic changes during estrus are those of increased cellular activity and edema. Edema is attributable to an increase in vascularity and congestion, causing movement of fluids from vessels into the interstitial area. The number of uterine glands per unit area becomes minimal during estrus, apparently because of the dispersing effect of the accumulating tis- sue fluid and a decrease in the tortuosity of the glands (864). During diestrus the edema subsides, and the endometrial glands become tortuous and therefore more cross-sections are seen in associa- tion with both endogenous (864) and exogenous (673) progesterone. Kenney (864) offered the opinion that several estrous cycles may be required before the endometrium overcomes the atrophy Characteristics of the Ovulatory Season 209 characteristic of the anovulatory season and that atrophic changes begin before the end of the ovulatory season. Others have concluded that histologic interpre- tation of biopsy of the endometrium may be affected by reproductive season (652, 1452). Britton (254) concluded that the endometrium presents a consistent histo- logic picture throughout the breeding sea- son. Based on specific staining tech- niques, the lumen of uterine glands contained glycogen and the luminal epithelia cells contained predominantly carboxylated acid mucins (542). The effect of irritating processes on these mucins and glycogens is noted in Section 12.3D (pg. 520). Electron microscopic studies (18, 1389, 859) indicate that ciliated cells are abun- dant on the surface of the endometrium during diestrus but vary in number and distribution during estrus. One study (859) found that the surface of the endometri- um was almost entirely devoid of ciliated cells during anestrus, with little secretory activity. It has been concluded that the patterns of secretory, as well as ciliary activity, in the uterine epithelium during the estrous cycle are similar to those observed in other large domestic species (1389). During estrus, many secretory cells were present; occasionally, pedunculated cells were noted and were apparently budding into the uterine lumen (apocrine secretion). The number of secretory cells declined rapidly during diestrus. Cervix. The cyclic histologic changes in the cervix have received little attention in the mare. The epithelial cells are taller, swollen, and filled with mucus during estrus and cuboidal during diestrus, with only a small amount of mucus in the lumen. Apparently, only certain cells pro- duce mucus during diestrus. Vagjna and vulva. The stratified squa- mous epithelium of the vaginal mucosa also undergoes rhythmic changes during the estrous cycle (85). There seemed to be more cornified surface cells during estrus, but prominent layers of flattened corni- fied cells were not seen at any stage. Similarly, vaginal smears contained few cornified cells, and the vaginal smearing technique was not useful for estimating stage of cycle. It was concluded that the vaginal epithelium during the first 40 days of pregnancy was similar to that observed during estrus. The activity of the vaginal epithelium seemed to increase during late pregnancy, but the data were limited. The mucosa of the vestibule has received little attention, but well-marked cornification of the epitheli- um and shedding of several layers during estrus have been reported (676). 6. 7B. Cyclic Changes Detectable by Transrectal Palpation Palpable changes in the tubular geni— talia can be of considerable clinical and experimental value (Table 6.4). Descriptions of the transrectal palpation characteristics of the uterus and cervix appear in reports on the clinical evalua- tion of the reproductive tract (644, 1362, 1664, 1499, 133, 106). The oviducts are not palpable but may become quite promi- nent in certain pathologic states. Dilated oviducts in a case of bilateral hydro- salpinx in a pony mare were initially mis- taken for ovarian follicles; using ultra- sonography, a fluid-filled oviduct was mistaken for a large follicle, but digital palpation indicated it was not part of the ovary (616). Uterus. During estrus the equine uterus is relaxed (flaccid; 697, 1664). Edema results in a spreading of the tis- sues, which enlarges the diameter of the horns. However, because of the dimin- ished tone, the horns feel flattened when compressed during palpation. The endometrial folds become especially dis- tended and can be felt slipping through the fingers when the hand is moved across the flaccid horns. During diestrus, when the edema subsides, the tissue 210 Chapter 6 TABLE 6.4. Clinically Detectable Changes in Tubular Genitalia during the Estrous Cycle Estrus Method of examination Organ Mid—diestrus Early Middle Late Transrectal Uterus Maximal tone <— Decreasing tone and thickness —> palpation and thickness Cervix Firm and distinct Beginning to Flatter, shorter Barely discernible; flatten and wider very flat Ultrasonic Uterus Minimal edema Increasing endometrial folding (nonechogenic imaging areas indicating edema) Cervix Echogenic <— Increasing nonechogenic areas ——> Palpation per Vagina Dry <——— Increasing in wetness —> vagina Cervix Firm, protruding Admits 1 Admits 2 Admits 3 fingers finger fingers or entire hand Speculum Vagina Viscous fluids, vaginal <—- Increasing fluid with decreasing -—> and walls stick together viscosity cervix Dull, yellow-gray Pink Bright pink Glistening red Decreased vascularity <— Increasing vascularity > Cervical Protruding, centrally Beginning Dropped below Near floor of orifice located, tight to drop center vagina and open Parting labia Vulva Wrinkled, pale, dry <— Increasing redness, moisture, —> (not reliable) and smoothness density increases, and the horns feel more tubular with increased compressed thickness, intermediate between the tone and thickness of estrus and early pregnancy. The extensive tone or turgid- ity characteristic of early pregnancy in this species occurs gradually between Days 12 and 25 and is discussed else- where (pg. 314). Cervix. Tone variation in the protrud- ing caudal portion of the cervix is a useful criterion for estimating stage of cycle. Cervical changes have been described as the most consistent and most marked rhythmic morphologic changes in the tubular genitalia (85). During diestrus, the cervix is comparable to a large thumb in size and consistency; it is constricted, firm, and readily palpated. A gradual relaxation of the cervix begins near the onset of estrus and maximum relaxation is reached as ovulation approaches. In this respect, the uterus and cervix respond in a similar manner to the pre— vailing hormonal milieu. With maximum softening as ovulation approaches, the cervix flattens readily with pressure, sim- ilar to the uterus. Tone gradually increas- es, and maximum cyclic tone occurs between days 5 to 10 of diestrus (85). Relaxation can be estimated by inserting fingers into the cervical os. During diestrus, one finger is admitted with diffi- culty; during estrus, the cervix opens and relaxes progressively until three or more fingers and sometimes the entire hand . can be inserted (644). Digital inspection of the caudal projection of the cervix can also disclose pathologic processes (e.g., lacerations). Characteristics of the Ovulatory Season 211 6. 7C. Cyclic Changes Detectable by Visual Inspection It has been concluded that the vaginal and cervical changes, as seen through a speculum, are important aids for detect- ing the ovulatory period (Table 6.4; Figure 6.18). This conclusion has been reached by many authors, and the early work in this area has been reviewed (85). A clinical technique is to determine if the condition of the ovaries (e.g., largest folli— cle) agrees with the condition of the cervix (133). Monitoring these events can be especially useful in the absence of a stallion and in mares showing weak behavioral changes. It is emphasized, however, that all of the changes described below are subject to variation to the extent that they may not be useful in individual cases. Vulva. The vulva may be inspected directly by parting the labia, and the vagina and the caudal portion of the cervix may be viewed through a speculum or endoscope. During diestrus, the labia may be small and wrinkled and the mucous membranes pale and dry. During estrus, the vulva may become more pen- dant or relaxed and smooth, with a red- dened moist mucous membrane. It is emphasized, however, that such changes during the estrous cycle may be quite subtle or undetectable. It has been con- cluded that variation in the vulva is too great for reliable detection of estrus (16). An objective evaluation has been made using color charts (85). The labia of most mares showed more color during estrus than during diestrus, but other mares showed little change. Some mares showed greater color intensity during diestrUs than others did during estrus. Some older mares with relaxed gaping labia showed high color intensity throughout the cycle. It was stated that individual labial varia- tion was too great for useful conclusions on the stage of the cycle. Vagina and cervix. In contrast with the appearance of the vulva, the vagina and cervix undergo visibly marked changes during the estrous cycle. Visual inspec- tion should be made immediately upon insertion of the speculum. Color intensity changes for the vagina were similar to those described above for the vulva (increasing intensity of redness during estrus with lowest intensity during mid- diestrus; 85). The color changes were not considered consistent enough for reliable use. Changes in the vascularity of vaginal and cervical mucosa, however, were more marked and consistent than in the vulva. The highest values were recorded during estrus, and a tendency was reported for maximal vascularity to coincide with the approach of ovulation. Vascular conges— tion decreased after ovulation to minimal values between Days 5 to 10 of diestrus. It was noted, however, that some individ- uals showed no vascularity changes. Endoscopes. Endoscopic examinations (Figure 6.18) are useful, not only to dis- play the visual cyclic changes, but also to detect pathologic processes (1796, 1797). The following account is based on a recent study (3). During estrus, the vagina and external cervical 0s glisten from a coating of thin mucus. Cervical folds are plump and jellylike; the most prominent folds are at the dorsal and ventral aspects. The dorsal fold may hang over the cervical 0s, and the ventral fold may lie loosely on the vaginal floor. The cervix does not protrude as prominently during estrus, and at maximum relaxation, the cervix lies on the‘ floor of the vagina and the vaginal fornix is shallow. The exter- nal folds are continuous with the internal longitudinal folds of the cervix which, in turn, are continuous with the longitudi- nal folds of the endometrium. During diestrus, the vagina and cervix are pale and the mucus coating is sticky. The cervix and vagina become more yellowish as the hyperemia (congested blood ves— 212 Chapter 6 sels) of estrus recedes. Prior to handling, the external os may lie on the vaginal floor; however, manually lifting the cervix breaks the surface tension between apposing sticky mucosal membranes, and the cervix then protrudes prominently into the vagina. The endometrial lining also may be visually inspected with a fiberoptic scope (944, 1793, 1796, 1794) or videoendo- scope (3). The following account is based on a videoendoscopic study (Figure 6.19; 3). During estrus, the endometrium is wet and glistening and the folds are promi- nent and plump. Even after dilation with air or saline, the endometrial folds pro— trude into the lumen; the folds can, how- ever, be flattened by overdistention With air. During diestrus, the folds are clearly identifiable, especially if saline, rather than air, is used for dilation. Air inflation causes the folds to collapse against the uterine wall. 6. 7D. Changes in Ultrasonic Morphology Echotexture. The ultrasonic anatomy of the uterus is influenced dramatically by the stage of the estrous cycle and is dependent on the prevailing circulating levels of ovarian steroids. During diestrus, individual endometrial folds are not discernible (Figure 6.21; 590, 614) and, compared to estrus, the uterine echotexture is ultrasonically homoge- neous. However, location of the lumen ESTRUS DIE STRUS often is identifiable due to a white line (bright reflection) when the uterus is viewed in the longitudinal plane. Because of the orientation of the uterus, the White line is most prominent in the uterine body. During estrus, the white line is not as visible or only short seg- ments are seen. The most profound change during estrus involves the devel- opment of individual endometrial folds, resulting in alternate and intertwining echogenic areas. The echogenic areas are attributable to the reflections of tissue- dense central portions of the folds, and the nonechogenic areas are attributable to edematous portions of the folds or to free estrous fluid between the folds. Occasionally, intraluminal fluid collec- tions are seen; these probably result from pockets of free estrous fluid. There seems to be a positive relationship between the size of the folds when pal- pated or viewed through a videoendo- scope and the prominence of the folds on the ultrasound image. Ultrasound scores also paralleled sexual behavior scores (6.99). Mean ultrasound scores (1=diestrus— like, 2=intermediate, 3=estrus—like) during interovulatory intervals in the summer and fall months are shown (Figure 6.22) for horse mares (699). A small mean surge in scores occurred dur— ing early diestrus in May to July but not during August to September. In addition, the September to October profile dur- ing estrus was broader due to an earlier FIGURE 6.21. Comparison of ultrasonical- ly detectable changes in cross-sections of uterine horns during estrus and diestrus. The image taken during estrus is larger in diameter, and the endometrial folds are far more prominent due to edema. Characteristics of the Ovulatory Season 213 Ultrasound echotexture 3 (n=20/group) Score -3 0 3 6 9 12 Number of days from ovulation 1518 0 3 FIGURE 6.22. Ultrasound uterine echotexture scores during interovulatory intervals in the sum- mer (May-June) and fall (September-October) in horse mares. Note the secondary early diestrus peak and the narrower peak during the periovulato- ry period in the summer group. The stars indicate days of significant differences between groups. Adapted from (699). increase and a later decrease in scores. These findings indirectly indicate that estrogen profiles may be influenced by month within the ovulatory season. A small surge in estrogens in early diestrus during the first half of the ovulatory season has not been reported, but the ultrasound studies indicate a need to look Uterine horn morphology (n=15) 32 30 E E 3-, 28 g Horn diameter m \ 5 i} N ‘ O) kirk 0 4 8 12 16 Number of days from ovulation for such a phenomenon. Perhaps the small early diestrus surge is related to the greater follicular activity during early diestrus at this time of year (pg. 183). The relationships among profiles for ultra— sonic follicular appearance, steroid con- centrations, follicular activity, and FSH levels for the early versus late ovu- latory season need to be elucidated. Diameter of uterine horns. Recently, the diameter of the uterine horns and endometrial echotexture during the interovulatory interval were character- ized by ultrasonography (647). A hyper- echogenic metal bead was sutured to each uterine horn to assure that measure- ments were taken consistently in the same location. The two end points were closely correlated; high values were obtained during estrus (Figure 6.23). The mean for both end points decreased a day before ovulation, corresponding to the expected decline in circulating estrogen concentrations (pg. 241). The large uterine horns during estrus were associated with edema (647). These characteristics (diame— ter, echotexture or edema, estrogen levels) are likely interrelated. A profile of uterine tone or compressed thickness during the equine estrous cycle corresponds to the profile representing circulating progesterone concentra- FIGURE 6.23. Mean diameter of a uterine horn as determined by ultrasound and endometrial echo- texture (score 1 = minimal folding to score 4 = maximal folding). Both end points were greatest during expected estrus and reced- ed the day before ovulation. There was significant correlation 0 between the two end points (r = 0.75). Adapted from (647). M 9.1008 leguawopua —L 214 Chapter 6 tions—low during estrus and high during diestrus. Studies on the hormonal basis of uterine changes in cycling and early preg- nant mares are discussed elsewhere (pg. 314). An enigma. The uterus during estrus is flaccid, as determined by rectal palpa- tion (pg. 209),whereas ultrasonic cross-sec- tions of the estrous uterus are circular. Also, the flaccid equine uterus during estrus contrasts with the turgid estrous uterus of cattle. Perhaps the two species differ in the nature of myometrial resis- tance to edematous expansive pressure during estrus. In cattle, the edema is resisted by tonic myometrial contraction and the horns feel turgid. In mares, the edema spreads the tissues and therefore expands the horns, but apparently the myometrium is relaxed and accommo— dates rather than resists the edema. Therefore, the horns feel flaccid despite the edema. This would also account for the enigma in estrous mares of a circular cross-sectional ultrasound image of a uterine horn, yet a flaccid uterine horn when felt during transrectal palpation. The flaccid estrus uterus in mares versus the turgid uterus in cattle may represent compatibility to the profound differences in ejaculate volumes and deposition sites in the two species. 6. 7E. Cyclic Secretory Activity Secretion of fluids during the estrous cycle is fundamental to the functions of the tubular genitalia. Presumably, the fluids are needed as a lubricant for intro— mission, as a medium for nutritive and mobility requirements of ova, sperm, and conceptus, and as a seal for the cervix. Uterine proteins serve as enzymes and as carrier molecules fOr hormones, vitamins, and minerals (cited in 1051). The nature and quantity of fluids from the caudal portions of the tract can be an aid in mon- itoring estrous cycle rhythm. Oviducts. A technique has been described for continuous collection of oviductal secretions in mares (478). The mean pH was 7.4, and no significant dif- ferences were detected during the cycle. Secretion rates were significantly greater during estrus than nonestrus. Average secretion rate for 12—hour periods varied from 1.6 to 3.2 ml during estrus. Maximal secretion rate was estimated to occur 1 to 4 days before ovulation, but critical data were not obtained. A gradual decrease occurred during early diestrus, reaching values of <1 ml by approximately seven days after the end of estrus. The free amino acid concentrations were signifi- cantly greater than in blood plasma or follicular fluid (477). Protein concentra- tions were greater for diestrus than for estrus. It has been reported that oviduc- tal fluid stimulated respiration of the sperm (476). The importance of this phe- nomenon is not known, but it was noted that studies in other species seem to indi- cate that rapidly respiring sperm fertilize more ova than controls. Recently (1693), the quantity of glycosaminoglycans (GAG) was reported to increase in the oviducts and uterus during the follicular phase; the authors suggested that GAG may have a role in the fertilization pro- cess, as has been reported for other species. Late entry. An initial in vitro study on synthesis and secretion of pro— teins by the oviducts has been reported (1864); the major proteins were identified according to molecular weight. Uterus. Techniques for collection of uterine secretions have been described (112, 1839). The quantity of uterine protein increased during the late luteal phase, followed by a rapid decline on Days 18 and 20 (1841, 1776, 1839). A number of unique proteins that apparently were not of serum origin were involved (1844). Albumin is a major component of uterine secretions in cyclic and pregnant mares. There was a marked increase in total acid phosphatase on Days 16 and 18 with a sharp decline on Day 20. These changes occurred at the end of diestrus after the time of uterine involvement in luteolysis. Characteristics of the Ovulatory Season Alternately, these cyclically appearing substances could be involved in the preparation for pregnancy. Uteroferrin (an acid phosphatase) is a progesterone- induced uterine specific protein in uterine secretions that may serve to carry iron to the developing conceptus (1843, 1051). Carbohydrate metabolism in uterine flushings has been examined in mares during the estrous cycle and early preg- nancy (pg. 317,- 1842). The concentrations of immunoglobulins in equine reproductive tract secretions also have been reported (1571, 1775, 1722, 1107). Lysosomal enzymes accumulate in the equine uterine lumen through steroid—modified processes (681). Their function is not known, but sugges- tions include zona pellucida removal and sperm capacitation. Vagina and cervix. The amount and viscosity of secretions recovered from the vagina and cervix have been estimated (85). Most mares showed rhythmic changes during the estrous cycle. Fluid drawn from the vagina and cervix varied in a’ comparable manner, and the volume and Viscosity were reciprocally related (as volume increased, viscosity decreased). There was an increase in the amount of fluid recovered from each organ one or two days before estrus, and the volume seemed to increase as ovulation approached. During days 3 to 5 of estrus, large amounts of a thin clear secretion with high lubricating properties were usually obtained. The fluid decreased in amount and increased in viscosity follow- ing ovulation, reaching minimal volume and maximal Viscosity during days 5 to 10 of diestrus. The increased viscosity during diestrus imparts an adhesive quality to the mucosa of the vagina. The walls stick together and make the intro- duction of an unlubricated speculum or arm difficult. In regard to pH, the following was con- cluded (85): l) Vaginal secretions tended to be more alkaline than cervical secretions, with a greater difference between the two during estrus; and 2) The more alkaline 215 secretions tended to occur on approxi- mately day 4 or 5 of estrus with a decrease in pH following ovulation. The pH on the day of ovulation (mean: 6.8) was significantly lower than at other days of estrus (approximate mean: 7.4; 1279). Vaginal pH, as well as pH of eye secre- tions, apparently decreased on day 4 of estrus (cited in 1279). Vaginal smears. As noted above, Andrews and McKenzie (85) concluded that vaginal smears were not useful for estimating stage of the estrous cycle. However, the properties of vaginal smears have been used extensively in Japan for detecting estrus and early pregnancy (1153). Macroscopic and cytologic changes occur, and fernlike crystals appear in vaginal smears during the estrous cycle. The fernlike crystals appear during estrus, but are not as prominent as in cat- tle. The report of Nishikawa (1153) can be consulted for detailed information on this subject. A brief discussion of pregnancy diagnosis by this technique is given else— where (pg. 332). The fernlike patterns of vaginal smears have been used to study the effects of teasing on vaginal mucus secretion (pg. 103). 6. 7F. Contractions Contractions of the smooth muscle of the oviduct and uterus surely play impor- tant roles. Movement and proper place- ment of sperm, ova, and conceptus and, ultimately, expulsion of the fetus all depend upon muscle activity of the organs and possibly the associated broad liga- ments. Contractions of oviducts. The contrac- tility of the mare’s oviduct is in need of study, especially in view of the unfertil- ized-ova retention phenomenon. Tech- niques have been described for monitor- ing the motility or intraluminal pressure of the oviducts in sows (717) and cows (177). Preliminary data suggested a higher level of activity in the oviduct ipsilateral to the active ovary in cattle. In a short 216 Chapter 6 communication (517), a technique for mon- itoring contractile activity of the oviduct was described for mares; it was stated that the following substances stimulated the tubal musculature: adrenalin, nora- drenalin, oxytocin, and PGan. Contractions of uterus. In vivo myo- metrial electrical activity has been stud- ied during the equine estrous cycle (1589). Three distinct patterns were reported: 1) estrus—well defined phases of closely grouped high-amplitude spikes separated by long periods of inactivity (10 to 45 minutes), 2) diestrus—diffuse phases with low amplitude spikes separated by variable periods of inactivity, and 3) lute— olysis—short and frequent bursts of activity. Patterns similar to those of lute— olysis also occurred 1 to 3 hours after injection of prostaglandin. Insemination, uterine manipulation, and nonspecific environmental stimuli (e.g., human voic- es) influenced activity at all stages of the cycle. The authors noted that the long periods of inactivity during estrus and the low amplitude activity during diestrus differed from the activity of the uterus of cows and sheep. In these species, intense activity occurs during estrus with near-absence of activity dur- ing diestrus. The importance of this species difference to the developing con- ceptus is profound (pg. 313 and pg. 439). Techniques for measuring uterine elec— trical activity and pressure. Pressure transducers or intraluminal balloons have been used to measure pressure in the uterus and vagina (630, 627, 958, 902, 628, 1442). Pressure in the uterus was usually higher than in the vagina and was influ- enced by urination, vocalizing, and respi- ration. Vaginal pressures were influenced by rectal peristalsis and stance of the mare. Oxytocin treatment caused rapid rise in intrauterine pressure that lasted about 20 minutes. Prostaglandin F201 increased the intrauterine pressure in about 10 minutes, and the pressure declined during the next 40 minutes. In another study (1441, 1442), it was reported that oxytocin administration increased intrauterine pressure in cycling mares but PGan did not. The contractions occurred within five minutes of the begin— ning of oxytocin infusion and declined within 60 minutes, indicating desensiti- zation or fatigue. Analogues of PGF2a caused increased uterine pressure in postpartum mares within 7 to 15 minutes (958). A circumferential strain gauge was developed (278) for monitoring uterine motility in response to oxytocin and PGan in ovariectomized estrogen-treated mares. Treatment with oxytocin resulted in low—amplitude high-frequency contrac- tions, whereas treatment with PGFZa resulted in high-amplitude low-frequency contractions. An abstract is available on physiologic and pharmacologic factors affecting uterine contractions, as deter- mined by electromyographic techniques and intrauterine pressure changes (839). In a recent study (970), it was concluded that placement of electrodes at multiple sites, compared to a single site, provided a more complete and accurate assessment of myometrial activity. In vitro studies. Isolated strips from the uterine musculature of mares con— tract when exposed to PGFZOL (274). Another in vitro study indicated that con- tractility of the mare myometrium is very sensitive to B—mimetic compounds and cal- cium antagonists (342). These workers showed that mare myometrial strips from both estrus and diestrus were endowed with remarkable and long-lasting (6 to 8 hours) spontaneous motility. Various aspects of the effects of drugs on sponta- neous uterine contractions are reviewed. Measuring uterine contractions by ultrasonography. The first experiments to use ultrasonography to assess uterine contractility were done in mares. Contractions were studied during the estrous cycle and early pregnancy (pg. 313; Characteristics of the Ovulatory Season 350, 646). The uterine body was viewed in longitudinal plane, and the extent of contractility was estimated on the basis of the streaming appearance of the endometrium caused by the movement of individual endometrial tissue reflectors. Uterine contractile activity during the interovulatory interval increased at Day 13, corresponding to the expected time of luteolysis (Figure 8.11, pg. 314). The effect of administration of estradiol, proges- terone, and PGFZa on contractility was studied using the ultrasound method- ology in seasonally anovulatory mares with minimal ovarian activity (349). Daily treatment with either estradiol or proges- terone resulted in increased activity with- in 4 and 14 days, respectively. Perhaps the rapid response to estradiol enables the uterus to prepare rapidly for sperm transfer, and the slow response to proges- terone prepares the uterus for intrauter- ine movement of the conceptus at about two weeks (pg. 305). A PGFZoc injection after 10 days of steroid treatment result- ed in an additional increase of activity in 30 minutes in progesterone-treated mares but did not further increase the high level of activity in estradiol-treated mares. The rapid response to PGFZa may be an accommodation to provide for uterine con— tractions for conceptus mobility. Summary. Mares, in common with other farm species, have active uterine contractions during estrus. Similarly, treatment of ovariectomized or anovula- tory-season mares with estrogens stimu- lates contractions. Presumably, such activity plays a role in semen transport. Mares differ, however, from other farm species during diestrus; the diestrous uterus of the mare is active, Whereas the uterus of other species apparently is quiescent. Long term administration (e.g., two weeks) of progesterone or a single injection of PGan in mares previ- ously treated with progesterone cause increased uterine activity. In cycling 217 mares, uterine activity is maximum at the time of luteolysis, suggesting that substances involved in luteolysis (PGFZoc or oxytocin?) play a role in the increased activity. The activity of the gravid uterus during the first few weeks after ovulation is of special interest and is dis- cussed elsewhere (pg. 314). Various mea- surement techniques have all indicated a positive uterine contractility response to oxytocin in nonpregnant mares. Several studies have also demonstrated uterine contractions in response to PGFZa, although one report (1442) concluded that PGonc was not uterokinetic in cycling mares. 6.8. Ovarian Irregularities This section will consider only those ovarian irregularities that occur within the ovulatory season of an individual ani- mal. An ovarian irregularity is defined as a deviation from the conventional dynam- ics of ovarian function. Dynamic (chang- ing) ovarian function reflects changes in a complex of hormonal interrelationships and balances. The most likely precarious period during the ovulatory season involves the transition in hormonal, bio- chemical, and biophysical events associat- ed with ovulation. Ovarian irregularities may represent hypofunction or hyper- function. Irregularities associated with ovulation or the follicular phase, for example, include multiple ovulations (hyperfunction) and failure of ovulation (hypofunction). Multiple ovulations, diestrous ovulations, failure of ovulation, hemorrhagic follicles, and prolonged luteal phases are discussed in this sec- tion. Variations in the expression of estrus during the follicular phase, includ- ing ovulation in the absence of behavioral estrus, are described in Section 3.5 (pg. 90). Ovarian tumors will not be considered; reviews and recent reports are available (133, 781, 1254, 227, 1535, 751, 1221). 218 Chapter 6 6.8A. Multiple Ovulations In the first edition (575), multiple-ovula- tion data from 14 sources were tabulated. There was wide variation in reported inci- dence, suggesting that certain intrinsic or extrinsic factors greatly influence the phenomenon. A source of variation in reported incidences was the nature of the data-gathering process. Slaughterhouse and necropsy determinations likely over- estimate the incidence since nonovulatory luteinized structures, or structures that may not be corpora lutea, may be counted as ovulations. As another example, slaughterhouse material may include specimens from mares with fetal loss but with supplementary corpora lutea, there— by inflating the apparent multiple ovula- tion rate. Palpation and ultrasonic deter- minations likely underestimate the incidence; double follicles or ovulations on the same ovary may be close together and difficult to discern by palpation (578). Even when ultrasound is used, a large follicle may be missed (590). The care given to establishing the occurrence of double ovulations varies widely. Some veterinarians, for example, discontinue palpating or scanning after a large follicle or an ovulation is detected, even though another large follicle is present. It is not surprising, therefore, that critical infor- mation on the factors affecting double ovulation rate did not become available until the 1980s. Definition. The definition of a double ovulation is seldom stated in reports on incidence. For example, two ovulations six days apart may be counted as double primary ovulations or as a follicular phase ovulation (primary) plus a dies- trous ovulation (secondary). Because of the difficulty in differentiating some double asynchronous primary ovulations from a primary plus a secondary ovula- tion, some workers define all double ovulations that are 1 or 2 days apart as asynchronous primary ovulations. This definition takes into account the occasional occurrence of a single ovula- tion after the end of estrus. Double ovula- tions that are more than two days apart should not be considered primary unless there are clear indications that the inter- im period was a part of the follicular phase (e.g., based on estrous behavior, ultrasonic uterine echotexture, and hor- monal analyses). Effects of breed. The 11 groups of horses tabulated in the first edition (575) had a higher incidence of multiple ovulations (4% to 43%) than the three groups of ponies (2% to 3%). It was concluded that the incidence of multiple ovulations is much higher in horses than in ponies. Further support for this conclusion comes from the reports that no multiple ovula- tions were detected in a herd of 12 semi- wild Korean ponies (1395) and that no twins were recorded in a herd of 25 to 30 ponies over 19 years (equivalent to approximately 522 mare-years; 823). Two slaughterhouse studies reported multiple ovulation rates in ponies of 10% (n=527; 1759) and 11% (n=127; 719). These data seem disconcerting when contrasted with palpation data. However, slaughterhouse studies have consistently yielded higher apparent ovulation rates in both horses and ponies. One slaughterhouse study found that the incidence in draft mares (24%) was significantly higher than in pony mares (11%, 719). Differences in ovulation rate can be expected among equine breeds and types on a comparative basis since there are wide differences in ovulation rates among breeds of swine and sheep. Data on ovu- lation-rate differences among breeds of horses (as opposed to ponies) were not reported until the 1980s. In a survey (607), veterinarians with experience with two horse breeds expressed the opinion that the incidence of double ovulations was higher in Thoroughbreds than in Standardbreds and was much higher in Thoroughbreds than in Arabians. The veterinarians and operators of two Arabian farms concluded that double Characteristics of the Ovulatory Season 219 ovulations and twin abortions were rare in Arabians. Subsequent studies substan- tiated these opinions. The following inci- dences of multiple ovulations were reported: Thoroughbreds, 25%, 22%, and 15%; Standardbreds, 15% and 13%; draft horses, 24%; Quarter Horses and Appaloosas, 10%, 8%, and 8% (review: 590). The propensity for multiple ovula- tions in Thoroughbreds was demonstrat- ed by a significantly greater incidence in Thoroughbreds than in Quarter Horses involving the same farm, season, and vet- erinarian (607). N 0 double ovulations were noted in eight Arabian mares monitored throughout the year in Iraq (498). In a recent report, the percentage of twin births recorded in stud books in Poland was 3% for Thoroughbreds and 0.8% for Arabians (404). Repeatability and heritability. Several veterinarians replying to a survey expressed the belief that the incidence of double ovulations and twin pregnancies was exaggerated in certain individuals or family lines (803). In mares with data for successive cycles, the probability of the occurrence of multiple ovulations for a given cycle was approximately doubled (significant) when the preceding cycle also had multiple ovulations. A research herd that was selected for a high multiple ovulation rate subsequently had a 56% incidence (1513). In an anecdotal account (607), a mare with a history of two sets of twin fetuses in four years had five ovula- tory periods during one season; all involved double follicles or ovulations. In another study (780), the incidence of mul- tiple ovulation was very high in one of 11 mares (multiple ovulations in 24 of 34 cycles), whereas, at the other extreme, another mare had one multiple ovulation in 24 cycles. Adequate anecdotal (780, 1513, 607) and statistically evaluated data (607) now are available to conclude, without reservation, that multiple ovulations are characterized by significant repeatability within individual mares. The involvement of genetic factors in multiple ovulations is indicated by the higher incidence in Thoroughbreds than in Quarter Horses, noted above, when the two breeds were under the same manage- ment program. The genetic or heritable aspects of twinning were suggested by examination of individual mare records (607). Among the mares on a farm with records for several (3 to 5) ovulatory cycles, two mares had an unusual predis- position for double ovulations and twin pregnancies. These two mares were the only mares on the farm that had three or more cycles with double ovulations and were the only mares in which twin preg- nancies were established twice during one breeding season. The two mares were dam and daughter. Such anecdotal infor- mation suggests that the occurrence of double ovulations and the establishment of twin pregnancies are heritable charac- teristics that could be selected against. Effects of reproductive status. On Thoroughbred and Quarter Horse breed- ing farms, approximately 50% lower inci- dence of multiple ovulations has been found in foaling (postpartum) mares than in barren and maiden mares (607; Table 6.5). The incidence of multiple ovulations was reduced in foaling mares for ovula- tory periods occurring during the first 80 days postpartum. N0 statistical evidence was obtained to involve month in this phenomenon. However, because some of the mares were apparently on lighting programs, the effects of month may have TABLE 6.5. Effect of Reproductive Status and Breed on Double Ovulation Rate Incidence of multiple ovulations Reproductive Quarter status Horses Thoroughbreds Foaling 26/358 (7%) 15/95 (16%) Barren 53/370 (14%) 17/57 (30%) Maiden 11/109 (10%) 2/44 (27%) 10% 24% Adapted from (607). 220 Chapter 6 been obscured. In this regard, in another study (989), the incidence of multiple ovulations for the first postpartum ovula- tory period was lower in January to March (7%) than in April to May (22%). The cause of the reduced number of mul- tiple ovulations, postpartum, is not known. The suppressive action of nursing on reproductive function, which occurs in mares (pg. 478) as well as in other species, is likely involved. Effects of season. An effect of season or month on multiple ovulation rate has been claimed by some workers and denied by others (review: 578). One report sug— gested a higher frequency for the spring (780), whereas another suggested a higher frequency for the fall (94); neither report presented statistical documentation. Most studies did not involve all months, the number of mares was small, or the data were not collected in a manner suitable for statistical testing. Four slaughter- house studies that involved all months and at least 500 mares did not find or did not look for a statistical effect of month on the proportion of ovulating mares that had multiple ovulations, except for one study (1759) in which a tendency (P35 mm) when the first ovula- tion occurred (617). The exception was a six-day interval in which the second follicle was 25 mm when the first ovula- tion occurred. Recent studies involving ultrasonic monitoring of individual folli- cles have demonstrated that double ovulations (synchronous and asyn- chronous) can originate from the same wave (1484, 598). Occasionally, however, asynchronous, and even synchronous, double ovulations can involve two sepa- rate major follicular waves (Figure 6.25; 598, 184). Approximately 50% of the double ovu- lations were synchronous (same day) and 50% were asynchronous (617, 1759, 1511). The number of days between double ovu- lations (11:67) in an ultrasound study in horses was 0 (55%), 1 (33%), 2 (9%), and 3 (3%; 591). In one study, 72% of asyn- chronous ovulations were only one day apart (1511), and in another study, the number of days between asynchronous ovulations ranged from 1 to 6 (mean, 2.3 days; 617). Unilateral and bilateral double ovulators did not differ in the number of days between ovulations (617, 1511). However, the results of an extensive slaughterhouse study (n=209 double ovulations) indicated that asyn- chronous ovulations occurred significant- ly more often in bilateral ovulators than in unilateral ovulators (719). The study based the age of the corpora lutea on gross appearance upon slicing, using the ratio of blood clot to luteal tissue as an indicator of age. Recent ultrasound stud- ies have shown that many developing cor- pora lutea do not develop blood clots (pg. 197). Thus, studies (719, 1759) that used Individual follicles Diameter (mm) M N O 01 .3 U1 0 5 10 15 20 250 5 Mare B 10 15 Number of days after ovulation 20 0 5 10 15 20 25 FIGURE 6.25. Examples of divergent origins of double ovulations. Mare A. Synchronous ovulations from a single ovulatory follicular wave. Note the preceding anovulatory wave during early diestrus. Mare B. Synchronous ovulations with one follicle originating from the early diestrous wave (secondary wave) and the other from the late diestrous wave (primary wave). Mare C. Asynchronous ovulations with both follicles originating from the primary wave. Note the anovulatory secondary wave during early diestrus. Adapted from (598 and 184). Characteristics of the Ovulatory Season 223 the proportionality of blood clots for assessing luteal age were based on a faulty premise. Hormonal relationships. The hormonal basis for a predisposition for double ovu- lations in some mares is not known. The circulating concentrations of LH and FSH were not different between mares that double ovulated and those that did not (1656, 1513). It has been suggested (1656) that the ovaries of some mares may be more sensitive to gonadotropins. Mares with double ovulations, and therefore two corpora lutea, have higher circulating progesterone concentrations (1216, 719, 1656, 1513). Similarly, mares with induced multiple ovulations also develop high cir- culating progesterone concentrations (1513). However, the concentration for dou- ble ovulations is less than twofold greater than for single ovulations (719, 1656). Perhaps the corpora lutea in double ovu- lators, like the follicles, are smaller than in single ovulators. Length of the estrous cycle was found to be four days longer in mares with double ovulations than in mares with single ovulations (719). Other workers reported a similar lengthening effect (1656), whereas some did not find an effect (780). 6.83. Diestrous Ovulations The above discussion concerns multiple ovulation (synchronous and asyn- chronous) over a short time span in asso- ciation with estrus (primary or follicular- phase ovulations). California workers (780) discovered the frequent occurrence of a second ovulation during diestrus (Days 2 to 15) despite high progesterone levels (1 to 8 ng/ml). These ovulations were termed diestrous ovulations, and they occurred in 10 of 11 mares (Quarter Horses and Thoroughbreds) over two years (incidence: approximately 21% of estrous cycles; 777). The diestrous ovula- tions were not accompanied by estrus, and the cervix remained pale, dry, tight, and sticky. The diestrous corpora lutea felt normal on palpation. Furthermore, in vitro conversion capacity (pregnenolone to progesterone) of diestrous and primary corpora lutea was comparable (483, 779). Oocytes from diestrous ovulations are fer— tilizable (777, 778). Equids are apparently the only animals that have a propensity for ovulating during a progestational state. The role of diestrous corpora lutea in prolonged luteal phases is discussed elsewhere (pg. 227). The occurrence of large preovulatory- sized follicles during diestrus, sometimes with ovulation, has been confirmed (1686), but the authors concluded that ovulation of a large mid—cycle follicle was rare. Maturation of such follicles is reportedly associated with intermediate levels of LH. A 3.6% incidence of diestrous ovula- tions was found in 55 estrous cycles in Trotter mares (718). Recently, diestrous ovulations were detected by ultrasound in 3 of 69 (4%) interovulatory intervals at 7, 9, and 11 days after the primary (follicu- lar phase) ovulation (617). The mares were of Quarter Horse and Appaloosa breed- ing. The incidence of diestrous ovulations in the latter two studies seems much lower than the 21% found in the original study (780). Perhaps, breeds with a high double ovulation rate (Thoroughbreds) also have a high incidence of diestrous ovulations. Further indications of a breed effect on the incidence of primary double ovulations and diestrous ovulations are the findings that both of these phenome- na are rare in ponies (double primary ovulations, pg. 218; diestrous ovulations, 575, 1759). In a transrectal palpation study in jennies, diestrous ovulations were not detected (1679). In regard to the possible effect of breed on the incidence of diestrous ovulations, recent information was obtained during ultrasonic monitoring of individual folli- cles (pg. 179). In a study in Standardbreds and Thoroughbreds, 3 of 5 mares with a major wave of follicular activity in early diestrus had a diestrous ovulation on Days 5 to 12 (total incidence including 224 Chapter 6 two-wave and one-wave intervals: 18%; 1484). In a study in Quarter Horses, diestrous ovulations did not occur in any of six mares with two major follicular waves (598). In both studies, rupture of a regressing (decreasing in size) large folli- cle occurred without the subsequent for— mation of a well-defined corpus luteum. In the first study, four instances were recorded (24%) in follicles that ranged from 17 to 28 mm. In the second study, one instance was recorded involving a for- mer dominant follicle in its regressing stage. The follicle decreased from a maxi- mum diameter of 48 mm to a prerupture diameter of 35 mm over 15 days. Apparently follicles undergoing atresia occasionally rupture and presumably do not contribute to the progesterone milieu (no detectable corpus luteum). Atresia was indicated by decreasing diameter of the follicle before rupture and the absence of detectable luteal development. This phenomenon (rupture of small or regressing follicles) needs further charac- terization and confirmation. 6.8C. Failure of Ovulation Failure of ovulation, despite expression of estrus and apparently normal follicular development, occurs in all the farm species. This section will consider ovula- tory failure that occurs within the ovula- tory season for a given mare. Although anovulatory estrus is very common during the anovulatory season (pg. 136), it is rare during the ovulatory season. This condition has been described as rare in Thoroughbreds (290); an inci- dence of 3.1% in 295 estrous periods was found in Thoroughbreds and Quarter Horses (780). Only two cases were found among 100 estrous periods in ponies (573). These findings indicate that mares are very dependable ovulators once the tran- sitional period between anovulatory and ovulatory seasons is complete; that is, once the first ovulation of the year has occurred. Apparently, the mare has devel— oped a very efficient system for triggering ovulation—a system that tolerates con— siderable leeway in concentrations of the ovulating hormone (LH), diameter of ovu- lating follicle, and other variables. 6.8D. Hemorrhagic Follicles The syndrome of cystic ovaries, compa- rable to what occurs in cattle, has not been documented as an entity in mares. A condition reported to be similar to cystic ovaries in cattle was induced in mares by treatment with a progestin (melengestrol acetate); it was proposed that such mares would make good teasers for collection of semen (236). , It was noted more than 40 years ago that mares examined during October and November sometimes had a large persis- tent follicle (261). A subsequent worker (1539) referred to such structures as autumn follicles and noted that they were hemorrhagic with liquid to gelatinous consistency. Four cases, apparently simi— lar, were reported in the first edition (575). The structures were removed and are shown (Figure 6.26). Hemorrhagic follicles were described recently in herds of horse mares (617, 285). In one study (617), 12 cases (4.7%) occurred in eight mares during 213 interovulatory intervals monitored by ultrasound. There was no significant effect of month on the incidence. One mare had five hemorrhagic structures (one during one interovulatory interval and four during the next). The hemor- rhagic follicles were detected on the day that another follicle ovulated (one mare), two days after an ovulation (two mares), on Day 10 of a 20-day interovulatory interval (one mare), or during what would have been an ovulatory period (five hem- orrhagic follicles in two mares). The latter hemorrhagic follicles resulted in pro— longed interovulatory intervals since the hemorrhagic structures were presumed to Characteristics of the Ovulatory Season 225 nnwnnzvu HEMORRHAGIC FOLLICLES FIGURE 6.26. Ovaries from 3 mares with a follicle that grew unusually large during estrus and then filled with blood without ovulating. A. The follicle was 80—100 mm at the end of estrus B. Hemorrhagic fo‘llicle on Day 5 after end of and 40 X 50 mm when the ovaries were removed on estrus. It did not contain a grossly visible wall of Day 11 after the end of estrus. The structure was luteal tissue. filled with clotted blood and contained an outer wall of luteal tissue, 2—5 mm thick. C. Enlarged ovary on Day 19 after end of estrus. On sectioning, the ovary contained follicles of 26, 20, and 20 mm and a 60 mm hemorrhagic follicle that had been present since estrus. Blood oozed from the hemorrhagic follicle during sectioning. 226 Chapter 6 be anovulatory. The formation of hemorrhagic follicles was initially indicated ultrasonically by scattered free-floating echogenic spots within the follicular antrum; the fluid swirled during ballottement of the ovary. The number of echogenic spots increased over the next few days; this was accompa- nied by enlargement of the follicle (range of maximum diameters: 60 to 90 mm). Thereafter, growth ceased and the con- tents became organized with apparent fibrinous bands. During ballottement at this time, the echogenic contents did not swirl and sometimes quivered, giving the impression of a gelatinous consistency. Most of the structures formed outer walls 4 to 7 mm thick that may have represent- ed luteinized tissue. The hemorrhagic fol— licles, thereafter, were firm (did not quiver during ballottement), gradually regressed, and were no longer detected after approximately one month. The ultrasonic appearance was consistent with previous descriptions (616). During studies on the nature of follicu- lar evacuation (1629), five cases of hemor- rhagic follicles were found (600). The mares were being examined frequently (at least every 15 minutes) and some- times continuously. All mares were treat- ed with hCG when the preovulatory folli- cle reached 35 mm. Three of the structures apparently were anovulatory since follicular collapse was not seen. These three structures were similar ultrasonically to those described above— floating specks for two days after expect- ed ovulation (ovulation was expected 48 hours after hCG treatment), gel-like con— sistency on the third day, and firm on the fourth day. The two other hemorrhagic structures, however, began to develop immediately (within 15 minutes) after complete evacuation of the follicle (ovula- tion) or after about 75% of the follicle had evacuated. These two structures devel— oped an apparent blood clot gradually over a few hours, but swirling of contents did not occur upon ballottement. These were dissimilar to corpora hemorrhagica, which begin to form at approximately 30 hours and develop gradually over the next 52 hours (pg. 197). Apparently, vascu- lar accidents can occur in association with failure of ovulation or during or immediately after follicular evacuation. Presumably, the amount of follicular fluid at the site of evacuation determines when the entering blood will coagulate. When hemorrhaging occurs into an unovulated follicle, coagulation does not occur for 2 or 3 days. When hemorrhaging occurs into a collapsed site or a site with inadequate residual follicular fluid, coagulation occurs immediately. In this regard, follic- ular fluid of nonequine species has antico- agulant properties (117). Equine follicular fluid has been reported to contain less fib- rinogen than blood plasma but is suffi— cient to form a clot after the addition of thrombin (1539). The anticoagulant prop- erties have been attributed to a heparin- like substance. The cause of hemorrhagic follicles is not known, and the apparent high inci- dence during the fall requires further doc— umentation. Aside from their importance as a cause of transient infertility when associated with anovulation, these struc- tures may provide a model for studying the mechanisms of ovulation. Hormonal as well as morphologic studies will be needed for elucidation of their cause and nature. The uterine echotexture associat- ed with their development and life span was often characteristic of the luteal phase (617). The gross and ultrasonic mor- phology indicated that some of them pro- duced progesterone. The possibility that the follicles that led to development of the hemorrhagic follicles were deficient in estrogen is raised by the absence of an estrogenic uterine ultrasonic morphology during expected estrus. Hormonal studies will be needed to confirm these observa- tions. A clinical report of structures, simi- lar to those described here, stated that they occurred in mares during the pro- duction of eCG after fetal loss (34). Characteristics of the Ovulatory Season 227 6.8E. Prolonged Luteal Activity Terminology. Prolonged luteal activity is characterized by maintenance of circu- lating progesterone concentrations in nonpregnant mares for longer than expected. Assigning appropriate terminol- ogy to such conditions is a challenge (599). Terms such as persistence of the corpus luteum imply that the life of a given luteal gland is prolonged. On the other hand, terms such as prolonged luteal phase do not clarify whether a given gland is prolonged or whether other glands developed sequentially, each of which may have either a normal or pro- longed life span. Nonluteal terms also are used sometimes to describe prolonged luteal activity (e.g., pseudopregnancy). The following considerations further com- plicate the choice of appropriate terminol- ogy: 1) Luteal tissue apparently can origi- nate from anovulatory follicles as well as from ovulatory follicles; 2) The corpus luteum that results from the follicular- phase ovulation (primary ovulation) is present during several reproductive states (diestrus, pregnancy, and embryo loss); and 3) New luteal glands may develop While another luteal gland is functional during normal progestational conditions (diestrus and pregnancy) and abnormal progestational conditions (e.g., prolonged luteal activity). The fascinating ability to ovulate dur- ing progesterone dominance (1533) is the main biologic obstacle to the study of pro- longed luteal activity in this species. A normal physiologic mechanism that is central to a discussion of prolonged luteal activity is the uterine luteolytic mecha- nism that becomes activated at the end of diestrus (pg. 270); if an embryo is present, the mechanism is blocked (pg. 438). The mechanism can be diminished or elimi- nated by loss of the appropriate compo- nents of the uterus; therefore, hysterecto- my (pg. 267) and severe uterine anomalies or pathology (pg. 522) can result in persis— tence of the corpus luteum. Current status. During a two-year study in seven Thoroughbreds and four Quarter Horses, a condition was discovered that has been termed spontaneous prolongation of luteal activity (1534), persistent luteal phase, and persistence of the corpus luteum (1532). Diestrous ovulations were also discovered during this long-term study and were detected during some of the pro- longed phases (pg. 223). The prolonged luteal activity occurred 12 times, and the mean length was 63 days (range: 35 to 95 days). The syndrome was described as sponta- neous because no uterine anomalies or infections were detected. The condition has been reported to be common (up to 25% of estrous cycles) and due to failure of the uterus to release sufficient PGFZa at the normal time (14 days postovulation; 1532). In another study (1136), persistent corpo- ra lutea were observed in three mares in which circulating concentrations of a prostaglandin metabolite, as well as pro- gesterone, were monitored. In one mare, prolonged luteal function was attributed to a diestrous corpus luteum which was immature at the time of PGan release and therefore nonresponsive. This inter- pretation is reasonable since immature corpora lutea do not regress when mares are treated with PGFZa (431) or one of its analogues (66). In the second mare, the pro- longed luteal activity was attributed to a persistent corpus luteum from failure of PGFZa release and to the formation of late diestrous corpora lutea that probably con- tributed to the maintenance of high pro- gesterone levels. The third mare had severe endometritis and developed a pro- longed luteal phase due to insufficient PGF2a release with incomplete luteolysis. Similarly, in another report, severe endometrial damage was associated with prolonged luteal activity due to delayed or inadequate PGFZoc release (782). In some of these mares, however, the corpus luteum regressed prematurely due to early PGonc release, presumably following an acute inflammatory reaction. Another group of workers monitored 228 Chapter 6 20 nonbred Trotter mares by transrectal palpation during 55 estrous cycles and found two cases (3.6%) of prolonged luteal activity (718). Secondary ovulation was detected subsequent to the follicular- phase primary ovulation in both mares. Corpora lutea were removed from one mare on the 52nd day of the prolonged luteal phase. One of the corpora lutea was smaller and seemed to be the original persistent corpus luteum. The term prolonged diestrus has been used to describe a condition that was the main cause of failure of nonpregnant mares to exhibit estrus during the breed- ing season (66, 70). The term included mares that had embryonic loss, mares that were not pregnant when examined at 19 to 45 days, and nonbred mares. Affected mares had progesterone concen- trations greater than 1 ng/ml and responded well to treatment with a prostaglandin analogue. The authors seemed to attribute the condition to per- sistence of the corpus luteum from the fol- licular-phase ovulation. Other workers (1106) also have used the prolonged diestrus terminology and have confirmed the common occurrence of this broadly defined condition on breeding farms. In a recent study (884), nonbred mares were defined as having spontaneous pro- longed corpus luteum syndrome when diestrous behavior persisted for at least 30 days without evidence of ovulation. Follicles were monitored by transrectal palpation. Seven of 40 estrous cycles (18%) developed the syndrome. Follicular growth was delayed until after Day 16 in the seven affected mares compared to Day 8 to 14 in normal mares. In addition, the estradiol production was not marked. Because failure of ovulation was based on transrectal palpation, this interesting study must be accepted with reservation. The literature on prolonged luteal activity, reviewed above, does not docu- ment unequivocally the occurrence of pro- longed life span of individual corpora lutea. The studies predated the use of ultrasonography as a luteal monitoring technique, and the nature of the studies precluded marking of luteal glands for identification. In addition, the transrectal palpation approach and schedule may have been inadequate for reliable detec- tion of ovulations subsequent to the follic- ular-phase ovulation (34). An illusion of persistence could have resulted if other progesterone sources developed from cor- pora lutea resulting from undetected sub— sequent ovulations or from luteinized anovulatory follicles. Spontaneous (no uterine pathology) persistence of the corpus luteum from the follicular phase ovulation has been accept- ed as a clinical entity by many reviewers, including the present author (575, 974, 718, 935, 1333, 133); however, it now appears that such an entity has been inadequately doc— umented and reevaluation is needed. Prolonged luteal activity in association with severe uterine pathology has been documented by prolonged progesterone profiles, but persistence of the life span of individual corpora lutea must be assumed on the basis of failure to detect PGFZoc release. However, one case has been recorded in which an individual corpus luteum persisted in association with endometritis; the corpus luteum (origin unknown) persisted throughout 41 days of daily ultrasonic monitoring in a mare with large intrauterine fluid collections (617). Pseudopregnancy. The terms pseudo- pregnancy, pseudocyesis, and spurious pregnancy are used by equine practition- ers to describe a syndrome in which non— pregnant bred mares do not return to estrus, and uterine tone is characteristic of pregnancy. The term pseudopregnancy was first used more than 50 years ago to describe greatly prolonged interestrous intervals in bred mares (291). It is general- ly considered that this syndrome results from embryo loss. A retrospective study, reported in the previous edition (575), con- cluded that pseudopregnancy can occur occasionally in nonbred mares, based on long interestrous intervals and failure to Characteristics of the Ovulatory Season 229 detect intervening ovulations by transrec— tal palpation. However, the monitoring system in this study was inadequate, and therefore the existence of pseudopreg— nancy in the absence of breeding should not be accepted as a documented entity. In a recent ultrasound study (604), pseu- dopregnancy was defined as development and maintenance of turgid uterine tone and the corpus luteum after embryo loss. In all of the 20 pseudopregnant mares, the location of the corpus luteum (left ver- sus right ovary) did not change during Days 11 to 40 and no ovulations, other than the follicular phase ovulation, were detected ultrasonically. Perhaps the term pseudopregnancy will be useful if further study demonstrates that turgid uterine tone is associated with prolonged luteal activity after embryo loss at the appropri- ate time, but that similar turgid tone is not associated with other types of pro- longed luteal activity. The occurrence of pseudopregnancy in association with embryo loss (pg. 528) and the hormonal basis for the turgid uterus (pg. 314) are dis- cussed elsewhere. Use of ultrasound to study prolonged luteal phases. Ovarian events and struc- tures were monitored by daily transrectal ultrasound examinations during 69 interovulatoy intervals in nonbred mares, primarily of Quarter Horse and Appa- loosa breeding (617). Diestrous ovulations were detected during three intervals (4%) 7, 9, and 11 days after the follicular— phase ovulations. Interovulatory inter- vals with diestrous ovulations on Days 7 or 9 were normal in length. However, the interval with the diestrous ovulation on Day 11 was prolonged (47 days). The orig- inal corpus luteum from the follicular— phase ovulation lost its detectability at the expected time. The diestrous corpus luteum was detectable from Day 12 to Day 36. That is, the prolonged portion of the luteal phase was attributable entirely to the secondary corpus luteum. In the absence of ultrasonic monitoring, the pro- longed luteal phase might have been attributed erroneously to spontaneous persistence of the primary corpus luteum. In addition to the single prolonged interovulatory interval due to the dies- trous ovulation, three prolonged intervals (34, 41, and 49 days) were attributable to the formation of hemorrhagic follicles that apparently failed to ovulate. Most of these structures formed outer walls 4 to 7 mm thick which may have repre- sented luteinized tissue (luteinized anovulatory follicles). Based on ultrasonic monitoring, the life span of the corpus luteum from the follicular-phase ovula- tion was not prolonged. Thus, of the four (6%) intervals that were prolonged for 34 or more days, none were attributable to persistence of the original corpus luteum. Perhaps the low incidence of die- strous ovulations in this herd, in turn, accounted for the low incidence of pro— longed luteal activity. This study demonstrated the advan- tage of ultrasonic monitoring for investi- gations that involve the life span of indi- vidual corpora lutea. With ultrasound, a second ovulation is less likely to be missed, and even if missed, the resulting corpus luteum may be detected later (590). In a study specifically designed to moni- tor individual corpora lutea, the intra— ovarian location, shape, and internal structure of each gland can be used to help assure that the same gland is being monitored. For these reasons, judicious use of ultrasound technology likely will be of immense value in future studies of pro- longed luteal activity. The technique should ultimately confirm or deny the existence and clarify the incidence of the syndrome known as spontaneous persis- tence of the corpus luteum. Suggested terminology for various types of prolonged luteal phases was pub- lished recently (599). It was noted that a term such as primary corpus luteum is essential in a discussion of prolonged luteal activity to define the luteal gland that results from the ovulation associated with the follicular phase. This is probably 230 Chapter 6 the most important consideration in com- munications on prolonged luteal activity. 6.9. Horses Versus Ponies As a result of a literature examination of genetic and physiologic differences, it has been concluded that the pony can be considered a small horse (536). However, several reproductive differences between the two mare types, especially when com- pared to certain horse breeds, are noted in this chapter and elsewhere in this text. Pony differences noted in this Chapter were the following: 1) longer estrus, diestrus, and estrous cycle; 2) shorter ovulatory season; 3) lower double ovula- tion rate; and 4) lower diestrous ovulation rate. French workers (928) have reported that ponies are less reliable than horses as recipients for transcervical embryo transfer; the pregnancy rate was 20 of 30 in larger mares compared to only 3 of 43 in ponies. The loss was due to a high inci- dence of PGan-induced luteolysis in the ponies, probably due to greater manipula- tion of the smaller cervix and greater uterine susceptibility to infection. Thus, the difference can be attributed, at least in part, to a smaller cervix. Other differ- ences, especially involving circulating hormone levels, can be attributed to a dif— ference in body size with a similarity in the mass of tissue producing the hor— mone. For example, eCG levels are high- er in ponies (pg. 421). This is attributable to similar total weight of endometrial cups. The similar weights of cups likely reflects the similar diameter of the embryonic vesicle and therefore bulk of the chorionic girdle at the time of inva- sion of the endometrium by girdle cells; girdle cells are the source of the eCG-pro- ducing cells of the cups. Later in preg- nancy (e.g., Day 100), the size of fetus diverges between the two mare types. Difference in fetal size is profoundly influenced by maternal body size and presumably uterine capacity (pg. 414). Recently (184), ovarian follicles, corpus luteum, and circulating progesterone lev- els were monitored daily in ponies and Quarter Horses. The diameter and growth profile over the seven days before ovulation did not differ between the two mare types (Day -1 mean diameter: 43 mm in ponies and 44 mm in horses). Similarly, the cross-sectional area of the ultrasonic image of the corpus luteum was not different. Progesterone results were equivocal, but there was a sugges- tion of greater concentrations in the ponies during mid-diestrus (approxi- mately 12.0 ng/ml in ponies and 10.5 ng/ml in horses); this difference, howev- er, was not in proportion to the difference in body weight. More study will be need- ed to reconcile the relationships among size of various endocrine glands, blood volume (body size), and circulating con- centrations of hormones. The question is important in attempting to study the effect of the concentrations of one hor- mone upon another. Reproductive differences among breeds within the same type (e.g., saddle horses) can also be marked. An outstanding example is the high multiple ovulation rate in Thoroughbreds (e.g., 20%) com— pared to an intermediate rate in Quarter Horses (e.g., 10%) and low rate in Arabians (e.g., 2%; pg. 218). Recent studies have suggested that there are breed dif- ferences in concentrations of hormones during pregnancy (e.g., estrogens, relaxin, pg. 431). The differences among types and breeds must be considered in extrapolat- ing research results. On the other hand, pony data, for example, should not be dis- missed because the research was not done in the breed of interest. Ponies are excel- lent and economical research models, and in most instances, the results will be a direct reflection of expected results in horses. Where doubt exists, the pony results provide direction for design of pro- jects dealing directly with a specific breed. Characteristics of the Ovulatory Season 231 HIGHLIGHTS: Characteristics of the Ovulatory Season Antral formation begins when the primordial follicles are 0.2 to 0.4 mm, and atre— sia is rare before 1 mm. One or two major follicular waves emerge during an estrous cycle. Each wave con— sists of a dominant follicle and several subordinate follicles. The primary wave begins at midcycle, and the dominant follicle is the source of the primary ovula— tion. Some mares have a secondary wave during early diestrus, and the dominant follicle becomes anovulatory or ovulates (secondary ovulation). The subordinate follicles of the primary wave begin to regress six or seven days before ovulation (physiologic selection of the ovulatory follicle). The selection mechanism can be overridden with gonadotropins. . L The first biochemical indicator of the selected ovulatory follicle, so far discovered, involves an increase in LH receptor content of the theca. Follicular evacuation during ovulation can occur abruptly (e.g., fluid 90% gone in 60 seconds) or gradually (e.g., 50% gone in 60 seconds). Ovulation occurs more often from the left ovary in maiden mares, but once preg- nancy has occurred, the asymmetry is lost. Ovulation usually occurs near the end of estrus. Multiple ovulation rate is affected by breed (high in draft mares and Thoroughbreds, intermediate in Quarter Horses, low in Arabians and ponies) and reproductive status (lower in postpartum mares). Unlike other species, mares frequently ovulate during progesterone dominance, especially certain breeds (e.g., Thoroughbreds). Sometimes a corpus hemorrhagicum forms in association with luteal develop- ment. The central area of the corpus luteum slowly fills with blood, beginning 20 to 30 hours after follicle evacuation. Compared to the uterus of diestrus, the uterus of estrus is thin, relaxed, and flac- cid as determined by palpation, yet edematous and large as determined by ultra- sonic imaging. In contrast to that of ruminants, the contractile activity of the equine uterus increases during diestrus and is at a maximum during luteolysis. The quantity of uterine secretions increases during late diestrus, followed by a rapid decline on Days 18 to 20. 232 Chapter 6 MILESTONES: Characteristics of the Ovulatory Season Extensive characterizations of the estrous cycle, ovulation, histology of tubu- lar genitalia, sexual behavior, and associated phenomena (85). Initial study of development of the corpus luteum (687). Characterization of uterine tone during the estrous cycle (1664). Discovery of diestrous ovulations (780, 1533). Discovery of prolonged luteal activity (1534). Scanning electron microscopy of the endometrium (1389). Characterization of follicular development by transrectal palpation, includ— ing the discovery that large follicles that are not destined to ovulate begin to regress by the beginning of estrus (575). 1979 Initial study on ultrastructure of the corpus luteum (951). 1980 Adaptation of transrectal ultrasonic imaging for examination of the repro- ductive tract, With initial characterization studies (1208). 1984-88 Detailed ultrasonic characterizations of cyclic changes in follicle populations (1264, 615); changes in shape, size, and echogenicity of the preovulatory folli- cle (1262); nature of follicular evacuation (1628); development, maintenance, and regression of corpus luteum (1629, 1261, 615); and cyclic changes in echogenicity of the uterus (614, 699). Development of ultrasonic technique for assessing uterine contractions With a description of the extent of uterine contractility throughout the estrous cycle (350). First report of ultrasonic monitoring of individual follicles during the estrous cycle (1484). -— Cfiapter 7—— ENDOCRINOLOGY OF THE OVULATORY SEASON The physiologic content of this chapter is developed in the following progression: 1) circulating concentrations of hor- mones, 2) regulation of the gonadotro- pins, 3) control of follicles and ovulation, 4) regulation of the corpus luteum, and 5) regulation of tubular genitalia. A final section will consider the applied aspects of artificial control of the estrous cycle. An introductory diagrammatic presenta- tion showing the organs that produce the reproductive hormones and the organs affected by the hormones is given in Figure 7 .1. 7.1. Concentrations of Hormones 7.1A. Luteinizing Hormone By the time techniques for quantitating equine LH were available, a rapid ovulato- ry LH surge was well documented in other species. Investigators in the equine field, however, found progressive increases and decreases in LH lasting many days, with the maximum concentration frequently occurring a day or two after ovulation. There was a one—year delay in publication of these findings, due entirely to the reluc- tance of manuscript reviewers to accept such a departure from the anticipated. Circulating levels. Early studies on cir- culatory concentrations of eLH were tab- ulated in the first edition (575). The abso- lute levels of LH in the tabulated reports varied as much as 191-fold among stud- ies. This was attributed to widely diver- § Portal 2 9 system 5 <6" 07’; &0 ’.G/}. E 55‘ 67?" 1 Q a e“ Arterial Venous system system E2- Estrogen P4 - Progesterone lH- Inhibin PGF Prostaglandin F20t RH- Releasing hormone LH- Luteinizing hormone FSH- Follicle stimulating hormone @l Capillary bed FIGURE 7.1. Simplistic presentation of hormonal control of the reproductive organs in the mare. Hormones are shown leaving the organ of produc- tion and entering the organs where they have their principal effect. In addition to exerting an effect through the hypothalamus, progesterone, estrogen, and inhibin may act directly on the pituitary. 234 Chapter 7 gent assay procedures, especially regard- ing the purity of the LH standards. The lowest values were obtained with the rel- atively purer McShan preparations (563, 1768). Except for differences in absolute values, however, the results of various investigations were reasonably consis— tent, and the mean LH profile may be summarized as follows (Figure 7.2): 1) remains low during mid-diestrus, 2) begins to increase a few days before the onset of estrus, 3) increases progressively thereafter to maximum values shortly after ovulation, and 4) decreases progres— sively over the next 4 to 6 days to the low diestrous values. Studies during the last decade have continued to support the ear- lier results (e.g., 828, 499, 827, 1348, 1146). No differences were found between 12 pony and 6 horse mares in the LH profiles dur- ing the estrous cycle (1092). Results of a study that encompassed the ovulatory season indicated that the postovulatory levels of LH were lower during the latter half of the ovulatory season (1644). Additional studies are needed on the effect of length of photoperiod on the LH surge and on LH bioactivity. Gonadotropins 1o (n=12> Concentration (ng/ml) -3 0 Number of days from ovulation 3691215182103 FIGURE 7.2. Mean concentrations of circulating FSH and LH during the estrous cycle of pony mares. Vertical bar (lsd) indicates the magnitude of the least significant difference (P<0.05) for each hormone. Adapted from (1092). Isoforms. The principal findings on cir- culating levels of eLH during the last decade centered on the divergence between immunoactivity and bioactivity due to LH isoforms (microheterogeneity; pg. 43). In one study (6), immunoactivity was examined by conventional RIA and bioactivity by an in vitro bioassay (mouse interstitial cell-testosterone assay). The LH profiles, as determined by the two assays, were similar during the luteal phase. During the follicular phase, the levels increased, but the relative increase for bioactivity was greater than for immunoactivity (Figure 2.1, pg. 44). This finding confirmed earlier studies (20) in that bioactive LH rose earlier than immunoactive LH during the periovula— tory surge. The importance of these changing isoforms of LH to regulatory mechanisms, if any, are not known. Investigators need to be especially aware of this phenomenon in attempting to determine, for example, the pathogene- sis of ovarian dysfunction (e.g., determin— ing the reasons for luteal dysfunction fol- lowing certain types of GnRH-induced ovulation). Pulsatility. A second recent emphasis in studies of circulating concentrations of eLH during the estrous cycle involves pulsatile release (pg. 45). It was found that pulses in the systemic circulation were detectable during the luteal phase but not during the high levels associated with the periovulatory surge (6, 511). In a more recent study (23), however, LH pulses were detected during the periovulatory period in pituitary venous blood. The fre- quency of pulses varied from approxi- mately one pulse per two hours early in the LH surge, to almost one pulse per half hour at the time of ovulation. The authors suggested that difficulty in detecting pulses in the peripheral circula- tion was because amplitude of each pulse was small. The long half life of eLH may result in a pool of sufficient magnitude to dampen the impact of individual rapidly occurring pulses. Endocrinology of the Ovulatory Season 235 The pulses of LH during diestrus occa- sionally will be detected even when sam- pling is done once each day. This again is attributable to the long half-life of eLH. Because many daily samples will miss an LH pulse, or a major portion of it, and the pulses occur at different times among mares, the daily means are low during diestrus, as shown (Figure 7.2). The pulses of LH are often synchronized with a pulse of FSH during diestrus, but not during estrus, when measured in the peripheral plasma (1602). However, con— comitant pulses can be detected during estrus in the pituitary effluent (pg. 46). In a recent study (184), daily values of LH and FSH during the interovulatory inter- val were examined in horse mares. Pulses of LH and FSH were located by pulsar analyses, and the FSH pulses were nor— malized to the LH pulses. The synchrony in pulses between the two gonadotropins was striking during diestrus, but the two gonadotropins were dissociated during late diestrus and estrus (Figure 7.3). The occurrence of LH pulses during diestrus may account for the diestrous-ovulation phenomenon in this species (pg. 223). Jennies. The circulating concentrations of LH during the estrous cycle of jennies were similar to those in mares (Figure 7.4; 1679). The peak LH value was on Day 1. However, the surge appeared to be longer than what has been reported for mares. The LH values of the surge were significantly elevated above baseline diestrous values for almost half of the estrous cycle. 7.13. Follicle Stimulating Hormone Circulating concentrations. Mean con- centrations of FSH are low during estrus, increase during diestrus, and decrease beginning approximately 8 days before ovulation (Figures 7.2 and 7.3). The mean FSH and LH profiles are approximately reciprocally related. On the average, FSH begins to rise before ovulation. Gonadotropins 25 20 15 10 U! N 01o Concentration (ng/ ml) N D d 01 10 0 4 8 12 16 20 2 Number of days from ovulation FIGURE 7.3. Synchrony in LH and FSH pulses (upper panel) and means for circulating concentra- tions of the two gonadotropins (lower panel). Data were obtained by daily blood sampling. To illus- trate the association between LH and FSH pulses, significant peaks of LH were identified statistically in individual mares. The significant LH peaks were designated 1°, 2°, and 3° according to prominence. The peak LH value and the daily value on each side of the peak were used to define each peak. The mean days of occurrence of LH peaks were calculat- ed, and the LH values of individual mares were normalized to the mean days of occurrence of peaks (1°, 2°, and 3°). The FSH values were then normal— ized to the LH values on the corresponding days. The stars indicate significant differences between the LH values and FSH values for each peak. Note that the magnitude of LH and FSH pulses was widely dissociated during the periovulatory LH surge (1°) but was closely associated during the first half of diestrus. Because of the occurrence of peaks on different days among mares, the 2° and 3° FSH and LH peaks were masked in the mean profiles for each hormone (lower panel). Adapted from (184). 236 Chapter 7 Gonadotropins in jennies 16 (n=14) 14 5|st ‘11 v w '1. E8 7‘“ H i ii 6) FSH H 5 a .5 6 a ‘5 E 8 C O o -12 -8 -4 0 4 8 12 Number of days from ovulation FIGURE 7.4. Daily plasma gonadotropin concen— trations in jennies before and after ovulation. The lsd bars represent the magnitude of least significant differences (P<0.05). Adapted from (1 6 79). FSH surges. Sometimes two or more surges of FSH occur. Two broad surges of FSH at 10- to 12-day intervals were described in the original studies by New Zealand workers; one occurred during late estrus and early diestrus and the other during late diestrus with a peak 10 to 13 days before ovulation (490, 526). The occurrence of bimodal or biphasic FSH profiles has been found by other laborato- ries (263, 1146). In a study in horse mares, 16 of 20 estrous cycles were judged to involve two surges of FSH; however, the surges were obscured in the mean profiles because of variation in occurrence time (1146). In another study (1092) using daily sampling, only 2 of 12 ponies and 1 of 6 horses had apparent bimodal FSH pro— files. Others (1606) commented that three distinctly different profiles or patterns of FSH levels were obtained in their study. These profiles were as follows: elevated FSH throughout diestrus (2 mares), an early diestrous surge (6 mares), and a late diestrous surge (2 mares). When averaged together, an apparent biphasic pattern was obtained. The profiles of cir- culating levels of FSH during the interovulatory interval apparently can exhibit various modalities (unimodal, bimodal, or trimodal) often due to surges detected during only one daily sample. When these patterns are averaged to- gether to produce an overall profile for a group, the mean profile can be a mislead- ing representation of FSH patterns in individual mares. Concentrations of FSH were examined every three days throughout the ovula- tory season in ponies (1644). On the aver- age, two surges of FSH occurred during estrous cycles early in the ovulatory sea- son, whereas one surge occurred late in the season. An effect of month of the ovu- latory season on FSH patterns has not been confirmed. The studies cited above indicate tremendous variation in the FSH profiles during the interovulatory interval as determined by daily sampling. The notion that FSH follows a consistent modality, as do the other hormones (e.g., progesterone and estradiol), is not justi— fied. The generalization of high mean di- estrous levels and low estrous levels is documented, but the high mean diestrous levels do not result from a consistent pat- tern among individuals. Perhaps the mar- vel is the tremendous variation that the mare can tolerate without an apparent effect on estrous cyclicity. Clearly, the complexities of FSH profiles during the estrous cycle have not been adequately resolved and probably will not be until experimentation takes into account the following: pulsatility (see next para- graph), repeatability within mares, breed or type of mares (e.g., horses, ponies), month of the ovulatory season, age, follic— ular profile, and other factors. The only available study in jennies indicated an average profile of three FSH surges, with maximum values at Day 3 and Day 9 Endocrinology of the Ovulatory Season 237 after ovulation and 10 to 12 days before ovulation (Figure 7.4; 1679). Clearly, more study also is needed in this species. Isoforms and pulsatility. As for LH, much experimentation with FSH during the past decade has involved microhetero- geneity and pulsatility. Unlike LH, the limited data on FSH microheterogeneity indicate that bioactive and immunoactive forms follow a similar profile during the estrous cycle, except that the low and high bioactivity values are lower and higher, respectively, than those for immunoactivity (475). In the same report, pulses of FSH were identified on Days 9 to 13 with a frequency of 1 every 8 to 12 hours and a mean duration of 4 hours. In other studies (1602, 184), pulses of LH and FSH occurred together and sometimes were tightly coupled, especially during diestrus. As noted above, synchrony of LH and FSH pulses has been detected during early diestrus even when samples were collected only once per day (Figure 7.3). Collection of local pituitary blood allowed detection of concurrent pulsati— lity even during the periovulatory period, (pg. 113; 23). Evans (481) detected FSH pulses in the peripheral circulation by sampling every 3 minutes and noted that sampling peripheral blood every 10 min- utes, as done by others (25), may not be adequate for characterization of FSH pulses. Pulses were detected in all of the time periods examined (Days 0, 3, 7 to 9, 15). Averaged over all days, the duration of peaks was 5.9 minutes and occurred every 15 minutes. The interval between peaks was longer for Day 0 than for Days 7 to 9 and Day 15 (equivalent to 3 pulses per hour during estrus and 5 pulses per hour during the middle and late luteal phase). Considerable additional study will be needed to clarify the nature and variation in FSH pulsatility during vari- ous reproductive statuses and phases. As noted above, pulsatility beclouds studies that are limited to infrequent sampling (e.g., once per day). 7.1 C. Progesterone Early studies. Sensitive methods for quantitating progesterone in the blood of mares were not reported until the 1970s. Methods available before that time (chro- matographic systems) permitted determi- nation of progesterone in blood only when concentrations were high (mid-luteal phase; 1460) or when measured directly in ovarian venous effluent (1464) or luteal tis- sue (1463, 1677). These earlier methods, nevertheless, led to the conclusion that progesterone discharge increases within the first 24 to 36 hours after ovulation (1464), and the concentrations found by these methods at mid-diestrus are compa- rable to those found with more modern techniques. Assay. Radioimmunoassay of equine progesterone is usually done in liquid phase by charcoal extraction. Direct RIA of progesterone in unextracted plasma is possible, especially since the labeled pro- gesterone is weakly bound by mare plas- ma (1026). Comparison of procedures for radioimmunoassay of progesterone using charcoal extraction versus solid phase has been done for several species, includ- ing horses (1529). Several reports are available on the effects of sample han- dling and storage on assay results (1159, 1802, 1178). Luteal progestins. Progesterone is, by far, the main steroidal product of the cor- pus luteum of the estrous cycle. In luteal tissue, ZOa—dihydroprogesterone was found, and the mean concentration was 7.0 ug/g in six corpora lutea from cycling mares (1677). An apparent high value of 40 ug/g was found in a corpus luteum that was believed to be regressing. Incidentally, 20a-dihydroprogesterone was not found in corpora lutea from preg- nant mares after Day 17. The importance of these findings on the production of pro- gestins, other than progesterone, by the corpus luteum is unknown; more study is needed. Within-animal comparisons of 238 Chapter 7 progesterone concentrations between luteal tissue and blood apparently have not been done, but the changes found in luteal tissue (1677) seem similar to those found in systemic blood by subsequent workers. Circulating concentrations. Results of systematic study through serial blood sampling were tabulated for 10 reports in the first edition (575). Study of the tabu- lated material suggested several general— izations. There were no obvious differ- ences among the breeds. Considerable variation was obtained, as indicated by the standard deviations or standard errors. Mares apparently have consider- able leeway in the amount of proges- terone required during diestrus and also, incidentally, during pregnancy (38). The amount of variation among days and among mares was too great to detect sig- nificant differences in one study which used 3 or 4 mares per day (1527); only the low estrous means and the high diestrous means were significantly different. The tendencies and interpretations across the many reports were consistent and permit- ted the following generalized description of the curve (Figure 7.5). The concentra- tions during estrus were well below 1 ng/ml, usually below 0.5 ng/ml, and approached or were below the lower lim- its of assay sensitivity. Concentrations increased within 24 to 36 hours after ovu- lation (discussed below) and thereafter increased progressively to the high di- estrous values by Days 5 to 7 after ovula— tion (range of means among 10 reports: 4 to 22 ng/ml). High values were main- tained, although somewhat erratically, until Days 13 or 14 (approximately 3 days before estrus). Thereafter, the values decreased rapidly until the low estrous values were reached. Studies during the past decade have continued to support the shape of the progesterone profile that was elucidated during the 1970s (e.g., 1010, 1591, 1091, 1808, 827, 1348, 1146, 1633). Similar circulating progesterone concen- Progesterone (n=11) —A J:- —L N .5 D co 0‘) Concentration (ng/ml) .h N 0 2 4 6 810121416 Number of days from ovulation FIGURE 7.5. Daily changes in plasma proges- terone concentrations. On the incline, a mean marked with a star is different (P<0.05) from the preceding mean. On the decline, a mean marked with a star is different (P<0.05) from the mean at Day 11. Adapted from (1633). trations during the estrous cycle have been reported for donkeys (1749). The peripheral circulating concentra- tions of progestins, other than proges- terone, and metabolites of progesterone during the estrous cycle have not been adequately investigated. Of the pro- gestins that are most likely to interfere with progesterone assay specificity, only 17a—hydroxyprogesterone was found in ovarian venous effluent and concentra- tions were relatively small (1464). In a more recent study (1091), concentrations of pregnenolone and the 17a-hyroxylated metabolites (170c-hydroxypregnenolone and 17a—hydroprogesterone) followed sim- ilar profiles (Figure 7.6). The concentra- tions of all of these progestins decreased at the time of luteolysis. Postovulatory rise. Progesterone does not increase until Day 3 after ovulation in cows and sheep but begins to increase immediately after ovulation in mares (1276) and swine (679). In a recent study (1633) in 12 pony mares, samples were Endocrinology of the Ovulatory Season 239 Circulating steroids (”=8) 40 Estradiol-17B i’l tr]- -- x i “i“ 20 ifl" E l [-1 ’r Estrone; E m 5 3- 10 i c 5 .2 — E E Dihydro- E 300 3 epiandrosterone 8 (DHEA) g 400 . O s 200 Androstenedione |'_ \/v+”i~ \~—'lIl \"+\\ :3 W i i l-i--|~-|~~l'i .-+--+-’r-\. wit , .- l-"|-+\i"i/¥ i $.10...“ I ‘|'-'| \ 3 10 Testosterone 6.0 f , Progesterone 2.0 j i Jul. 0.8 ,' +~.IL.i E 0.4 i“ i E ‘ + 1 70L—hydroxy- .5 0.1 |-- ll“l--[”l progesterone E E 8 6.0 C o 0 2.0 4,’l‘*‘+~l--l--I 0.8 |_+ 4’4" 04 I“ III R ' ‘1.—r-r—T-,--|" 170L-hydroxy- . pregnenoione 0.1 QV -8-6-4-2024 68 Number of days from ovulation FIGURE 7.6. Concentrations of various steroids during the periovulatory period. Adapted from Mienecke et al. (1091). taken at half-hour intervals in mares with precise information (monitored con— tinuously) on the time of follicular evacu- ation. The half-hour samples extended from three hours before until two hours after the end of follicular evacuation. Additional samples were taken at 5, 8 and 12 hours and every 12 hours there- after. Progesterone concentrations were below 0.5 ng/ml for three hours before and five hours after follicular evacuation (Figure 7.7). In 9 of 12 mares, proges- terone increased between 8 and 12 hours and in the remaining mares did not increase until between 12 and 24 hours (2 mares) or 24 and 36 hours (1 mare). The first significant increase averaged over all mares occurred between 12 and 24 hours. Timing of the study was more precise than in earlier work (1276, 1271). Results, however, are compatible among studies. Ultrasonic luteal morphology (presence or absence of central blood clot; 1633) or mat- ing (1271) did not alter the characteristics of the post-ovulatory progesterone rise. Progesterone ‘5 (n=12) —l N Concentration (ng/ ml) 00 0 24 48 72 96 120 Time relative to end of follicle evacuation (hours) FIGURE 7.7. Changes in peripheral progesterone concentrations beginning within one-half hour of the defined end of evacuation of the ovulatory folli- cle. A mean marked with a star is different (P<0.05) from the preceding mean marked with a star. Adapted from (1633). 240 Chapter 7 Effect of month. Progesterone concen— trations were compared between the summer (May to July) and fall (August to October) in riding-type horse mares (1633) There was no significant effect due to season (May to July versus August to October) or a day by season interaction. A study of slaughterhouse specimens con- cluded, however, that mean concentration of progesterone during the luteal phase increased markedly during April to September (1176). In contrast, in another study in light horse mares (886), a decline from cycle to cycle in the diestrous levels of progesterone occurred over the course of the ovulatory season in 14 of 20 mares that subsequently entered an anovulatory state. Six mares maintained a consistent level of diestrous progesterone and did not become anovulatory during the ensu- ing winter. Results of these studies (1633, 886, 1176) seem divergent, indicating the need for additional study. A decrease in the diestrous progesterone plateau after the middle of the ovulatory season would be consistent with the declining magni— tude of LH surges (1644) and with a luteotropic effect of LH (pg. 265). Diurnal and pulsatile effects. Several authors have commented on the appear— ance of two progesterone peaks in mares at Days 5 or 6 and again at Days 8 to 10, but such a phenomenon has not been demonstrated convincingly. Levels do appear to be more erratic during the max- imal luteal phase than can be accounted for by assay error. The possibility of diur- nal variation in progesterone concentra- tions was indicated by the conclusion that morning samples were as much as 3 ng/ml higher than evening samples (1674). It also has been concluded that large dif— ferences in progesterone concentrations can occur over a 48—hour period; however, concentrations tended to be higher in the afternoon than in the morning (313). Clarifying studies of diurnal effects are needed. Recently (482), pulsatile release of progesterone was described for mares. The pulses were detected in 3-minute and 15-minute blood samples. The mean num- ber of pulses ranged from 3.5 to 6 per 8 hours for the various days studied. The author commented that the observed pulses explain the day to day variation obtained by other laboratories for daily blood samples. The appearance of a small progestin peak just before ovulation has been sus- pected (1674). It was suggested that this progestin might be 17a—hydroxyproges- terone, based on comparative considera- tions (apparently such peaks occur in women) and because of the findings of others that mature follicles contain con- siderable quantities of this steroid prior to ovulation. The authors pointed out that this observation was preliminary and required confirmation. Progesterone in milk and in other specimens. The pattern of progesterone profiles in milk during various repro- ductive states is similar and highly cor- related with the pattern in blood (784, 124, 909, 659, 1565, 462, 911). Radioimmunoassay was used in most studies, but enzyme- immunoassay also has been used (982). Lower levels and less variation are obtained if the milk is defatted (659). Milk progesterone values may have clinical application in early diagnosis of pregnan- cy and in detection of estrous cycle irreg- ularities (784, 659); breeding programs have been proposed based on monitoring milk progesterone at intervals of 2 or 3 days (784,659). Progesterone concentrations can be measured in uterine—rinsing fluid (981), but it is not clear whether the procedure might have some diagnostic or research value over measuring progesterone in the blood or milk. Luteal function can also be monitored in mares by determination of 200(- hydroxylated progestins in feces (1411). This technique would be useful for feral populations. In one study, values ranged from 30 to 50 ng/g during the follicular phase and 140 to 260 ng/g during the luteal phase. Progesterone, in contrast to Endocrinology of the Ovulatory Season 241 the ZOa-hydroxylated progestins, did not show cyclic patterns in feces. 7.1D. Estrogens Assay. Estrogen metabolism and the excretion of urinary estrogens in mares have been extensively studied for many years (pg. 65). The excretion of estrogens in the urine is believed (730) to provide a rea- sonable index of secretion rates, based on the concept that estrogens are not stored in the body. Assay technology has advanced to the point where characteriza- tion of concentrations of circulating estro- gens can be done. Assay of circulating estrogens is complicated by the many types of estrogens that can cross-react with the RIA antisera. Some researchers have used chromatographic separation techniques to circumvent this problem, whereas others have studied levels of total estrogens. A hydrolysis and extrac- tion technique for preparing plasma sam- ples for RIA of combined conjugated and unconjugated plasma estrogens has been described (1219). Values thus obtained were 100 times higher than for specific measurement of estradiol-17B. It was con- cluded that such measurements are sim- ply performed and give a good indication of follicular activity. One assay approach (RIA) used a separation technique that allowed direct estimation of estrone sul- phate concentrations in ethanol extracts of plasma (1010). A recent report (529) includes a discussion of equine estrogen assay techniques. Circulating concentrations. Initial stud- ies on the changes in circulating or uri- nary excretory levels of estrogens were tabulated in the first edition (575). Reports during the last decade include an assess- ment of peripheral changes in estrone sulfate concentrations during the estrous cycle, with data normalized to the day of ovulation (1010). Several other groups of workers have reported on circulating lev- els of estradiol (6, 1091) and estrone sul- phate (529, 910). The following generaliza- tion seems to be a reasonable summariza- tion of the results of initial, as well as recent, reports on circulatory estrogen concentrations (Figure 7.8). Beginning 6 to 8 days before ovulation, or at approxi- mately the beginning of estrus, circulat- ing concentrations of estradiol-17B or estrone sulfate increase progressively and reach a peak approximately two days before ovulation. By the time ovulation occurs, concentrations have been decreas- ing and reach basal diestrous values near the end of estrus or Within a day or two after ovulation. Similarly, the concentra— tions of estrone and estradiol-17B (deter- mined by RIA after extraction) increased significantly on Days -4 and -3, respec- tively, and decreased on Day -1 (Figure 7.6; 1091). Plasma concentrations of estra- diol-17B have been reported for jennies (1837, 1749); the profiles seem similar to those described for horses. Urinary concentrations. Urinary estro- gen concentrations during estrus report- edly are higher early in the breeding sea- son (spring) than late in the season (fall). This may be another source of variation among studies. In one study (730), excre- tion rates for both estradiol and estrone seemed to follow a similar pattern during Estrone sulfate (n=12) 1000 800 O) O O h C) 0 Concentration (pg/ ml) [0 O O -8 -4 0 4 8 12 Number of days from ovulation FIGURE 7.8. Concentrations of estrone sulfate in daily samples of peripheral blood. Adapted from Makawiti et al. (1010). 242 Chapter 7 fall and spring, and relative urinary con- centrations of the two hormones were similar. However, absolute levels were higher for both hormones during the spring. A significant monthly difference in preovulatory urinary estrogens has been reported (1216). The significance seemed attributable to higher levels dur- ing estrus early in the year. The monthly changes in concentrations of excreted estrogens seem positively related to the monthly changes in length of ovulatory estrus or the follicular phase (pg. 174). However, it was stated in a review by one of these authors (1202) that their previous finding (1216) of seasonal variation in pre- ovulatory estrogen levels may have been an artifact due to variations in urine pro— duction since a seasonal pattern was not found in plasma. The effects of month of estrogen production should be reevaluat- ed. Strong but indirect indications of diminished estrogen production in the later portion of the ovulatory season are the seasonal effects on endometrial ultra— sonic echotexture (pg. 212). Secondary peaks. Only a preovulatory peak has been reported for the estrous cycle of mares. In contrast, cattle and sheep have more than one peak (679). Inspection of Figure 7.8 suggests that a secondary peak may have occurred at Day 6, but the authors (1010) did not com- ment. Specific studies are needed, using frequent sampling or a more sensitive assay, to look for secondary peaks. An early diestrous peak can be hypothesized on the basis of the following: 1. Some interovulatory intervals have considerable follicular activity during the first half of diestrus (pg. 178); 2. Ultrasonic uterine echotexture stud- ies have revealed the presence of a slight but significant increase in an estrus—like echotexture in early diestrus during the first half of the ovulatory season but not during the second half (pg. 212); and 3. A one-day increase in uterine tone has been reported to occur in early diestrus (Day 6; 650). Late entry. The dynamics of urinary and plasma estrogen conjugates and plasma estradiol-17B were found to be in parallel (1860). A threefold increase in mean urinary and plasma estrogen con- jugates occurred during the ovulatory peak. Most noteworthy, urinary and plas- ma estrogen conjugates, but not plasma estradiol, showed a secondary surge over Days 4 to 7. This observation is consis— tent with the initial indications of a sec- ondary postovulatory peak noted above. The secondary estrogen surge may reflect follicular growth that occurs in some mares in early diestrus or may reflect estrogen production by the developing corpus luteum. Luteal estrogens appar— ently are produced during the production of eCG (pg. 446). Perhaps the high post- ovulatory LH concentrations in this species stimulate estrogen synthesis by the developing corpus luteum. A reduc- tion in estrogen conjugates to baseline levels occurred at the end of the luteal phase (1860), further suggesting estrogen production by the corpus luteum. 7.1E. Androgens Role. Androgens are generally associ— ated with reproductive function in males. Various androgens, however, have been detected in the peripheral cir- culation of females, including mares. The possible physiologic importance of androgens in the female includes a role in sexual behavior (pg. 96), development of preantral follicles, follicular atresia, regulation of FSH (pg. 250), and, together with estrogen, determining whether a large follicle will regress or ovulate (1252). As noted below, two and possibly three androgens (androstenedione, dehy- droepiandrosterone, possibly testos- terone) increase in the circulation of mares during estrus. Because in some estrous cycles there is evidence for a surge of growth of follicles immediately after ovulation (pg. 178), the androgens may function in bringing the follicles to Endocrinology of the Ovulatory Season 243 an antral stage prior to this time. In regard to atresia, it is noted elsewhere (pg. 182) that large nonovulatory follicles regress during estrus at a time when androgens are increasing in the circula- tion. However, direct effects of androgens on antral follicles and atresia are specu- lative. Nevertheless, there are good indi- cations that androgens have an FSH reg- ulatory role in females, and studies in this area have been extended to mares in the past decade (pg. 250). In follicular fluid. Testosterone has been identified in the follicular fluid of mares (1468). The concentration of testos— terone in follicular fluid was twice as high as in the peripheral plasma, sug- gesting that the follicles may contribute to circulating testosterone levels. A recent study (1469) found that testos- terone levels in the follicles of mares remained constant during follicular growth and the early stages of atresia. In another recent study (1737), a significant increase in testosterone was found in the follicular fluid from the time the preovu- latory follicles were 32 to 34 mm until 28 to 32 hours after reaching 35 mm. An injection of hCG at the time the follicle reached 35 mm did not significantly increase the testosterone levels of follicu— lar fluid but did increase (significant) the progesterone and possibly (not signifi- cant) the PGan levels. Estradiol levels did not increase with or without hCG treatment. During atresia there was a decrease in steroidogenesis without an accumulation of androgens (1469). Circulating concentrations. Testoster- one levels in plasma reportedly were higher at estrus in 4 of 6 mares, with another peak occurring 11 days before ovulation (1470); the data were limited and must be accepted with reservation. In other studies (1091, 1126), a nonsignifi- cant rise in testosterone occurred during estrus (Figure 7.6). Androstenedione is an estrogen precursor in the biosynthet- ic pathway (Figure 2.7, pg. 62). A signifi- cant elevation in androstenedione in PGonc-treated mares was found on the day before the post-treatment ovulation (548). Other studies (1091, 1126) also sug— gested that a rise may occur during estrus but further study is needed. The follicular content of androstenedione (1469, 1091) and testosterone (1737) increased as ovulation approached, giv- ing support to the report of circulatory increases. A preovulatory rise of dehy- droepiandrosterone also has been report- ed (1310). Measurable quantities of this androgen have been extracted from fol- licular fluid of mares, and it has been proposed that the hormone is part of an alternate, though minor, metabolic pathway between pregnenolone and androstenedione (Figure 2.8, pg. 63). Max- imum dehydroepiandrosterone (DHEA) concentrations were observed prior to ovulation in 10 of 12 estrous cycles in pony mares but were already in decline when ovulation occurred (1310). Peak values before ovulation ranged from 300 to 720 pg/ml. Other workers (1091) found a significant increase by Day -6, with a maximum mean value on Day -1 (Figure 7.6). No association was found between lev- els of DHEA (1310), testosterone, or androstenedine (1126) on the expression of covert versus overt estrus. A possible association between the appearance of the hormone and estrous expression was not tested adequately, however. One mare was treated with dexamethasone, and the peak levels of dehydroepiandro- sterone during estrus appeared to be reduced. The authors hypothesized that the steroid may be of adrenal origin since the corticoid dexamethasone seemed to suppress circulatory levels and only small amounts of the androgen were found in ovarian follicles. Other workers (1091) have also concluded that the increase in DHEA during estrus is not from the ovarian follicles. According to an abstract (1837), plasma testos- terone in jennies was at highest levels on Days 6 to 10. 244 Chapter 7 7.1F. Cortisol Plasma cortisol levels in the mare fol- low a profile similar to progesterone dur- ing the interovulatory interval (104). Extraction of samples was used to elimi— nate assay interference from progesterone and its metabolites. Plasma values of cor- tisol were high during diestrus and low during estrus, reaching the lowest value two days before ovulation. A single burst of cortisol was detected at the expected time of luteolysis in 6 of 7 cycles. Perhaps these spikes were a result of PGFZoc pro- duction by the uterus. Decline in cortisol during estrus may be necessary for the events that accompany follicular growth and ovulation. In this regard, administra— tion of dexamethasone, a synthetic gluco- corticoid, significantly reduced estrous behavior, LH concentrations, preovulato— ry follicular growth, and the incidence of ovulation (98). Ovulation was not blocked when only a single injection was given (450). Curiously, a significant mean decrease (45%) of circulating cortisol lev- els occurred during sexual stimulation and copulation in mares (1151). A recent study in ovariectomized mares (1071) found suppression of LH levels after dex- amethasone treatment; concentrations of cortisol also were reduced. These studies raise questions about the role of adrenal steroids in ovarian function and whether abnormal production of such steroids plays a role in stress—mediated interfer- ence with ovarian function. The effects of transportation on certain reproductive end points were examined in mares transported for 24 hours during preovula- tory estrus (163). Transportation increased cortisol levels but did not alter the time of ovulation, duration of estrus, or preovula- tory surges of LH and estradiol. 7.1G. Prostaglandins Exogenous PGonc is a potent luteolysin in mares (pg. 271), as well as in other species. In the original study of prosta- glandins in the blood of mares, the con- centrations of the F-series of prosta- glandins were measured in the uterine veins during the estrous cycle in anes- thetized mares (433). Concentrations in uterine venous plasma increased and peripheral progesterone decreased between days 10 and 14 of diestrus (Figure 7.9). Presumably, the concentra- tions of the prostaglandin-F series reflect- ed primarily PGFZoc concentrations. In another study (1840), an increased level of prostaglandin-F was obtained from uter- ine flushings in Thoroughbreds and Quarter Horses on Day 14 postovulation, the approximate time of expected luteol— ysis. Other workers have studied PGonc involvement in luteolysis by measuring a PGFZoc metabolite (15—keto-13,14—dihy- dro-PGFZa) commonly known as PGFM. The metabolite has a prolonged half-life in the peripheral circulation and there- fore is widely used as an indicator of PGFZa production (880). In the example presented by these workers for the mare, a small increase in PGFM occurred Progesterone & PGF 25 (n=3 or 4) -l M 01 0 Concentration (ng/ ml) 3 Day 7 2 6 1o 14 18 0f estrus Number of days after estrus FIGURE 7.9. Progesterone concentrations in jugu- lar vein and PGF concentrations in uterine vein in cycling anesthetized ponies. Within each end point, means with no common letters are significantly dif— ferent. Adapted from (433). Endocrinology of the Ovulatory Season simultaneously with the first decrease in progesterone (Figure 7.10). Release of the metabolite continued for about 36 hours after luteolysis was completed. Half-life studies of this metabolite and another metabolite found in urine have been reported recently (632). Results of the various studies cited above are compati- ble with the hypothesis that PGonc plays a role in luteolysis at the end of diestrus in mares. A study has been done on the concen- trations of prostaglandins (PGE2 and PGan) in the equine corpus luteum (1741). Dispersed cells were incubated for 24 hours, and hormone concentrations were measured in the medium. The pre- dominant prostaglandin released by the early corpus luteum was PGE2 in contrast to PGFI in other species. Secretion rate of prostaglandin decreased from early diestrus (Days 4 or 5) to later in diestrus (Days 8 or 9 and Days 12 or 13); however, the ratio of PGonczPGEz increased. It was suggested that the changing ratio 245 may be important in controlling corpus luteum life span. Day of diestrus did not affect progesterone production by the incubated cells. Tissue culture of di- estrous equine endometrium has indicat- ed that cultured endometrial cells produce PGEZ, as well as immunoreactive PGFZOL after 1 to 24 hours of incubation (1736). 7.1H. Inhibin Circulating concentrations of immuno— reactive inhibin during the estrous cycle were determined daily in horse mares (185). The highest mean values occurred on Day 0 at both ends of an interovulato— ry interval (Figure 7.11). Other than the consistently high levels on Day 0, the concentrations were highly variable among individuals, as shown in the two examples in the figure. However, a sig- nificant decrease in mean concentration occurred between Days 0 and 1. Mean concentrations then remained low, fol- lowed by a significant increase between Progestins & PGFM 12 _L D co Progestins concentration (ng/ ml) 03 -2 0 2 4 6 8 10 240 200 160 120 80 (m1 /6d) uonenuaouoo IAHEJd 40 .mu-o-o-o 0-0 05-0; 0 ov 12 14 16 18 0 2 Number of days from ovulation FIGURE 7.10. Peripheral plasma concentrations of 15—keto-13,14-dihydro-PGF20c (PGFM) and progestins during an estrous cycle of a single mare. Adapted from (1136). 246 Chapter 7 Days 7 and 12. Concentration in a 38 mm preovulatory follicle was approximately 9,000—fold higher per milliliter than in the plasma. Viable equine follicles have been reported to have more bioac— tive inhibin-like activity than atretic follicles (296). FSH & Inhibin Mare A 25 . ir-inhibin 20 15 10 5 0 12 Mare B FSH concentration (ng/ml) and relative amount of ir-inhibin -4 0 4 8 12 16 Number of days from ovulation 2002 FIGURE 7.11. Concentrations of immunoreactive inhibin (ir-inhibin) and FSH in two representative mares and mean concentrations based on daily blood sampling. Mean concentrations of the two hormones were reciprocally related. The vertical bar (lsd) indicates the magnitude of the least significant difference (P<0.05) for each hormone. The examples (Mares A and B) illustrate the wide variation among mares. Adapted from (185). 7.2. Regulation of Gonadotropins 7.2A. Temporal Relationships Among Hormones A common method for studying the effects of one hormone upon the circulat- ing concentrations of another is to com- pare the interrelationships of concentra— tions over time. If one hormone increases in concentration when another decreases, the two changes may be causally related, especially if one change follows the other. Conclusions on cause-and-effect relation— ships based solely on such studies must be tempered. Such observations do, how- ever, provide a reasonable basis for devel- oping hypotheses for critical testing. Estradiol and progesterone. In gener- al, the principal ovarian steroids (estra- diol and progesterone) are reciprocally related. This is entirely expected since progesterone characterizes diestrus or the luteal phase, and estrogen character- izes estrus or the follicular phase. LH and FSH. Means of the two gonadotropins, LH and FSH, also seem to be primarily reciprocally related (Figures 7.2, 7.3, and 7.11). Data for LH and FSH were normalized for individual mares according to highest LH value and lowest FSH value (1092). The reciprocal relation- ship between LH and FSH means became dissociated between the day of the lowest FSH value (mean: 2.8 days before ovulation) and the day of the high— est LH value (mean: 1.6 days after ovula- tion). During this time (the few days encompassing ovulation) both LH and FSH increased significantly. At all other times of the cycle, mean LH and FSH changes were in opposite directions. These relationships apply to averages; as noted above, pulses of the two gonadotropins occur concomitantly dur- ing diestrus. Endocrinology of the Ovulatory Season Estradiol and LH. Both estradiol and LH increase gradually over approximate- ly the same time, except that the estradi- ol peak occurs days before the LH peak (Figure 7.12; also compare Figures 7.2 and 7.8; 1162, 1249). The two hormones decrease gradually over several days. Progesterone and LH. Progesterone production by the new corpus luteum begins just before the reduction in circu- lating concentrations of LH (compare Figures 7.2 and 7.5; 82, 1145). Similarly, progesterone reduction at the end of diestrus precedes the LH increase (Figure 7.13). At the beginning of diestrus, pro- gesterone increased significantly one day prior‘to a significant decrease in LH. At the end of diestrus, progesterone decreased significantly two days before a significant increase in LH. These rela- tionships raise the question whether cir- culating levels of progesterone have a negative influence on the circulating lev- els of LH. The association between increasing prostaglandin and decreasing progesterone was noted earlier (pg. 244). Estradiol & LH 15o (n=8) —L O O 150 LH concentration (ng/ ml) 01 O 100 50 (nu/5d) uonenueouoo Z3 -15 -10 -5 0 5 10 Number of days from LH peak FIGURE 7.12. Mean peripheral estradiol and LH concentrations. Adapted from ( 1249). 247 Progesterone & LH (n=7) . ‘ Progesterone ’ Concentration (ng/ ml) M O -12 -10 -8 Number of days from ovulation FIGURE 7.13. Mean peripheral progesterone and LH concentrations in pony mares. For each end point, a star indicates the mean with the first sig— nificant decrease or increase. Adapted from ( 5 75). Estradiol and FSH. The temporal rela- tionships between FSH and the ovarian steroids have not been adequately charac- terized. However, the period of FSH— decline before ovulation corresponds roughly with the rise in estradiol, sug— gesting estradiol may have a negative effect on release of FSH. Furthermore, FSH begins to increase near the end of estrus at approximately the time that estradiol decreases. Progesterone and FSH. Temporal asso- ciations suggest a positive feedback effect of progesterone on FSH. The temporal relationships between progesterone and FSH were studied by normalizing data according to first increase in progesterone above 1 ng/ml and first decrease below 1 ng/ml (1092). At the end of estrus, FSH increased before progesterone increased (Figure 7.14). At the end of diestrus, the two hormones began to decrease on the same day, but progesterone decreased more rapidly. Circulating levels of FSH, therefore, cannot be attributed solely to a positive feedback of progesterone since FSH increases at end of estrus before a detectable increase in progesterone. 248 Chapter 7 Progesterone & FSH .1 D on CD Concentration (ng/ml) J) -4-2024-4-2024 Days from P4 >1 ng / ml at end of estrus Days from P4 <1 ng / ml at end of diestrus FIGURE 7.14. Changes in concentrations of FSH when data were normalized according to the day of the first increase in progesterone above 1 ng/ml (left) and the first decrease in progesterone below 1 ng/ml (right). The vertical bars (lsd) indicate the magnitude of the least significant difference (P<0.05) for each hormone Within each period. Adapted from (1092). Inhibin and FSH. In a study in horse mares (185), mean immunoreactive-inhib- in (ir—inhibin) and FSH concentrations were inversely related (negative correla- tion of the means: r = —0.55; Figure 7.11). The significant decrease in mean concen- trations of ir-inhibin on Days 0 to 1 was associated with an increase in FSH between Days 0 and 5. Mean FSH concen- trations then remained high, whereas mean ir-inhibin concentrations remained low. A significant mean increase in ir— inhibin on Days 7 to 12 was associated with an FSH decrease on Days 11 to 14. However, as shown in the figure, the mean profiles of both hormones were poor representations of the respective values in individuals. The number and time of occurrences of pulses, as determined from daily samples, were highly variable but demonstrated adequate consistency for significant mean profiles (Figure 7.11). The results were consistent with the hypothesis that inhibin reduces the con- centrations of FSH. However, the rela- tionship between the two hormones was not tight in individual mares. Additional study is needed, especially involving more frequent sampling and monitoring of fol- licular dynamics. 7.2B. Experiments on Altering Circulating Levels of LH Progesterone and estradiol treatments. Progesterone treatment decreased and estradiol treatment increased the circu- lating concentrations of LH in ovariec- tomized pony mares during the summer (Figure 7.15; 555). The effects were main- tained until treatments were discontin— ued. Estradiol plus progesterone had a greater retarding effect than did proges- terone alone. However, in ovarian-intact mares, exogenous estradiol in the pres— Effect of steroids on LH (n=3/group) 7 5 \ 2 E2 5 3 U) 5 S: .9 1 E E Control 0 5 / s *H' 0 , ._ P ' ‘N / 4 o J, E 3 :F.“.-.‘.‘.“.’.’ ~.-.-. . O... P a_ P4 + E2 ’ 1 '03 .. .. ‘ I—— Treatment ——1 13 5 7 9111315171921 Number of days from start of treatment FIGURE 7.15. Effect of daily treatment with estra- diol (E2) or progesterone (P4) on concentration of LH in long-term ovariectomized mares in the sum— mer. The estradiol treatment (upper panel) was done in a separate experiment. Adapted from (555). Endocrinology of the Ovulatory Season 249 ence of endogenous progesterone did not depress the LH levels below those associ- ated with endogenous progesterone alone (1479). The authors commented that basal levels of endogenous estradiol may have caused maximum LH suppression in syn- ergism with the endogenous proges- terone; therefore, further depression did not occur. Treatment of ovariectomized mares during the winter, a time when LH concentrations are very low, did not result in further depression by proges- terone (555); estradiol caused an increase in LH but not as effectively as it did dur- ing estrus or in ovariectomized mares during the summer. The suppressing effect of progesterone and the positive effect of estradiol on circulating levels of LH in ovariectomized mares has been confirmed (1598). It also was shown that estradiol not only increased the secretion of LH but also increased pituitary LH storage and extent of response to GnRH. In ovarian-intact horse mares, daily treatment with estradiol caused elevated LH levels after the corpus luteum regressed (263). In a subsequent study (495), estradiol alone did not consistently alter LH levels, but toward the end of treatment (Days 25 to 28) an increase was observed. Exogenous progesterone or progesterone plus estradiol reduced LH concentrations for the duration of treat- ment (28 days; 495). The combination of the two steroids was more effective than progesterone alone, a result that also was obtained in ovariectomized mares. In other investigations, progesterone sup- pressed LH concentrations when proges— terone was given after ovariectomy (1071, 1053, 1598). However, administration of progesterone, sufficient to raise blood lev- els to 3 to 4 ng/ml, did not depress LH in long-term ovariectomized mares (1071). The authors concluded that the amount of progesterone was insufficient. A single injection of estradiol in ovariectomized mares resulted in an immediate decrease in LH levels, but this effect was transient and was followed by an increase (1095). In other species, estradiol stimulates LH (1375), and an initial inhibitory effect pre- cedes the stimulatory effect (cited in 1095). The studies cited here support the hypothesis that estradiol has a positive and progesterone a negative feedback effect on circulating LH concentrations in mares. The positive effect of estradiol also was demonstrated in a recent study of cultured pituitary cells (137); estradiol caused a dramatic increase in the LH response of the cells to GnRH. Results of this in vitro study also suggested that conjugated estrogens may play a role in LH regulation. Androgen treatments. Administering androgens (dihydrotestosterone) to anestrus or ovariectomized mares did not affect circulating levels of LH (1596, 561). Initial treatment of ovariectomized mares with progesterone did not decrease the LH levels; subsequent treatment of the progesterone-primed mares with dihy- drotestosterone did result in decreased LH levels (1071). Furthermore, plasma LH tended to be reduced (P>0.08) by testos- terone propionate, and this effect was not altered by immunization against estro- gens (556). It appears that the effect of testosterone propionate on circulation levels of LH is a direct androgenic effect. In contrast, the effect on FSH involves, in part, conversion of the androgens to estrogens (pg. 251). Passive immunization against androgens did not alter length of estrus but did result in a more prominent LH surge (1610). It appears that andro- gens play a negative role on the LH surge which may be secondary to a primary pos- itive function involving pituitary produc- tion of FSH. The effects of androgens on FSH are described in the next section. Role of isoforms. Studies on LH micro- heterogeneity indicated that estradiol not only enhances circulating levels but increases the biologic potency of LH dur— ing the periovulatory period (pg. 44; 21). Estradiol and GnRH apparently interact to produce the desired biologic potency by altering prevailing LH isoforms. 250 Chapter 7 7.20. Experiments on Altering Circulating Levels of FSH Progesterone and estradiol treatments. The nature of ovarian regulation of circu- lating FSH concentrations is more com- plex than for LH and is a greater experi- mental challenge. Temporal associations raise the possibility that progesterone has a positive effect and estrogens have a negative effect. In some studies, FSH con- centrations were not altered significantly by administration of estradiol or proges- terone to ovariectomized mares during the anovulatory or ovulatory seasons (552) or by various steroid treatments in ovarian-intact horse mares (495, 263, 1092). When suppressive effects occurred, they seemed to be related to the development of large follicles. However, more recent studies have demonstrated that exogenous estradiol can cause a depression in FSH circulating concentrations. Estradiol injections sup— pressed the elevated FSH that occurred in control mares on Day 5 (263). In anoth— er study (1479), exogenous estradiol given during diestrus caused an apparent sup- pression of FSH concentrations until close to the time of luteolysis. Furthermore, in ovariectomized mares treatment with estradiol, as well as androgens, decreased the secretion of FSH (1598). A single injec- tion of estradiol to ovariectomized mares resulted in an immediate (within 3 to 8 hours) decrease in FSH followed by an increase (1095). These short-term changes would not have been detected by the daily sampling procedures used in earlier experiments. A negative effect of estradiol benzoate on circulating levels of FSH in mares has been reported (560, 1785). Acute suppression of FSH secretion by estradiol has been shown in other species (160, 1375). In contrast to earlier studies, treatment of ovariectomized mares with proges- terone increased the daily FSH secretion and increased the FSH response to exoge— nous GnRH (1071). The recent study involving ovariectomized mares in June found that exogenous progesterone depressed the circulating levels of FSH (1598). In an earlier study, however, pro- gesterone treatment of ovarian-intact anestrous mares had no such effect (1596). A treatment regimen with a synthetic progestin (altrenogest) plus estradiol resulted in reduced FSH levels (1205). In summary, although early studies on the effects of progesterone and estradiol on FSH produced negative or equivocal results, several recent studies have shown a suppressive effect of estradiol and a positive effect of progesterone. Role of androgens. In a series of cre- ative studies by Thompson and associ- ates, it was initially noted that testos- terone propionate treatment during late estrus and early diestrus resulted in decreased secretion of FSH (cited in 1606). Challenge of androgen-primed mares with GnRI-I, however, resulted in FSH release. A subsequent study (1606) showed that testosterone given during estrus did not affect the length of estrus or diestrus or the level of FSH during estrus; however, a postovulatory surge in FSH occurred. In mares treated with the progestin altrenogest, testosterone propi- onate reduced the FSH circulating levels by 50%; this was followed by a rebound (1600). These results led to the hypothesis that androgens cause FSH to accumulate in the pituitary during estrus and that the accumulated FSH is then released during diestrus. Androgen treatments suppressed the circulating FSH levels by causing FSH to build up in the pituitary; a challenge with GnRH then caused a release of the FSH (Figure 7.16). Many subsequent studies by this group have confirmed that testosterone propi- onate increases the FSH response to GnRH (1324, 1609, 560, 1785, 556). However, it was not known whether the effect of andro— gens was due to testosterone or to one of its metabolites; estrogen is a metabolite of androgens through the process of aromati- zation (pg. 62). Therefore, testosterone propi- onate was tested in mares that were first Endocrinology of the Ovulatory Season 251 Effect of androgens and GnRH on FSH .5 O O N U! 01 O No androgen treatment FSH concentration (ng/ml) GnRH GnRH GnRH i l i 0 4 8 12 Time (hours) FIGURE 7.16. Concentrations of FSH in plasma of control and androgen (testosterone propionate) treated mares administered GnRH at 0, 4, and 8 hours one day following the last androgen treat- ment. Adapted from Garza et al. (560). actively immunized against estrogen (556). The estrogen immunization reduced the response of FSH to the androgen by 52%. The authors concluded that the effects of testosterone propionate involved both androgenic and estrogenic (through metabolism) activities. It was noted that testosterone can be metabolized to estro- gens by many tissues, including the hypothalamus and pituitary (cited in 556). In the most recent study (1598), treatment with various steroids increased FSH stor- age in the pituitary, but only the andro- gens increased the FSH response to GnRH. These and other studies by this group of investigators provide strong indi- cations for a positive role of androgens on the diestrus surge of FSH. Androgens are produced by the follicles during estrus and apparently are discharged into the circulation since circulating concentra- tions of androstenedione, and perhaps testosterone, increase during estrus (pg. 243). On a comparative basis, andro— gens also have been implicated in regula- tion of FSH in ewes and female rats (cited in 1606). A recent report (1783) indi- cated the existence of a direct positive feedback effect of androgens on FSH at the level of the pituitary in rats. Effects of a proteinaceous fraction of follicular fluid. Studies on the effects of whole follicular fluid and its proteina— ceous and steroidal fractions on FSH con- centrations have been made in ovari- ectomized mares. Injections of whole follicular fluid had a pronounced FSH— depressing effect. Depressed FSH con- centrations also occurred in ovariec- tomized (1094) and ovarian-intact (181) mares treated with follicular fluid from which the steroids were removed by char- coal extraction. In a subsequent study (1095), it was found that estradiol adminis- tration enhanced the FSH-suppressing effect of charcoal-extracted follicular fluid. Similarly, whole follicular fluid (containing estradiol) was more effective in suppressing FSH than a proteinaceous fraction of the follicular fluid (1094). Treatment of ovariectomized mares with a single injection of a proteinaceous frac- tion of follicular fluid resulted in a disso- ciated response between FSH and LH that lasted for 48 hours. The FSH concen- trations initially decreased without alter- ing LH; however, 24 hours after the injec- tion, concentration of LH increased while FSH remained decreased. Follicular fluid treatment during late diestrus depressed circulating FSH concentrations and follic- ular development (pg. 260). These results indicate the presence of a proteinaceous FSH-inhibiting factor in follicular fluid that could account for the close associa- tion between pulses of the two gonad- otropins during diestrus and the promi- nent dissociation during estrus (pg. 235). On a comparative basis, at least one of the proteinaceous substances in follicular fluid that depress circulating FSH levels is inhibin (pg. 245). There are also indirect indications that inhibin-like substances play a profound role in the control of fol- licular waves and in the preovulatory decrease in circulating levels of FSH; 252 Chapter 7 a recent study has demonstrated a recip- rocal relationship in the circulating con- centrations of FSH and immunoreactive inhibin in mares (pg. 248). In nonequine species, many newly dis- covered proteinaceous substances, other than inhibin, have been isolated from fol- licular fluid (review: 1626). The roles, if any, of these proteinaceous substances in follicular development and ovulation ver- sus atresia have not been elucidated. It does not seem productive to review cur- rent knowledge and speculation on these substances in this text, especially since mares have not yet become a serious part of this emerging research area. 7.2D. Role of GnRH The feedback effect of ovarian hor— mones on the pituitary hypothalamic area is complex and the details are beyond the scope of this text. Effects can be direct on the hypothalamus and higher centers, which in turn result in altered GnRH pro- duction, release, or pulsatility. The GnRH is the final link in the cascade of events leading to an effect on the pituitary. At other times, the ovarian products appar- ently can act directly on the pituitary to influence pituitary production of a gonadotropin or to alter the response to GnRH. Administration of a certain ovari— an hormone can reduce the levels of a cir- culating gonadotropin—but it may do so by increasing the storage of the gonadotropin. Therefore, a later challenge with GnRH can result in a surge of the gonadotropin in the circulation. Thus, negative response to the ovarian hormone in regard to circulating levels of the gonadotropin may be negative only in terms of release—it may be positive in terms of synthesis and in its overall role. This phenomenon has been well illus— trated by the androgen-FSH studies described above. Pulsatility of GnRH and gonadotropins in pituitary effluent. The circulating lev— els of GnRH are not detectable, but the technique of cannulation of pituitary venous blood has allowed study of GnRH output (23). During the periovulatory period, secretion of GnRH, LH, and FSH occurred continuously with synchronous pulses superimposed on the tonic back- ground. About 90% of the GnRH pulses were associated with a pulse of the gonadotropins. The pulse frequency, as indicated above, was about one pulse per two hours early in the LH surge and one pulse per half hour at the time of ovula- tion. The interval between pulses was 33 minutes on the day of ovulation in two mares. It appears that synchrony of pulses among the hormones occurs throughout the estrous cycle as demon- strated by the high levels in pituitary effluent. However, when the hormones are diluted in the general circulation, GnRH is not detectable, and the syn- chrony of prominent FSH and LH pulses (as detected by daily sampling) is most obvious during the first half of diestrus (pg. 235). In cerebrospinal fluid. Both estradiol and progesterone increase in the cere- brospinal fluid in accordance with increases in plasma (421). The potential importance of this finding can be appreci- ated by studying Figures 1.32 and 2.3, showing the close relationship of the third ventricle (containing cerebrospinal fluid) and the median eminence (proximal end of hypothalamic-pituitary portal system). Estradiol concentrations were 80% of the plasma concentrations, whereas progesterone concentrations were only 10%. It is possible, therefore, that the cerebrospinal fluid plays a role in mares in the feedback effect of ovarian steroids, especially estrogens, on the cen- tral nervous system. The mechanisms involved in the release of GnRH involve Endocrinology of the Ovulatory Season 253 complex interactions between neurotrans- mitters and neuroendocrine tissue (907) and are beyond the scope of this text. It also should be noted that since cere- brospinal fluid bathes the hypothalamic- median eminence area, the fluid might be involved in an ultrashort regulatory path- way between pituitary and hypothala- mus. In this regard, LH levels in the cere- brospinal fluid of mares are elevated on the day of ovulation, similar to what occurs in the blood (26). Studies on the nature of the cells lining the third ventri— cle over the median eminence are noted elsewhere (pg. 121). Estradiol and GnRH. Results of studies involving administration of GnRH and estradiol during early estrus illustrate the interplay between an ovarian hor- mone and GnRH. A single injection of GnRH during early estrus caused LH release, and this effect was enhanced by prior treatment with estradiol (553). The GnRH also was infused continuously for 24 hours beginning on Day 2 of estrus. Plasma LH concentration increased gradually, reaching high val- ues at six hours. The high values were maintained throughout infusion. This result contrasted with an approximately two-hour response to a single injection of a comparable dose. These findings indicate that the prolonged LH rise dur- ing estrus results from continued GnRH release from the hypothalamus. Furthermore, LH release is apparently facilitated by estradiol which also con— tinues to rise at this time. The enhanc- ing ability of estrogens on the effect of GnRH on LH also may involve an altering of LH isoforms. The ratio of bioactive to immunoactive forms increased when estradiol and GnRH were given together but not when either was given alone (21). The positive effect of estradiol on LH response to GnRH has been confirmed (1598). Immunization against GnRH. Recently the effects of active immunization against GnRH on gonadotropin secretion were studied in ovariectomized mares (559). The immunization series began in November, and the mares were ovariec- tomized in June. On the day of ovariec— tomy, concentrations of LH and FSH were lower in the immunized mares than in controls. Furthermore, the concentra- tions did not increase after ovariectomy. Treatment of controls with testosterone propionate decreased LH and increased FSH response to GnRH challenge, as expected (pg. 250). These effects did not occur in immunized mares. This study directly indicated a role for GnRH in the high levels of LH and FSH in ovariec- tomized mares and further demonstrated the involvement of GnRH in the effects of androgens on gonadotropins. GnRH effect on LH and FSH. Previ- ously discussed indications of a role of GnRH in both LH and FSH secretion in the mare include the following: 1. Exogenous GnRH causes a rapid increase in LH and FSH and can induce ovulation and multiple ovulations; 2. Pulses of LH, FSH, and GnRH are synchronized, as indicated by assays of the hormones in pituitary effluent and push-pull samples from the median eminence; 3. Seasonal fluctuations in pituitary LH are correlated with concentration of GnRH in the hypothalamus (690); and 4. GnRH immunization resulted in 95% to 99% reduction in the LH response to inducers of gonadotropin secretion with- out a reduction in FSH response (557). Other aspects of the role and the mode of action of GnRH are given else- where (portal system and chemical aspects of GnRH, pg. 53; role in reproduc- tive seasonality, pg. 120; and exogenous GnRH for terminating the ovulatory season, pg. 166). 254 Chapter 7 7.2E. Interactions of Ovaries and Season The control of gonadotropins is com- plex in mares. The intrinsic regulatory factors must interact with the mecha- nisms modulating reproductive seasonali— ty, not only during the anovulatory sea— son, but throughout the year. The interaction of seasonal pressures with the ovaries in the regulation of LH and FSH has been reviewed (576), and the subject is updated in this section and elsewhere (pg. 121). Luteinizing hormone. In a series of experiments in ponies (540, 539), profiles for LH were normalized in the spring according to the day that LH concentra- tions rose above 2 ng/ml (Figure 7.17). LH (n=4 /group) _L N _L D co 0') .b Concentration (ng/ml) -24 -16 -8 0 8 16 24 32 Day 0 is the day concentrations first rose above 2 pg/ml FIGURE 7.17. Concentrations of LH in ovariec— tomized and intact mares at onset of ovulatory sea- son. Data were normalized according to the day (day 0) when LH first rose above 2 ng/ml; mean date on which this occurred is indicated for each group (not significantly different between groups). Concentration of LH for intact mares for the sample closest to the day of ovulation is significantly differ- ent between the two LH surges. Adapted from (539). The day on which this occurred was not different between intact and ovariec- tomized mares, suggesting that the initial rise in LH results from environmental influence. Second, LH concentrations rose more rapidly in the ovarian-intact group, indicating a subsequent positive ovarian influence presumably due to estradiol (pg. 148). Third, during the first month of the ovulatory season, LH concentrations increased progressively in the ovariec- tomized mares, and the maximum LH concentrations in successive ovulatory surges in intact mares also increased pro— gressively. These results demonstrated a continuing influence of both ovarian and environmental factors as the ovulatory season progressed. A study in horse mares examined the LH concentrations resulting from ovariec- tomy at various stages of the estrous cycle (499). Ovariectomies were done on Day 14, first day of estrus, and fourth day of estrus. The results confirmed that post- ovariectomy increases in LH did not reach the levels found in intact mares during the periovulatory interval (Figure 7.18). The rise in the Day 14 group was similar to that seen at the time of luteoly- sis. This indicated that the initial LH rise was related to release from progesterone inhibition and that continuation of the periovulatory LH increase was due to a positive ovarian influence (presumably estradiol). Follicle stimulating hormone. A com- parison of circulating FSH concentrations between ovariectomized and intact mares during the middle of the ovulatory season is shown in Figure 7.19. For all days, means for ovariectomized mares were higher than the means for intact mares. Results indicated that at all stages of the estrous cycle ovarian factors depressed the FSH concentrations from what would have been obtained if the ovaries were not present. The study involving ovariec- Endocrinology of the Ovulatory Season 255 Effect of ovariectomy at Day 14 on gonadotropins 16 FSH N++i++i 12 ,ixl_.+’i Ovariectomized (n=4) Ovarian intact (n=1 1) D _A 0‘) Concentration (ng/ml) 12 0 2 4 6 8 10 12 14 Number of days after ovariectomy FIGURE 7.18. Gonadotropin response to ovariecto- my on Day 14 in horse mares. The post-ovariectomy FSH levels rose immediately and remained above any of the mean levels during the estrous cycle. The LH levels did not rise as high as during the periovu- latory LH surge in intact mares. Data courtesy of J. E. Fay and R. H. Douglas {499). tomy during the estrous cycle (499) con- firmed that circulatory levels of FSH in ovarian-intact mares do not at anytime approach the high levels observed in ovariectomized mares. Following ovariec- tomy, FSH levels increased above those of ovarian-intact mares regardless of the FSH (n=5/group) 3o ’ ‘ A[Ovariectomized 25 i i l "i """ i it E \m 20 ; 5 C .915 ‘5 §1o C 8 5 Ovarian intact -16 -12 -8 -4 o 4 8 Day 0 is day of ovulation in ovarian-intact mares FIGURE 7.19. Concentrations of FSH in ovariec- tomized and intact pony mares during the middle of ovulatory season. Data for intact mares were nor- malized according to day of ovulation. Concentra- tions of FSH are significantly greater for all days in the ovariectomized group than in the intact group. Adapted from (539). day of ovariectomy (Figure 7.18). The maximum mean concentrations during the estrous cycle occurred on Days 2 to 14 (approximate means: 7 to 9 ng/ml); the mean levels were much higher after ovariectomy. The interactions of photoperiod with ovarian influences are summarized in Figure 7.20. 256 Chapter 7 SUMNIARY: Gonadotropin Control by Ovarian-Seasonal Interactions Anov. season Ovulatory season Anov. season Ovariectomized mares (Influence of photoperiod only) Ovarian-intact mares (Influence of ovaries and photoperiod ) 2v Direction of ovarian influence FIGURE 7.20. An overall concept of the manner in which length of photoperiod and ovarian hormones interact to regu- late circulating conCentrations .of FSH and LH. Conceptually, the level of circu— lating gonadotropins is held in check by melatonin during the anovulatory season and is further modified by prevailing ovarian products during the ovulatory season. Seasonal effects. In ovariectomized mares (free of ovarian influences), the circulating levels of FSH and LH follow a seasonal profile under the influence of length of photoperiod (broken lines). During the anovulatory season in intact mares and the corresponding season in ovariectomized mares, the levels of gonadotropins are suppressed by a sys- tem involving increased nightlength and the resulting production of melatonin and suppression of GnRH. During the months corresponding to the ovulatory season in intact mares, melatonin levels are reduced, and in ovariectomized mares the GnRH—gonadotropin system is at maximum potential productivity. OV=Ovulation Follicle stimulating hormone. Inovari— an—intact mares, ovarian factors continu- ally suppress seasonal influence on the FSH levels as shown; however, the nega- tive impact of ovarian products is greater during estrus than during diestrus. Two . ovarian products that have been shown to have a negative effect on FSH levels are estrogen and a proteinaceous product. Luteinizing hormones. The mean LH levels also are continually high in ovariectomized mares during the months corresponding to the ovulatory season. However, unlike FSH levels, the LH lev— els are intermediate between the low lev— els of diestrus in ovarian-intact mares and the high levels of estrus. That is, the \ influence of the ovaries on seasonally controlled LH levels is negative during diestrus (due to progesterone) and posi- tive during estrus (due to estrOgen). See the summary on page 156 for dis- cussion of the regulation of gonadotropin control during the anovulatory season, including the transitional periods between seasons. Endocrinology of the Ovulatory Season 7.3. Regulation of the Follicles and Ovulation 7.3A. Preantral and Intrafollicular Controls Preantral. The early formation of antral follicles from the reserve of primor- dial follicles has been discussed (pg. 176). Comparative studies of the hormonal con- trol of the development and atresia of fol- licles have been reviewed (645). The earli- er concept was that prior to the formation of follicular fluid or follicular steroids, follicles develop without the aid of gonadotropic hormones. It appears that more recent experiments in some species have not entirely supported this concept. These reviewers concluded that early fol- licular development (before the antral stage) is influenced by gonadotropins, but the matter has been beclouded by subjec- tive and anecdotal evaluations and by confusing interpretations and presenta- tions of quantitative data. Clearly, much more comparative and critical research, as well as specific research in fillies and mares, will be needed before the important question of preantral control is resolved. At present, only limited study even remotely exam- ined this question in mares (pg. 176). Particularly lacking, except for a limited slaughterhouse study (1759), is informa- tion on early follicular development in the newborn and prepubertal filly (pg. 490). Studies of preantral and antral follicular development in fillies with and without inhibition of gonadotropins (e.g., gonado- tropin antisera) are needed. The mare may offer a good model for studies on the roles of FSH versus LH since the mean concentrations of the two hormones tend to be reciprocally related (dissociated) during the estrous cycle. Intrafollicular. Research on the roles of estrogen, progesterone, and androgens within the follicles in the nonequine species has been the subject of recent reviews on intragonadal regulation of 257 follicular maturation and ovulation (1626). The principal thrust of current writings seems to be that intragonadal factors (steroids and nonsteroids) affect the development and atresia of follicles by inhibiting, enhancing, or altering the response of the follicles to gonadotropins and not by solely changing the circulating levels of the gonadotropins. In addition to steroids, substances that have been iso- lated from follicular fluid and are now being studied include inhibin, activin, fol- listatin, glycosaminoglycans, oxytocin, GnRH-like protein, renin-angiotensin, substance P, luteinizing inhibitor, gonad- otropin binding inhibitors, growth factors (insulin, epidermal growth factor, and TGFB), plasminogen activator, and oocyte-controlling substances (e.g., oocyte maturation inhibitor). These substances and their possible roles will not be dis- cussed here, but the reader should be aware that the regulation of follicles, especially the intragonadal aspects, is very complex, is poorly understood, and goes far beyond the roles of the conven- tional follicular steroids. Results of a recent study (744) involving follicular fluid aspiration in mares suggested that follic- ular fluid of immature preovulatory folli- cles also may contain a luteinization inhibitor; the aspirated follicles luteinized as indicated by an increase in peripheral progesterone. To summarize a recent nonequine review (1626), FSH binds to granulosa cells, including those of preantral folli- cles and stimulates the production of estradiol. The estradiol, in turn, stimu- lates the production of more granulosa cells and increases the sensitivity to the gonadotropins. Thus FSH, through estrogen, is a potent follicular stimu- lant. Estradiol also has a positive feed— back effect to ensure the necessary gonadotropin surge for growth of the preovulatory follicle. That is, estradiol has two positive feedback roles—one at the pituitary and one within the ovary. Progesterone and the androgens also have 258 Chapter 7 intrafollicular and pituitary feedback roles. As the follicle matures, the intrafol- licular proteins (e.g., inhibin) begin to come into play. Thus, as a generality, gonadotropins control the follicular microenvironment by regulating steroids, which in turn modulate the production of intracellular proteins as well as the sensi- tivity to the gonadotropins and circulat- ing levels of the gonadotropins. All of this seems extremely complex, partly because it is and partly because it is poorly under- stood. Ovulation. Ovulation is clearly depen- dent on LH in mares as in other species. However, little else is known about the process at the level of the follicular wall in this species. Current concepts on the endocrine and biochemical processes involved in the mechanism of ovulation in the nonequine species have been reviewed (965). The preovulatory changes within the follicle wall involve a complex series of biochemical reactions in which LH, cyclic AMP, prostaglandins, steroids, and proteolytic enzymes are among the implicated substances. Of current interest to equine biologists is the involvement of PGan in the pro- cess since treatment by analogues of PGFZa are being advocated as a method for ovulation induction during estrus (pg. 282). Indications for a role for PGFZa in the ovulatory process in nonequine species include the following (review: 1397): 1) PGFZOL increased in the ovula- tory follicle as ovulation approached; 2) Indomethasone (inhibits an enzyme in the synthesis of prostaglandin) and an antiserum against PGFZoc blocked ovula- tion when injected into the follicle; and 3) PGFZoc overcame the ovulation- inhibiting effect of indomethasone. In mares, it is known that indomethasone will interfere with ovulation (pg. 244). It has been reported that daily injections of PGFZa in mares interfere with ovulation rather than hasten it (434). Injections began on Day 13 and were given for 10 days. The interference did not seem related to depression of LH or depression of follicular growth. This study needs confirmation in view of the emerging use of PGan analogues for practical induc- tion of ovulation during estrus. 7.3B. Experiments on Follicle Suppression Gonadotropin antisera. Several studies have been done on the role of gonado— tropins in follicular dynamics in mares treated with an antiserum against an equine pituitary fraction; the antiserum presumably included antibodies against all pituitary proteinaceous hormones in addition to the gonadotropins (1268, 1270, 1269). Antiserum was given on days 2 to 6 of estrus and ovaries were excised the next day. Mean length of estrus was reduced in antiserum—treated mares (3.8 days), whereas control mares were still in estrus on the day of ovarian removal at 7 days. The largest follicle was affected by the antiserum as indi— cated by reduced volume, weight, and diameter. General structural features of the follicular wall of the largest follicle were poorly defined, and the follicle was undergoing degenerative changes in all antiserum—treated mares. Results indi— cated that the development of the largest follicle during estrus is dependent on gonadotropins. Furthermore, a reduction in gonadotropins results in degenerative changes (atresia) in the preovulatory fol- licle. This point is noteworthy because it indicates that the mysterious phe- nomenon of atresia can occur when the gonadotropin stimulus for the large pre- ovulatory follicle is blocked. Numbers of subordinate follicles in various classifi- cations (>20 mm, 10 to 20 mm, and >2 to 10 mm) during estrus were not sig- nificantly affected by antiserum treat- ment. That is, the mechanisms for atre- Endocrinology of the Ovulatory Season sia apparently were triggered prior to the second day of estrus (onset of antibody treatment). This finding is compatible with more recent findings that the large follicles (subordinate follicles) that accom- pany the ovulatory follicle (dominant fol- licle) up to approximately the beginning of estrus, begin to undergo atresia 6 or 7 days before ovulation (pg. 182). It appears from studies in other species that a combination of FSH, LH, and estrogens is involved in atresia. When LH reaches a certain level, the fol- licles that are not sufficiently influenced by FSH and estrogens undergo atresia. When antiserum was given during early diestrus (days 1 to 4 of diestrus) and late diestrus (days 7 to 10), but not during mid-diestrus (days 4 to 7), the number of follicles larger than 10 mm and the diameter of largest follicle were reduced (Figure 7.21). These results are compati- ble with gonadotropic stimulation during early and late diestrus at times that cor- respond with the emergence of the two follicular waves: 1) the wave during early diestrus that sometimes gives ori- gin to a diestrous ovulation, and 2) the wave that gives origin to the primary ovulatory follicle (pg. 178). Similarly, the lack of response on days 4 to 7 of diestrus is compatible with the concept that, on the average, follicular waves do not emerge at this time. On the basis of circulating levels of LH and FSH (pg. 233 and pg. 235), the suppressed gonadotropin was primarily FSH during early and late diestrus and LH during estrus. Unfortunately, these projects preceded the availability of technology for assay- ing the gonadotropins. Ovarian steroids. Gonadotropins also have been suppressed by administra- tion of estradiol and progesterone. The more recent approaches were accompa- nied by measurement of circulating gonadotropins. Daily progesterone treatment appeared to inhibit the daily 259 Effect of pituitar antiserum on largest ollicle (n=4/group) 25 Control N 0 Diameter (mm) a 10 Days of diestrus FIGURE 7.21. Follicular effect of an antiserum against an equine pituitary fraction. Mares were treated for four days prior to necropsy on the indi— cated day of diestrus. Means which are significantly different between groups on a given day are indicat- ed by a star and significant differences within a group are indicated by different superscripts. Adapted from (1268). increase in LH during estrus, and the preovulatory-size follicles failed to ovu- late (495). Treatment with a combination of progesterone and estradiol had an even greater suppressing effect on LH and suppressed the development of large follicles. The steroid treatments did not appear to affect FSH concentra- tions directly. Although the aspects of the experiment that involved FSH were not readily interpretable, a relationship between increasing LH and the growth of an ovulatory-sized follicle was shown. In a more recent experiment (1265), the largest follicle averaged only 12 mm after a regimen of daily progesterone and estradiol, but the gonadotropin lev- els were not determined. Mares treated daily with estradiol, beginning on Day 1 after ovulation, had suppressed follicu- lar growth during the time of the next 260 Chapter 7 estrus and failed to ovulate (1808, 263). Levels of FSH were suppressed only dur- ing early diestrus but apparently not during the time when follicles did not develop. The depressed follicular growth, therefore, was not attributable to decreased FSH levels. The LH levels during the time of follicular suppression were comparable to estrus levels. Follicular fluid. A course of charcoal- extracted follicular fluid (proteinaceous fraction) was given, beginning on Day 10 for 5 days, and an injection of PGFZoc was given on Day 10 to regress the corpus luteum (181, 180). The levels of FSH were immediately suppressed, and the length of the interovulatory interval was pro- longed (Figure 7.22). The follicular growth that normally begins at mid- diestrus was suppressed, as indicated by diameter of largest follicle. The follicle was significantly larger in the controls by Day 14 and continued to grow over the next four days. In treated mares the largest follicle remained at the mid- diestrus diameter. This experiment demonstrated the role of FSH in the for— mation of the follicular wave that pro- duces the ovulatory follicle. 7.3C. Experiments on Follicle Stimulation Gonadotropin preparations of equine and nonequine origin can stimulate development of multiple follicles and induce ovulation of a preovulatory folli- cle in mares. These experiments have demonstrated the general relationships between the gonadotropins and follicu- lar development and ovulation. The availability of highly purified prepara- tions of FSH and LH should soon allow more detailed studies on the relative roles of each gonadotropin in follicular regulation. Eguine pituitary extracts. Pituitary extracts have been used to stimulate Effect of follicular fluid on FSH and follicles (n=3/group) Charcoal-extracted follicular fluid 00 O O }_|_ _A O C 01 O C) FSH (percent change) ('11 o 32 Largest follicle 28 24 20 16 Diameter (mm) ; r r ~ I r~~~l 12 PG F2oc l— Treatment —l 10 11 12 Number of days from ovulation 13 14 15 16 17 18 FIGURE 7.22. Percentage change in concentrations of FSH and diameter of largest follicle in mares treated with saline (control) or charcoal-extracted follicular fluid (proteinaceous fraction with minimal steroids). Concentrations of FSH are expressed as a percentage of the pretreatment concentration (con- centration on Day 10 = 0%). A star indicates a dif— ference (P<0.05) between groups for each day. Adapted from (181). follicular development and ovulation during the anovulatory season (pg. 165), to stimulate multiple ovulations during the ovulatory season (.933, 422, 1815, 1513, 1505), and to study the time of physiologic selection of the ovulatory follicle (Figure 7.23). Daily regimens of pituitary extract Endocrinology of the Ovulatory Season 261 that began during the last half of the estrous cycle were effective, although those that began late (e.g., Day 19; 1815) had a reduced response. All of these reg- imens likely involved the follicles of the follicular wave that begins at mid- diestrus (pg. 180). Studies apparently have not been done during the first half of diestrus when a major follicular wave (sometimes ovulatory and sometimes anovulatory) occurs during some estrous cycles. The effects of extract treatment on number of ovulations summed over several experiments are shown (Figure 7.24). An injection of hCG when the first follicle reached 35 mm resulted in synchronization of mul- tiple ovulations, and an injection after the first ovulation increased the number of ovulations. The calculations of Irvine (801) can be consulted for information on the amount of LH and FSH expected to be in the equine pituitary extracts used in these studies. Regimens of pituitary extracts can be used repeatedly. In a recent study (1205), extract treatments were started four days after withdrawal of a progestin synchronization regimen, and the treat- ment course was repeated at least six times per mare during a season. The overall mean number of ovulations was FIGURE 7.23. Ovaries of a mare treated with equine pitu— itary extract during the ovulato- ry season. Ovaries were sec— tioned midsagittally and opened like a book. There are two corpo- ra lutea (dark structures) on the left ovary and one on the right ovary. Adapted from (933). 1.8 from a mean of 3.3 preovulatory- sized follicles. Levels of both FSH and LH were much higher in treated mares than in control mares. It has been con- cluded that an interrupted extract regi— men resulted in more synchronized growth of groups of follicles; prolonged extract administration resulted in stim- ulation of ovulation but the follicular Effect of pituitary extract on ovulation rate 40 Number ovulations i per mare |:|Controls 47 1.1 i0.1 00 O -Treated 112 3.0 :02 Number of mares N O _L o 0 1 2 3 4 5 6 7 8 Number of ovulations FIGURE 7.24. Ovulation rate in mares treated with an equine pituitary extract. Adapted from (1815). 262 Chapter 7 growth did not occur in waves (756). A porcine—FSH preparation also stimulated follicular development in mares (801, 1505) but at the doses used, was less effective than equine pituitary extract (mean num- ber of ovulations per mare: 1.6 versus 2.2, respectively; 1505) Gonadotropin—releasing hormone. Ovulation also can be induced by injec— tions of GnRH during estrus. In an early study, the duration of estrus was reduced and day of ovulation tended to be hastened by an injection of 1 mg on day 2 of estrus (809). Daily injections of 2 mg were more effective and significant- ly reduced the interval to ovulation. In an abstract (1724), other workers conclud- ed that a single injection of 4.5 mg was ineffective for this purpose. More recent- ly, it was found that pulsatile delivery of GnRH (one 5-second 20 ug pulse/hour) beginning on Day 16 induced an earlier increase in circulating LH levels and reduced the number of days to ovulation by an average of three days (827). A recent abstract (1516) reported that nei— ther pulsatile or constant delivery of GnRH beginning on days 6 or 7 of estrus resulted in an increased rate of multiple ovulations. By this time, however, subor- dinate follicles would have been commit- ted to atresia. Other studies have shown that termination of the anovulatory sea- son with a GnRH protocol can result in multiple ovulations (pg. 166). The stimula- tory effect of GnRH on gonadotropin release during estrus (pg. 253) and the applied use of GnRH for the ovulatory season (pg. 281) and anovulatory season (pg. 166) are discussed elsewhere. Human chorionic gonadotropin. It has been known for 60 years that an injection of hCG during early estrus will shorten estrus and hasten ovula- tion. Ovulation usually occurs within 48 hours after treatment. Mean diame- ter of the preovulatory follicle 12 hours before ovulation was not different between hCG-treated and control mares (1576). In another study (1507), the diame- ter of the preovulatory follicle (Day —1) was not different between hCG-treated and control mares (40 and 42 mm), even though hCG shortened the interval to ovulation by an average of 1.3 days. Since hCG reduced the interval to ovula- tion, these results indirectly suggest that the preovulatory follicle grows more rapidly in hCG-treated mares. A systemic injection of hCG caused an immediate fourfold increase in proges— terone in the follicular fluid within 28 to 32 hours; hCG was given when the folli- cle surpassed 35 mm (1737). Intrafollicu- lar concentrations of testosterone and estradiol were not greater than in the controls. Concentrations of PGFZoc increased but not significantly. Much of the incentive for the work with hCG stems from its widespread use in breed- ing programs. This subject is presented elsewhere (pg. 279), including informa- tion on the interval from hCG treat- ment to ovulation and the formation of antibodies against hCG. Human menopausal gonadotropin can be used for induction of multiple ovulations in mares (871, 1594). 7.3D. Selection of Dominant Follicles As described in Chapter 6 (pg. 180), the primary ovulation originates from the dominant follicle of the primary follicu- lar wave. The wave emerges at mid-cycle and follicles grow in synchrony for a few days. A selection mechanism eventually dissociates the follicles into dominant and subordinate positions. Selection is manifest at late diestrus. A similar selection process occurs in some mares in early diestrus in association with a Endocrinology of the Ovulatory Season 263 secondary wave. The endocrinologic dynamics presaging selection have not been defined for any species. In mares, the occurrence of selection under differ- ent hormonal circumstances (early and late diestrus) could be utilized to unravel some of the mysteries surrounding this fundamental process. As noted above, the selection mechanism can be overrid- den by the administration of pituitary preparations that result in multiple ovu— lations—that is, failure of atresia of sub- ordinate follicles. These findings suggest that a decline in a gonadotropin, presum- ably FSH, plays a role in the selection process. The temporal relationships between the time of selection and circu- lating hormone levels are as follows: Estrogens. The concentrations of estro- gens apparently increase at approximate- ly the time of follicle selection for both the secondary wave (pg. 242) and primary wave (pg. 241). Progesterone. Progesterone levels are increasing during the approximate time of‘selection in the secondary wave and decreasing during the primary wave. However, for both waves progesterone is low during the time of selection. Q. There are lingering high levels of the ovulatory LH surge into early diestrus and occasional early-diestrus pulses of LH at the presumed time of selection during a secondary wave. During the primary wave, the ovulatory LH surge is increasing during the time of selection. On a temporal basis, there- fore, LH remains a candidate for a role in follicle selection. FS_H. Studies in cattle have indicated that a decline in FSH is an integral com- ponent of the selection mechanism(3). Decreasing FSH could be involved in selection during the primary wave, as indicated by temporal relationships and the ability of FSH to override the mecha- nism. It is not yet known whether a decline in FSH is temporally associated with selection during a secondary wave. The associations among FSH surges, emergence of major waves, and selection of a dominant follicle need a major re- search effort. 7.3E. Temporal Relationships Time associations between hormonal changes and structural changes are compatible with the concept that FSH is the gonadotropin associated with follic- ular growth except for final growth of the large preovulatory follicle in associ- ation with increasing LH levels. Growth of follicles at the beginning of diestrus, however, is in the presence of decreas- ing, but high, LH levels as well as increasing FSH levels. The associations of FSH levels and extent of follicular activity have been demonstrated during the inactive and resurging phases of the anovulatory season (pg. 150). However, studies characterizing the associations between FSH profiles and follicular activity within mares are lacking for the ovulatory season. The interrelationships among follicu- lar and gonadotropin profiles are sum- marized in Figure 7.25. 264 Chapter 7 SUMMARY: Regulation of Follicles during the Estrous Cycle 6 8 10 12 14 16 18 20 Number of days from ovulation FIGURE 7.25. Interrelationships among gonadotropin levels, folliculogenesis, and ovu- lation. The hormone profiles are a representation of means for many mares. Surges of gonadotropins, especially for FSH, occur Within individual mares. Since these surges occur on different days among mares, they are masked in the mean profiles. A. Poorly understood period during early diestrus when the FSH and follicular pro- files vary Widely among interovulatory intervals. B. FSH stimulation of emergence and initial development of the follicular wave that gives origin to the primary ovulation. C. Inhibition of circulating FSH levels by combined action of estradiol and a proteina- ceous inhibitor from the follicles. In addition, androgen from the follicles favors the build-up of pituitary stores of FSH, which are then available for GnRH—stimulated release of FSH when the inhibitors decline. D. Selection mechanism wherein one follicle becomes the ovulatory follicle and the other follicles of the wave begin to regress. Nature of the mechanisms is not known but probably involves the FSH decline. E. Positive effect of follicular estradiol on LH levels. The occurrence of luteolysis before this time has removed the LH inhibitory effects of progesterone. F. Final growth phase of the preovulatory follicle in association with increasing lev- els of LH. G. Induction of ovulation by the high levels of LH. Endocrinology of the Ovulatory Season 7.4. Regulation of the Corpus Luteum Many recent reviews on the mechanisms controlling luteal function in nonequine species are available (e.g., 115, 704). Unless otherwise stated, this section refers to research done specifically in mares. A com- parative basis will be used, however, to develop the concept that the uterine control of the corpus luteum in mares is through a systemic pathway rather than through the local pathway of other farm species. 7.4A. Role of Gonadotropins Ovulation signals the initiation of growth of the corpus luteum and produc- tion of progesterone. Mean FSH levels are increasing during this time, and mean LH remains quite high for several days after ovulation. Presence of gonado- tropins during this time, especially the protracted high mean concentrations of LH and sporadically occurring LH pulses (pg.>235), is likely important for early luteal development. The prominent LH surge in mares, with highest levels occur- ring after ovulation, may account for the early post-ovulatory rise in progesterone levels in this species. Experiments with antibodies to gonado— tropins. The hypothesis that pituitary hormones play a role in luteal develop— ment and maintenance can be tested by blocking the endogenous hormones with antibodies. This approach has been wide- ly used in many species. Antibodies to LH, for example, cause luteolysis during diestrus in cattle and sheep (679). The technique has been used to demonstrate the dependency of the equine corpus luteum on pituitary hormones (1268, 1270). Mean weight of corpus luteum was less for antiserum-treated mares than for control mares following all three treat- ment periods of diestrus (days 1 to 4, 4 to 7, and 7 to 10). In another project, corpora lutea of antiserum-treated mares were small, hard, and pale to intense yel- 265 low and their general macroscopic appearance was that of regressing corpo- ra lutea (1270). Corpora lutea of control mares were large, soft, and pink to orange-red, and their general macroscopic appearance was that of functional luteal tissue. These results indicate that endogenous pituitary factors, inhibited by the antiserum, are necessary for develop- ment and maintenance of the corpus luteum in mares. The experiment was not designed to identify the active pituitary hormone. Administration of pituitary hormones. Another approach involves administra- tion of pituitary hormones to determine whether they will extend luteal life or prevent the luteolytic effect of other treat- ments. In an initial trial, 2,000 units of hCG were given subcutaneously to six pony mares daily on Days 11 through 16 (575). Length of diestrus did not differ sig- nificantly from controls. In another exper- iment, hCG (1,500 units) or an equine pituitary extract was given daily to pony mares on Days 9 through 17. Significant reduction in progesterone values occurred by Day 13 in control mares but not until Day 17 in hCG-treated mares. Mean pro- gesterone did not decrease in the pitu- itary extract-treated mares, indicating possible maintenance of the corpus luteum. In other trials, the same dose of hCG and pituitary extract appeared inef- fective in overriding the luteolytic effect of intrauterine infusion of saline solution or injection of PGan. Results of the above described attempts to maintain the cor- pus luteum in mares with gonadotropic preparations have been encouraging but equivocal. A recent study indicated that injections of hCG on Days 3, 4, and 5 (100 iu/day) increased peripheral proges- terone concentrations on Days 7 to 14 in cycling mares; in pregnant mares, the ele- vation continued on Days 15 to 30 (863). Exogenous GnRH injections in diestrous mares can induce a short-term increase in serum LH values (833, 22). The luteotropic effects of LH also may be involved since 266 Chapter 7 administration of GnRH to diestrous mares caused increased progesterone production (833). More study is needed, especially with ultrasonic monitoring or recovery of ovaries, to be certain that the primary corpus luteum is maintained; secondary ovulations or luteinization of follicles can complicate studies of this type. Administration of hCG in early diestrus is especially suspect in terms of ovulating or luteinizing a follicle of a major follicular wave. In vitro studies have demonstrated that luteal cells increase the production of progesterone when exposed to either eLH or hCG (863). However, the increased production of progesterone by LH in a tissue culture system was not confirmed in other studies (1741, 333). Catechola— mines (epinephrine and norepinephrine) have an in vitro-stimulating effect on luteal cells in cattle but apparently not in horses (333). Receptors for LH have been described for equine luteal cells (1557, 1348, 1350). The number of LH receptors increased 21-fold between Day 1 and Day 14 and the recep- tor affinity increased fivefold. Receptor number and affinity were highly correlat- ed with concentrations of circulating and luteal progesterone. As the serum LH concentrations declined to basal levels, the number of luteal LH receptors increased. This may partly account for the luteotropic ability of low levels of LH. Several phenomena are reviewed in this text that indirectly suggest a positive association between LH levels and luteal progesterone productivity. These indirect indicators are as follows: 1. The amplitude of the ovulatory LH surge diminishes toward the end of the ovulatory season (1644), and at least one study indicates that progesterone levels also are less marked at this time (pg. 240); 2. Poor progesterone productivity of corpora lutea resulting from GnRH- induced ovulation during the winter was associated temporally with reduced post— ovulatory LH levels (pg. 168); 3. A secondary postovulatory estrogen peak may be a result of luteal stimulation by the prolonged postovulatory portion of the LH surge (pg. 242); and 4. The farm animal with the earliest postovulatory increase in circulating pro— gesterone (mares) is also the animal with considerable circulating LH during the time of luteal development. Conclusion on role of gonadotropins. Taken together, the above studies with pituitary antiserum, gonadotropins, GnRH, and in vitro culturing and the listed indirect indicators are strong rationale for the hypothesis that mainte— nance of the equine corpus luteum dur- ing diestrus requires a continuous sup- ply of circulating gonadotropins. On a comparative basis (1156), the most likely gonadotropin with luteotropic activity is LH. During mid—diestrus, LH concentra- tions are low, but low levels may be all that are required for luteal maintenance. In this regard, the occurrence of occa- sional LH pulses during diestrus may also be important. 7.4B. Role of the Uterus Convincing information has been avail— able for many years and many species, including mares, that demonstrates that the uterus is the pivotal organ in regres- sion of the corpus luteum in the absence of pregnancy. Removal of the uterus results in maintenance of the corpus luteum, whereas stimulation of the uterus by insertion of a foreign substance causes early luteolysis (reviews: 566, 572, 577, 574). Naturally occurring cases of uterine impairment also may cause luteal persistence in various species. For exam- ple, in cattle, congenital absence of a uterine horn or endometrial glands (568) or destruction of endometrium by pyome- tra (1333) may be accompanied by a per- sisting corpus luteum. Similarly, uterine infections may cause either early termi- nation or extension of luteal life in mares (pg. 522; 782). Endocrinology of the Ovulatory Season Hysterectomy studies. An initial exper- iment in mares tested the hypothesis that the uterus is necessary for regression of the corpus luteum (609). The experiment preceded availability of a technology for measuring circulating levels of equine progesterone or monitoring corpus luteum morphology (ultrasound). There- fore, the corpus luteum was marked with ink at the time of hysterectomy two days after the end of estrus. At 30 days, the marked corpus luteum was large and appeared to be functional in all hysterec— tomized mares. The corpus luteum was small and far regressed in all sham-oper- ated mares. In another study (1536), hysterectomy of Thoroughbred mares resulted in maintenance of the corpus luteum, as indicated by levels of circulat- ing progestins and results of ovarian palpation. These studies demonstrated an important role of the uterus in terminat- ing luteal life in the absence of pregnancy in the mare. Luteal life (pg. 443) and follic- ular development (pg. 448) in hysterec— tomized versus pregnant mares are dis- cussed on the indicated pages. Uterine irritation. An alternate method of studying uteroluteal relationships, as noted above, utilizes placement of foreign material into the uterus. When this is done early in an estrous cycle in cattle and sheep, it causes early luteolysis (review: 566). Historically, this technique was an important early research tool. It permitted study of uteroluteal effects by early activation of the mechanism, as opposed to removal (hysterectomy) or inhibition (pregnancy). Many years before the first studies on the luteolytic effect of uterine foreign bodies in any species, equine practitioners noted that estrus can be induced in mares by infusing isotonic saline into the uterus (reviews: 613, 1135). The phenomenon was known as early as 1935 (cited in 1680), although it was not realized for many years that the proce- dure exerted its effect by stimulating luteolysis. This early veterinary observa- tion should have directed attention to 267 uteroovarian relationships. It also should have provided impetus for utilization of the mare as a model for research on the effect of the uterus on the ovaries, but it did not. Infusion of 250 to 1,000 ml of isotonic saline causes early regression of the cor- pus luteum when done during mid- diestrus but not when done in early diestrus (Days 0 to 4). A recent study demonstrated that low pH of the infused saline was the main factor in stimulating PGan release, whereas increased tem- perature and osmolarity of the fluid had no effect on PGFZoc release (1239). Thus, phosphate—buffered saline may have neu- tral properties and may not cause short- ening of the cycle; this is a consideration in collection of intrauterine embryos (202). It appears that mares are similar to sheep and cattle in that any procedure which irritates the endometrium can lead to luteal regression. In this regard, endometrial biopsy and collection of spec- imens for bacterial culture, stimulated early luteolysis in mares (787, 883, 788, 789, 565, 135). This effect did not carry over into subsequent cycles (788). Furthermore, digital dilation of the cervix alone (788) or intracervical electrical stimulation (660) also resulted in an early return to estrus. Cervical dilation may have exerted its effect through secondary uterine irrita- tion or infection, however, rather than as a result of cervical stimulation per se. A more recent study found that cervical dilation, alone, did not alter luteal func- tion (1786). Furthermore, it has been con- cluded that procedures such as biopsy probably are not sufficient to cause luteol- ysis unless there is an already existent endometritis or the procedure introduces bacterial contaminants (1381). Introduc- tion of stallion semen into the diestrous uterus also has a luteolytic effect in mares (1805). It seems that most, if not all, uterine manipulative procedures (e.g., biopsy, culturing, fiberoptic examination) in mares may be followed by early luteal regression (1025), especially if bacterial 268 Chapter 7 contamination occurs. A peculiar effect of uterine biopsy procedure on sexual behavior and steroid concentrations has been noted (1158); signs of estrus and increased circulating progesterone lev- els were reported to occur following uterine biopsy even though the mares had been previously ovariectomized. 7.4C. Local versus Systemic Uteroluteal Pathways A series of studies has demonstrated that the regulatory functions exerted by the uterus on the corpus luteum involve, at least in part, a local uteroovarian pathway in all farm species except the mare. As described below, because sev- eral experimental approaches all failed to demonstrate a unilateral relationship in mares, it is likely that the uterine control in this species is exerted through a systemic, conventional, or whole—body pathway. A list of contrasts between mares and cattle that indicate that the uteroluteal effect is systemic in mares and local or unilateral in cattle is given in Table 7.1. The differences between mares and other species has been useful in studies of the uteroovarian pathway. Unilateral hysterectomy: In cattle (625) and sheep (567, 1115), when the uterine tissue on only one side is removed, regression of the corpus luteum occurs more readily when the retained uterine tissue is ipsilateral to the corpus luteum. Moreover, in unilat- erally hysterectomized cattle and sheep, the premature luteolysis that is induced by exogenous hormones occurs more readily when the retained uterine tissue and the corpus luteum are on the same side. This has been demonstrated by administration of oxytocin in cattle (625), progesterone in cattle (1825) and sheep (567), and estradiol in sheep (17). In contrast, a unilateral hysterectomy study in mares failed to find a local relationship between uterus and ovaries (609). The corpus luteum was maintained in 2 of 5 mares and in 3 of 5 mares in which the relationship between the retained uter- ine tissue and the corpus luteum was TABLE 7.1. Indications that the Pathway from Uterus to Ovaries for Uterine-induced Luteolysis is Local in Heifers and Systemic in Mares Item Heifers Mares 1. Partial hysterectomy removed 2. Intrauterine device 3. Route of administration of PGFZOL 4. Minimal dose of PGFZOL 5. Anatomy of uteroovarian vasculature 6. Unilateral relationship between embryo and corpus luteum 7. Relationship between conceptus and uterus during critical period Luteal maintenance when ipsilateral uterine horn is Stimulates luteal regression if ipsilateral to corpus luteum IU much more effective than IM in inducing luteolysis High (e.g., 25 mg) Ovarian artery and uteroovarian vein in close apposition Crucial ipsilateral relationship Conceptus expands into ipsilateral horn No relationship between side of removal and luteal maintenance Stimulates luteal regression regardless of side No differential effect whether given IU or IM Low (e.g., 5 mg) Ovarian artery and utero- ovarian vein not in apposition No ipsilateral relationship Conceptus travels throughout the uterus Endocrinology of the Ovulatory Season contralateral and ipsilateral, respective- ly. This study suggested the absence of a unilateral uteroovarian pathway and also suggested that presence of over half of the uterus was a critical amount for luteolysis. Unilateral insertion of an intrauterine device (IUD): Early regression of the corpus luteum occurs more readily when an IUD is inserted ipsilateral to the cor- pus luteum in cattle (624) and sheep (619). In mares, however, surgical fixation of an IUD into the horn ipsilateral or con- tralateral to the corpus luteum or intrauterine infusion of physiologic saline caused luteolysis as determined by progesterone concentrations (575). The rate of progesterone decline was not sig- nificantly different among the three groups. Route of administration and dose of PGFZoc: In sheep, the minimal effective luteolytic dose of PGan was less when given into the uterus than when given systemically (431). Comparisons of reports from several laboratories indicate that the minimal luteolytic dose of PGFZoc in cattle is approximately 20 times greater when given systemically than when infused into the uterus. In a critical study, the minimal systemic dose was 15 to 25 mg (cited in 577). However, intrauterine doses as small as 1 mg were effective, and administration into the ipsilateral horn was more effective than administration into the contralateral horn. In the early days (1960s) of re- search into luteolytic effects in cattle, supplies of PGan were scarce. There- fore, intrauterine administration was routinely used so that a much smaller dose would be required. In contrast to results in sheep and cattle, no significant difference between the intrauterine and intramuscular routes of PGan adminis- tration was found in mares (432). Anatomy of uteroovarian vascular sys- te_m. Morphologic studies were done to determine whether there were differences in the uteroovarian vascular anatomy 269 between species in which a local pathway' has been demonstrated and species in which specific study has failed to demon— strate a local pathway. Results of these studies have been reviewed (571, 572), including an extensive series of colored photographs for 13 species (574). To summarize, in the three farm species under consideration (cattle, sheep, and horses), the uterus and ovaries are drained by a common vein (uteroovarian vein), but there is a species difference in the location of the ovarian artery relative to the vein (Figure 7.26). In the species with a local pathway (sheep and cattle), the ovarian artery is tortuous and is in close apposition to the wall of the uteroovarian vein. In the species with a systemic pathway (horses), the ovarian artery does not have major contact with the uteroovarian vein; the artery contacts the uterine branch of the uteroovarian vein only in a limited area where it passes obliquely across the vein. The anatomical difference could account for the presence or absence of a local uteroovarian pathway for a given species. An extensive series of studies involving surgical anastomoses of uterine and ovari- an arteries and veins in nonpregnant and pregnant cattle and sheep has demonstrat— ed that an adequate local pathway through which the uterus regulates the corpus luteum in cattle and sheep is venoarterial, involving the veins that drain a given side of the uterus and the ipsilateral ovarian artery (reviews: 574, 572). Unilateral pregnancy. In sheep (1114) and cattle (397),‘ the establishment of pregnancy on one side of the uterus by surgical isolation of uterine horns results in luteal maintenance on the gravid side more readily than on the nongravid side. In superovulated cattle, induction of unilateral pregnancy by tying the oviduct and sectioning the adjacent uterine horn resulted in a greater proportion of regressed corpora lutea on the nongravid side than on the gravid side. In uterine-intact cattle, embryo survival at Day 30 was greater 270 Chapter 7 SHEEP t... Ovarian artery‘ @é Ovarian vein 2% HORSE Ovarian artery Ovarian vein FIGURE 7.26. Comparative diagrammatic presentation of arteries (clear) and veins (cross-bars) of a uterine horn and the adjacent ovary in a sheep and a horse. In sheep, which have a unilateral luteolytic uteroovarian pathway, the ovarian artery is tortuous and closely applied to the ovarian vein which drains most of the uter— ine horn. In horses, which do not have a unilateral luteolytic effect, the ovarian artery is relatively straight and caudal to the ovarian vein. when the embryo and corpus luteum were in the ipsilateral position than when they were in the contralateral position (396). In intact mares, fixation of the conceptus is as likely to occur on the contralateral side as on the ipsilateral side, indicating that a unilateral relationship between corpus luteum and embryo is not important in this species (pg. 311). Anatomy of the conceptus. The equine conceptus is spherical and mobile and is able to contact all parts of the endometrium many times per day during the time luteolysis must be blocked. In contrast, the trophoblast in cattle expands but covers only the uterine epithelium on the side of the corpus luteum by the critical time for blocking luteolysis (pg. 439). 7.4D. Role of Prostaglandin F20: The discoveries on uterine control of the corpus luteum led directly to the now widespread use of PGFZcx and its ana- logues for regulation of reproductive cyclicity, termination of pregnancy, and termination of persisting luteal activity in farm species, including mares (pg. 284). In addition, it provided an explanation for the pathogenesis of luteal dysfunction in association with uterine inflammation and anomalies. History. The increasing indirect evi- dence during the 1960s for the exis— tence of a uterine luteolysin prompted several laboratories to begin searching for the postulated substance through iso- lation and characterization attempts. Endocrinology of the Ovulatory Season 271 Systematically, crude and refined prepa- rations of endometrial tissue from several species were shown to be luteolytic in vitro and in vivo (review: 84). The sys- tematic approach eventually gave way to a direct approach when, in 1969, it was reported that PGonc produced pro- nounced luteolytic properties in pseudo- pregnant rats (1256). It was therefore sug- gested that because PGan is produced by the endometrium, the postulated lute- olysin might be a prostaglandin. Sub- sequently, workers in many laboratories in the early 1970s demonstrated that prostaglandins were potent luteolysins in many species. Mares were included in this» wave of activity in 1972 when it was shown that a single subcutaneous injec- tion of PGFZoc caused a return to estrus in approximately three days (427). There was some controversy during the early 19708 as to whether PGonc was the uterine luteolysin. Most of the opposition against the postulate that PGFZoc is the uterine luteolysin has waned, and the two terms uterine luteolysin and PGFZoc are used synonymously. The luteolysin in mares. This section will present the basic aspects of PGFM as a uterine luteolysin in mares. The more applied aspects are discussed in Section 7.6C (pg. 284). The following two initial and distinct lines of research sup- ported the postulate that PGan is the uterine luteolysin in mares: 1) Concen- trations of the PGF series increased in the uterine venous blood prior to the decline in circulating progesterone in diestrous mares (pg. 244); and 2) Exo- genous PGFZa caused prompt luteolysis in cycling (427, 1161, 1177), pseudopreg- nant, early pregnant (906), and hysterec- tomized mares (426). The temporal asso- ciation between luteolysis and the detection of prostaglandin has been well- confirmed using quantitation of PGan or PGFM in peripheral blood (881, 1136), uterine lumen (1699), and cultured and noncultured endometrial tissue (1700, 883, 1736). Release of PGFZoc also has been shown in association with uterine patho- logic processes (1136, 782) and uterine manipulations (189, 202). Administration of phenylbutazone, an inhibitor of PGFZoc synthesis that is widely used in horses as an anti-inflammatory therapeutic, did not alter luteal function in initial experi- ments (93, 465). However, phenylbuta- zone appeared to prevent induced luteol- ysis associated with uterine biopsy (466). It also has been shown that endotoxemia (e.g., from colic, laminitis) or injected endotoxins induced prostaglandin-medi- ated luteolytic effects in mares and other species (538). Minimal dose. The minimal effective luteolytic dose of a single injection of PGFZOL in mares is extremely small, as noted above. In ponies (148 to 195 kg), the minimal dose was approximately 1.25 mg of the free acid (427). In horses (350 to 500 kg), the minimal dose was approxi- mately 5 mg of the PGan THAM salt (equivalent to 3.75 mg of free acid; 1192). Using the median body weights given in these reports, the minimal effective dose was 8.8 ug/kg for horses and 7.3 ug/kg for ponies or approximately 8 pig/kg over the two types of mares. The minimal effective dose in sheep is 6 mg or 144 ug/kg (429). Thus, mares are about 18 times more sen- sitive than sheep to the luteolytic effects of systemically administered PGan. Furthermore, doses considerably below the 8 ug/kg level are often effective in mares. The number of horse mares that apparently responded to doses of 2, 3, 5, and 10 mg was 3 of 5, 3 of 5, 5 of 5, and 6 of 6, respectively (1192, 1193). Others (1500) obtained a response in 87%, 87%, and 78% of mares with doses of 2.5, 5.0, and 7.5 mg/day, respectively. In pony mares, doses of 0.25 and 0.75 mg resulted in at least a partial response (431, 432). 272 Chapter 7 SUMMARY: Luteal Regulation Days 0 to 5, period of luteal develop- ment: Initiation of luteal development occurs immediately after ovulation with luteinization of the granulosa cells and production of progesterone. The high level of LH present at the time plays a role in luteal development and may account for the increase in progesterone levels immediately after ovulation. The corpus luteum regresses when an anti- serum against pituitary gonadotropins is given. Days 5 to 14, period of luteal mainte- nance: Gonadotropins (most likely LH) are needed to maintain the corpus luteum. The high affinity for LH by the luteal cells may allow binding of LH molecules despite the low mean levels of LH in the blood during diestrus; occasion- al LH pulses also may be important. The luteal—maintenance effects of gonado- tropins are indicated by the apparent (not adequately demonstrated) ability of gonadotropic hormones to prolong the life span of the corpus luteum and especially by the ability of antiserum against pitu- itary extracts to cause regression of the corpus luteum. Day 14 to beginning of estrus, period of luteal regression: In the absence of an embryo, a luteolysin (PGFZoc) is released by the uterus. The luteolysin travels through systemic routes to the ovary where it causes luteal demise. The role of the uterus is indicated by maintenance of the corpus luteum when the uterus is removed and by early regression of the corpus luteum when foreign material is inserted into the uterus. The involvement of PGFZu as the uterine luteolysin is sup- ported by the luteolytic effects of exoge— nous PGFZu and by the increase in endogenous PGonc during the time of luteal regression. Rapidity of action. The rapid drop in progesterone caused by a single injec— tion of PGFZu during mid-diestrus is shown (Figure 7.27). This rapid decline has been found in many studies (e.g., 432, 1163, 833, 1350). Mean intervals from treat- ment to estrus have been reported as 2 or 3 days (means and SEs from several experiments: 2.2 i0.4; 3.3 i0.6; 2.5 i0.4; 2.3 i0.3; 2.7 $0.3). Behavioral, ovulatory, and hormonal events during post-treat- ment estrus are indistinguishable from those in nontreated mares, and the effects do not carry over into subsequent estrous cycles (1146, 1161, 1196). Transient increase in hormones. An interesting phenomenon associated with administration of PGFZu or an analogue (1146, 1163, 1350) is a transient elevation in LH, FSH, progesterone, and estradiol prior to the precipitous decrease in pro— gesterone. Progestins increased from 8 to 10 ng/ml within 10 minutes of treat- ment and then decreased to 5 ng/ml by one hour; estradiol increased nearly twofold at one hour and then decreased (1163). The concentrations of gonado— tropins increased within 45 minutes and returned to baseline over the next few Effect of PGF on progesterone (n=6) _| 00 a _L 01 _L N (D 0') Progesterone concentration (ng/ ml) 03 1 6 Number of hours after treatment 12 24 FIGURE 7.27. Effect of a single intramuscular injection of PGFZOL on peripheral progesterone con- centrations. Means with no common superscript let- ters are significantly different. Adapted from (432). Endocrinology of the Ovulatory Season 273 hours. The transient rise in LH and FSH may represent a direct action of PGFZoc at the hypothalamic-pituitary area or a decline in receptors on the luteal cells. The rise in estradiol and LH may be interrelated. The increase in both estradi- 01 and progesterone may be related to the recent finding that the corpus luteum of mares produces estrogens as well as pro- gesterone (pg. 446). The rise in proges- terone may be from rapid release of luteal progesterone and estrogen in association with luteolysis. In a recent study (833), concurrent treatment with PGan and GnRH did not overcome the luteolytic effects of PGan. In another study (1283), luteal tissue was subjected in vivo to regression by a PGFZoc analogue; in vitro incubation of the regressing luteal tissue resulted in transient PGFZoc production. Data were consistent with the possibili- ties that luteal PGonc production is a component of the luteolytic mechanism or that the released PGFZa is a consequence of tissue degeneration. Site of action. The effectiveness of dif- ferent routes of administration (into mus- cle, uterus, or corpus luteum) has been examined in mares (432). There were no significant differences among the three routes in intervals from treatment to estrus or to ovulation, length of post— treatment estrus, or length of interovula- tory interval. Results of progesterone analyses also indicated that local admin- istration into the uterus or corpus luteum did not improve the luteolytic efficacy of PGFZa over systemic administration (intramuscular). Failure to obtain a greater response in progesterone decline when PGFZa was given into the corpus luteum was unexpected. Perhaps the PGFZoc passed directly into channels (e.g., lymphatic) that removed it from the ovary before a local effect could be exert- ed. In this regard, it has been demon- strated (425) that the injection of PGan into the ovarian artery was more effective in causing luteolysis than injection into the carotid artery. This result indicates that the principal site of luteolytic action of exogenous PGFZoc in the mare is at the ovarian level rather than the hypothala- mic-pituitary level. One is left with an enigma upon accepting the following strong indica- tions: 1) the uterine luteolysin is PGFZoc, 2) PGan acts principally at the ovarian level, and 3) PGFZoc arrives at the ovary through the general circulation. From limited studies in other species, it appears that most (90%) of the prosta- glandins released into the circulation are cleared in one passage through the lungs. In sheep, for example, the local utero- ovarian venoarterial pathway would bypass the lung clearance mechanism. Without evidence for a local pathway in mares, one must assume that lung clear- ance is less effective or that only extremely small quantities of PGFZa are required at the level of the ovary. As noted earlier, mares are many times more sensitive than ewes to the luteolytic effects of PGan when given by systemic routes. It has been concluded that the affinity for PGan of cellular membranes prepared from mare corpora lutea is approximately 10 times greater than that of membranes from cow corpora lutea (876). High affinity of mare corpora lutea for PGFZoc binding, compared to reported affinity in other species, has been con— firmed (1699). Greater affinity for binding PGan may partly explain the high sensi- tivity of the mare to the luteolytic effects of PGan; this may negate the need for a local uteroovarian PGFZoc-concentrating mechanism. These ramifications suggest a good comparative research area involv- ing lung clearance rates in mares versus other species (e.g., sheep). Resistance of new corpus luteum. It is noteworthy that the newly develop- ing corpus luteum (first 4 days after ovulation) resists the luteolytic effects of PGan in mares (428, 1193, 431) as it does in other species. The reason for this refractory period has not been determined. The corpora lutea tested for 274 Chapter 7 binding of PGan were not taken during the period that mares are resistant to the luteolytic effect of PGan (Days 0 to 4: 876). Also, in a subsequent study (1699), the binding of receptors of luteal cells was done from Day 4 onward and therefore probably missed the most crucial days for determining if failure of the early cor— pus luteum to respond to PGFZa was due to lack of receptors. Nevertheless, the binding of PGan increased from Day 4 to Day 12, so perhaps the refractoriness of early corpora lutea is due to lack of ade- quate receptors for PGan. 7.4E. Mechanism of PGFZa Production A fundamental aspect of the control of luteal life in the mare concerns the mech— anisms that trigger release of the uterine luteolysin in the absence of an embryo. On a comparative basis, the ovarian steroids are most likely involved through a priming action on the endometrium. That is, progesterone and estrogens may be required to act on the endometrium for a certain interval before the luteolysin is released. Progesterone. Many studies have been done in many species on the effects of exogenous estrogens and progestins on the life span of the corpus luteum (review: 679). Administration of proges- terone to cows and sheep in early diestrus shortens the life of the corpus luteum (1825), exerted, at least in part, through the unilateral uteroovarian pathway (566). In contrast, administration of proges- terone in mares in early diestrus did not shorten the interovulatory interval (570). In mares and sows, the corpus luteum starts secreting progesterone shortly after ovulation, whereas in sheep and cattle, a few days elapse before circulating proges- terone concentration rises (pg. 238). Administration of progesterone after ovulation in mares and sows, therefore, does not alter the period during which circulating progesterone is present. In contrast, exogenous progesterone would cause exposure of the endometrium in cattle and sheep to progesterone a few days earlier than normal. Triggering of the release of uterine luteolysin may depend upon prior exposure of the endometrium to progesterone for a given interval. Therefore, progesterone treat- ment during the estrous cycle in cattle and sheep, but not in mares and sows, may hasten release of luteolysin due to the earlier exposure to progesterone. Ovariectomized mares treated with pro- gesterone for at least 14 days had increased levels of PGan in the uterine lumen compared to nontreated or estro- gen-treated mares (1845). Estrogen. The role of estradiol in the regulation of luteal life has been studied extensively in nonequine species. Estro- gens have been implicated as one of the hormones that, along with progesterone, is involved in triggering luteal regression. For example, destruction of follicles in sheep causes luteal maintenance, and administration of estradiol late in diestrus causes luteal regression (569, 679). Furthermore, PGonc release is preceded by a small estrogen peak in the blood. In sows, however, there apparently is no estrogen peak prior to release of lute- olysin, and exogenous estrogen given late in diestrus causes maintenance rather than regression of the corpus luteum. Studies in mares indicate that estrogen does not alter the life span of the corpus luteum (263, 1808, 885, 439). The interovula- tory interval may be extended after pro— longed estradiol treatment, apparently due to an effect on the follicles rather than on the corpus luteum. A preliminary report (136) suggested that exogenous estradiol given early in the luteal phase depressed the growth of follicles, whereas administration late in the luteal phase stimulated follicle growth. An earlier study indicated that estrogens extended luteal life (178), but results of this study are questionable in View of the more recent findings. Endocrinology of the Ovulatory Season 275 Increased equine endometrial produc- tion of PGFZa occurred in vitro in the presence of estradiol but not progesterone (1700). Estrogen appeared to stimulate incubated endometrial tissue from proges- terone-primed mares. Stimulation oc- curred when endometrial tissue was from late diestrus; stimulation did not occur when endometrial tissue was from early diestrus or from progesterone-primed ovariectomized mares. Administration of estradiol to ovariectomized mares after 14 days of progesterone treatment result- ed in the highest rates of PGF20L produc- tion. Long-term progesterone treatment of ovariectomized mares without use of estradiol did not stimulate PGFZa produc- tion in tissue culture (1700). One group of workers (885) has suggested that the role of estrogen in luteolysis is permissive (that is, must be present for luteolysis to progress); tamoxifen, an estrogen antago— nist that binds to estrogen receptors, slowed or prevented luteolysis. Late entry. Two experiments indicated that estradiol- 1713 enhanced the exogenous oxytocin— induced stimulation of PGFZoc (1862); a fourfold greater PGFM response to oxy- tocin occurred when mares were first treated with estradiol. Considerable follicular growth occurs in mares before the time of expected luteoly— sis (pg. 180). The growing follicles appar- ently produce estradiol. A recent study (884) showed that circulating estradiol increased before luteolysis. Estradiol con- centrations were significantly elevated 24 to 72 hours before endometrial PGan production occurred (Days 12 to 14). These results demonstrated that the temporal relationship between estradiol production and PGan production are compatible with a role of estradiol in trig- gering production of PGFZa. However, at this stage (approximately Day 12), follicle diameters do not differ between nonpreg— nant and pregnant mares (pg. 322); study is needed to determine if estradiol pro- duction also occurs in pregnant mares at the corresponding time. It has been proposed (1700) that proges— terone priming involves production of the prostaglandin synthetase enzyme system or the sequestering of precursors. Elabor- ation of the enzyme system or recruit- ment of precursors may be a slow process and therefore requires long-term expo- sure. Estrogen may activate the enzyme system and therefore requires only short- term exposure. Oxytocin. During the past decade, a third player, oxytocin, has been proposed to have a role in PGonc production by the endometrium at the crucial time for lute- olysis at the end of diestrus. Results of studies in ruminants suggest that oxy- tocin (of luteal origin) stimulates the release of PGan from the endometrium and that PGan, in turn, stimulates fur- ther pulses of oxytocin. This positive feed- back loop mutually reinforces the two secretions. Because PGF2a is luteolytic, the loop self-destructs as the corpus luteum regresses. One line of supporting research is that exogenous oxytocin caus- es early luteolysis in cattle through a local uteroovarian pathway (625). The pros and cons of this hypothesis have been reviewed (999, 515). An early study (570) found that administration of oxytocin to mares did not induce luteolysis, and this finding has been confirmed (1137). More recent studies have found, however, that exogenous oxytocin induced an increase in circulatory levels of PGFZa metabolite (PGFM) when given toward the end of the luteal phase (202). A subsequent experi- ment showed that the release of PGan in response to oxytocin was maximum at the time of luteolysis (633). This increase in response was not obtained in the preg- nant mare. Other recent research results that are compatible with a role for oxytocin in lute- olysis in mares are the following: 1) Production of PGFZa by endometrial tis- sue cultures was enhanced by oxytocin (883); 2) Circulating oxytocin concentra- tions were highest at the time of luteolysis (1593); and 3) Oxytocin-binding sites were 276 Chapter 7 greatest in the endometrium and myo- metrium on Days 14 to 17 (1572). These recent findings are compatible with the hypothesis that an increase in circulating oxytocin and endometrial oxytocin recep- tors in the late luteal phase stimulates synthesis and secretion of PGan; the increase in oxytocin secretion is due to estrogen stimulation (1593). The oxytocin was released into the circulation in puls- es, and the pulses have been partly char- acterized. Based on studies in nonequine species, it has been postulated that the increasing concentration of estradiol has a stimulatory effect on oxytocin—binding sites (cited in 1572). The above studies in mares have not considered Whether oxy— tocin has a role in the increased uterine contractility that occurs at the approxi- mate time of luteolysis (pg. 217). In this regard, the interval from initiation of pro- gesterone treatment in ovariectomized mares to a response is about 14 days for the following two end points: onset of uterine contractions (pg. 217) and PGan production (pg. 274). In addition, the num— ber of oxytocin-binding sites was three- fold greater in the myometrium than in the endometrium, and the number of sites increased in the myometrium, as well as the endometrium, near the time of luteolysis (1572). The events involved in PGFZa produc- tion are diagrammed in Figure 7.28. 7.5. Regulation of Tubular Genitalia The rhythmic changes in the reproduc- tive tract, including gross, histologic, con- tractile, and secretory changes, are under the control of the ovarian steroids. In gen- eral, changes during estrus and diestrus result from the influence of the ovarian steroids that predominate during a given phase of the cycle (estrogens and proges- terone, respectively). This principle has been derived from extensive studies in species other than the mare, and there is considerable literature on this subject in the nonequine species. For purposes of continuity, the follow- ing is a brief summary obtained from reviews (248, 639) for nonequine species. The steroid hormones are preferentially concentrated in the target tissues by solu- ble proteins called receptors, and the steroids reach the receptors by entry into the cells. Apparently the cell membrane provides little or no barrier to the diffu- sion of steroids into cells because of their lipophilic properties. In contrast to the intracellular (nuclear) location of steroid receptors, the receptors for the peptide hormones are located in the cell wall. The receptors themselves are influenced by the fluctuations in ovarian steroids. For example, there is considerable evidence that estrogens stimulate the synthesis of the receptors for ovarian steroids within the target tissue and that progesterone antagonizes this effect. The mechanism through Which the steroid-receptor com- plex exerts its effect on target tissues includes stimulation of specific RNA syn- thesis. Messenger RNAs carry informa— tion necessary for specific protein synthe- sis which, in turn, promotes physiologic change (e.g., contractions of smooth mus- cle, secretions of glands, hyperplasia and hypertrophy of cells, enzyme induction, changes in intracellular and extracellular fluids). These principles are based on studies in nonequine species, especially rodents. The only published information on steroid binding in mare tissues that the author has found was a short report on estrogen binding affinity of the uterus in various species (454) and an abstract (1624) on estrogen and progesterone receptors in the endometrium. Numbers of receptors were high during estrus, peaked in early diestrus, and then declined. Basic studies should be conducted in mares on the changes in receptor content or steroid- binding capacity of the tubular geni- talia, especially the uterus. Endocrinology of the Ovulatory Season 277 SUMMARY: Mechanism of PGFZa‘ Releaso :5 Follicle Estrogen Progesterone Oxytocin FIGURE 7.28. Diagrammatic presenta- tion of the hormonal control leading to release of uterine PGan and resulting in luteolysis. In nonequine species, the cor- pus luteum is a source of oxytocin, but L the source of oxytocin has not been defined for mares. Many of the indicators for involvement of progesterone, estrogen, and oxytocin in the production of PGFZa by the endometrium is from nonequine species. ‘Recent studies, however, indicate that mares also utilize these three hormones in triggering release of PGFZa. Progesterone. Ovariectomized mares treated with progesterone for 14 days had increased levels of PGFZoc in the uter— ine lumen. It is believed that such long- term exposure of the endometrium to progesterone is needed to prime the endometrium to PGan release, perhaps by production of a prostaglandin syn— thetase enzyme system or recruitment of precursors. Estrogens Endometrial tissue Efrem » progesterone primed mares produced" increased PGonr in vitro in the presence ' of estradiol. Treatment of progesterone— primed ovariectomized mares with estra-‘g _ ‘diol resulted 111 higher increases in PGFZa ‘ production Estrogen is thought to be aLL component of the PGFzLa triggering mech- ' anism perhaps by turning on the enzyme system. This may accennt for the require- ment of only short-term exposure. _ L 933%. Oxytocin receptors increase- in the endometrium in the late luteal \ phase, endometrial tissue responds to PGFZa production in vitro in the presence of oxytocin, and circulating levels of o‘xy» tocin increase at the time of luteolysis. The nature of oxytocin's apparent role in PGFZa release in mares has not been defined. 278 Chapter 7 Insight is needed on the essential con- trol of the genitalia during the estrous cycle and the manner in which such con— trol changes with the establishment of pregnancy and pseudopregnancy. The mare may be an excellent model for such studies because of the dramatic response of the genitalia to ovarian steroids. For example, the extremes in uterine tone between estrus and early pregnancy and the degree of cervical relaxation during estrus versus diestrus are profound. Furthermore, such changes are readily monitored in the mare. Concurrent mea- surements are needed on levels of steroid receptors, plasma steroids, and tissue steroids during the course of morphologic and physiologic responses. It seems an entire area in equine reproductive research is being neglected—an area with considerable contributory potential from both the basic comparative View and the applied equine industry—oriented View. The associations between progesterone and estrogen and uterine secretions are receiving attention in mares. Uterine secretions are probably most important in early pregnancy. Progesterone from the corpus luteum is crucial during early pregnancy, and the early conceptus is a source of estrogens (pg. 66 and pg. 429). Administering progesterone resulted in increased total protein, uteroferrin (1053), and acid phosphatase (743) in the uterine secretions of ovariectomized mares. Giving progesterone plus estradiol was even more effective; the estradiol was effective when given systemically or directly into the uterus. Ultrasound scanners offer a convenient research tool for study of the regulatory aspect of uterine contractility, and cur- rent knowledge on hormonal control of equine uterine contractions is reviewed elsewhere (pg. 313 and pg. 217). The func— tions of uterine contractions have taken an added dimension with the discovery of embryo mobility (pg. 305). 7.6. Artificial Control of the Estrous Cycle In some farm species, the development, maintenance, and regression of the cor- pus luteum primarily accounts for the rhythmicity of the estrous cycle because of the short follicular phase. In cattle, artificial synchronization of ovulations can be done by controlling only the luteal phase. In the mare, however, approxi- mately a third of the total length of the estrous cycle consists of the follicular phase (estrus). Therefore, artificial con- trol of the equine estrous cycle reflects the operator’s success in controlling both phases. This species difference will be apparent in the following discussion of artificial control of equine reproduction. The goal of artificial control of time of estrus or ovulation is a major motivator in equine reproductive research. The pur- poses for ovulation control can be placed into three categories: 1) to enable breed- ing of an individual animal at a predeter- mined time (appointment breeding), 2) to allow breeding of a group of animals within a narrow predetermined time (synchronized breeding), and 3) to space ovulations in a group of animals over a relatively prolonged time that is most compatible with a given set of managerial conditions (dispersed breeding). These three purposes require methodology that will cause ovulation at a predetermined time. Ideally, if control of ovulation time is adequate, breeding can be done with no need to detect estrus. Furthermore, the emphasis has been toward development of methods that can be initiated at any stage of the estrous cycle or in concert with the onset of the natural or artificial— ly hastened breeding season. The follow- ing three methods of control and their combinations will be discussed: 1) induc- ing ovulation during estrus, 2) delaying ovulation until mares are released from the treatment regimen, and 3) terminat- ing the luteal phase by administration of PGFZa or one of its analogues. Endocrinology of the Ovulatory Season 279 This section is limited to control of ovu- lation time in nonlactating cycling mares. A number of reviews on estrous and ovu- lation control have been published in recent years (1524, 36, 976, 975, 1512). One reference (975) lists the doses and sources of products that are currently available in the United States. Discussion will be found elsewhere on methods of ovulation stimulation or control during the anovu- latory season or in concert with the onset of the ovulatory season (pg. 158) and during the postpartum period (pg. 487). 7.6A. Induction of Ovulation during Estrus Induction of ovulation is widely used in the equine industry because of the long and variable length of estrus and, more practically, the long and variable interval from onset of estrus to ovulation. Human chorionic gonadotropin. The use of hCG to induce ovulation at a pre- dictable time in mares was first reported by workers in the Soviet Union in the 1930s, and this and other early work has been reviewed (190, 993, 1090). The results of several studies on the effects of hCG on hastening ovulation are shown (Table 7.2). Administration of hCG on day 2 of estrus shortened the mean length of estrus approximately 1 to 3 days in the various studies. There is general agree- ment among many studies that duration of estrus and interval from onset of estrus TABLE 7.2. Effect of a Single Injection of hCG on Interval to Ovulation Response No. hours to Treated Controls Ref. ovulation % n % n (993) Within 48 89% 34 (1576) 24 to 48 74% 35 27% 40 ( 600) Within 48 80% 55 (450) 24 to 48 73% 145 . . . . 24 to 48 84% 31 18% 32 to ovulation are reduced in mares treated with hCG and similar compounds. The variation in length of estrus also was sig- nificantly reduced (9.93). Hastening oocyte maturation with hCG has become a common practice for recov- ery of oocytes for research purposes, including in vitro fertilization. The hCG is given when the preovulatory follicle is approximately 35 mm, and the follicle is usually aspirated 28 to 36 hours later (1212, 740, 887). Certain aspects of the efficacy of hCG were studied recently in conjunction with a study on postovulatory breeding in ponies (1824); the hCG results are report- ed here. The hCG was given as a single 2,000 iu intramuscular injection when the largest follicle reached 35 mm. Each treated mare was paired with a nontreat- ed mare that had a follicle of the same diameter as in the treated mare at the time of treatment. Frequency distribu- tions were prepared for the interval from treatment to ovulation (Figures 7.30 and 7.31). The hCG injection resulted in ovu- lation within 1 or 2 days in 80% of mares (significantly different from controls) in agreement with studies cited above. Ovulations occurred in 36 to 72 hours in 65% of the treated mares. As expected, the mean number of days from treatment to the day of ovulation was shorter (P<0.0001) for treated than for nontreat- ed mares (2 versus 4 days, Table 7.3). The interval to ovulation was not dependent on diameter of the preovulatory follicle at the time of treatment. The number of mares with follicles less than 35 mm in diameter at the time of treatment, howev- er, was small; the minimal antral diame- ter was 31 mm. More study of the mini- mal diameter of follicles that will respond to hCG is needed. There is some question, however, about the efficacy of hCG when given in succes- sive estrous periods. Some workers indi- cated that refractoriness does not develop from repeated treatments (381, 993). One study reported, however, that hCG treat- 280 Chapter 7 Effect of hCG on interval to ovulation 80 hCG treated mares Nontreated mares n= 55 n: 55 50 Mean: 2.0 days Mean: 4.2 days SD: i0.9 SD=i1.7 Frequency (%) J: O N 0 12345671 ment was effective during the first cycle, but treatment during the next estrus was ineffective in reducing the interval to ovu- lation or the length of estrus (1576). More recently, it was found that anti- bodies to hCG formed after 2 to 5 conven- tional injections for ovulation induction in 5 of 12 mares (1351). The half-life of the antibodies ranged from one to several months. The authors calculated that the level of antibodies in these mares could Effect of hCG on interval to ovulation 7O 60 50 g; ; 40 hCG-treated mares E’ (n=55) S a 30 2 L 12-18 24-30 36-42 48-54 60-66 6-12 18-24 30-36 42-48 54-60 284 Interval from treatment to ovulation (hours) FIGURE 7.31. Frequency distribution for interval from hCG treatment to ovulation based on examina— tion for ovulation every 6 hours. From (600). 2 3 4 5 6 7 8 9 Interval from treatment to ovulation (days) FIGURE 7.30. Frequency distri— bution for interval from hCG injection to ovulation. Each treated mare was paired with a nontreated mare that had a folli- cle of the same diameter as the treated mare at the time of hCG injection. From ( 600). precipitate 2 to 10 times the amount of hCG in an ovulatory injection. However, the antibodies did not prevent hCG- induced ovulations. All treated mares ovulated within 48 hours, regardless of antibody titers. During the subsequent breeding season, five mares that had developed antibodies were treated again for 3 or 4 consecutive cycles (1349). A hyperimmune reaction occurred in 3 of 5 mares. In 35% of the ovulatory periods (6 of 17), ovulations occurred later than 48 hours; however, there were no controls for comparison. More recently (1792) 2,500 iu TABLE 7.3. Effect of hCG on Interval from the Day the Preovulatory Follicle was a Given Size to Day of Ovulation No. days from indicated Diameter follicle size to ovulation follicle at time No. of treatment mares Treated Controls 31-34 mm 6 1.8 i0.2 5.2 i0.9 35-37 mm 27 2.1 i0.2 4.3 $0.3 38—40 mm 19 1.9 i0.1 3.9 i0.4 >40 mm 3 1.7 :03 2.7 i0.9 TOTAL 55 2.0 $0.1 4.2 i0.2 No significant difference within hCG-treated groups in number of days to ovulation. Difference (P<0.0001) between treated and nontreated mares in number of days to ovulation. From (600). Endocrinology of the Ovulatory Season 281 of hCG was given to induce ovulation over five successive cycles in one year and dur- ing at least two cycles during the next year. All of 14 mares developed antibodies. However, no effect of the immune response on ovulation time or pregnancy rate was observed. The expected shortened time to ovulation did not occur consistently, but the mares did ovulate, conceive, and foal in the presence of anti-hCG antibodies. No significant binding of the anti-hCG to eCG (1792) or eLH (1792, 1351) was detected in vitro, and no difficulties in the ovulatory response to eLH was observed in vivo (1351). In summary, it has been well demonstrated that anti-hCG antibodies form in response to clinical doses of hCG. However, the antibodies apparently do not cross-react with eLH and therefore do not interfere with the normal ovulatory response to the natural LH surge. Efficacy of hCG treatment over several cycles may be reduced, and hastening of ovulation may not be as effective. However, interfer- ence with ovulation or with pregnancy establishment and maintenance have not been observed in recent studies. Injection of an ovulatory dose of hCG (2,000 iu) was not effective in mares in which hCG antibodies had been induced deliberately (200 iu hCG/day for 20 to 30 days; 450). Treatment with a corticoid (dexamethasone) did not suppress the immune response to hCG. Treatment with an equine pituitary extract effective- ly induced ovulations (86% of 14 mares responded in 24 to 48 hours), and the extract effect was not blocked by the hCG antibodies. In addition, because it is not a foreign protein, the extract probably did not stimulate an immune response. Further studies are needed on the immune response to hCG, especially when considering that hCG treatment is sometimes used repeatedly, especially during the spring transitional period. Workers in the Soviet Union confirmed the stimulatory effects of hCG on ovula- tion (1455). Interestingly, these authors found pregnancy rates of 62%, 62%, 50%, 12%, and 12% for doses of 0, 1,500, 3,000, 4,500, and 6,000 iu, respectively (n=8 per group). It was concluded that the high doses (4,500 and 6,000 iu) caused re- duced pregnancy rates (also see pg. 452). This detrimental effect was attributed to excessive production of estradiol-17B, based on limited study of estrogen excre- tion rates. This phenomenon needs to be confirmed and further investigated. The estrogen stimulation may be related to the luteal stimulatory effect of eCG (pg. 446) and possibly eLH (pg. 242). Various workers have found pregnancy rates of 60% to 82% in mares treated with hCG (993, 1090, 1719). In all studies, the pregnancy rates were not lower than in controls. Thus, hCG administration does not affect pregnancy rate adversely. It improves breeding efficiency because fewer services are required in a given estrus due to advancement of time of ovulation. Gonadotropin releasing hormone. Single injections of GnRH will increase LH levels, but the induced LH peak is inadequate to hasten ovulation (1090, 622, 809, 1196, 1282, 1725). However, daily admin- istration of 2 mg, beginning on day 2 of estrus, significantly shortened the dura— tion of estrus and the interval to ovula- tion (809). In the treated group, 67% of the mares ovulated within 48 hours. Similarly, 40 ug of a GnRH analogue given every 12 hours was effective; an average of 3.8 injections was used (1507). Other workers reported that a single 40-ug injection of an analogue was effec- tive (1218). Other) reports are available on the use of GnRH to induce ovulation When given alone (711, 1724, 715, 1101) or in combination with PGan (221, 1725). In summary, multiple injections, continuous delivery, and pulsatile delivery of GnRH have effectively induced ovulation but a single injection has not, except for one report involving a potent analogue. It is expected that longer-acting forms will become available, and a single injection will suffice. When it does, GnRH will likely be an alternative to hCG. 282 Chapter 7 Prostaglandin F20: analogues. In the past few years, a prostaglandin analogue (fenprostalene) has been tested for has- tening the time of ovulation during estrus (1397). It was noted that fenprostalene has a 24-hour half-life in cattle, but the half- life is not known for horses. Fenprosta- lene was given as a single injection when mean diameter of the largest folli- cle was 43 mm. Ovulation occurred with- in 48 hours in 81% of 16 mares compared to 31% of 16 controls. The interval to ovu— lation and duration of estrus were also significantly reduced. Another PGan analogue, luprostinol, caused elevated levels of circulatory LH and FSH during the spring transitional period (825). Release of GnRH occurred after the gonadotropin release and prob- ably contributed to a sustained release of the gonadotropins. The authors (825) cited several publications that indicated that PGonc analogues may be able to induce estrus or ovulations in mares without a corpus luteum or with very low progesterone concentrations (see also 774). That is, the beneficial effect was not related to induction of luteoly- sis. The authors commented that the LH- and FSH-releasing properties of PGFZoc analogues deserve additional study. In a more recent study (1507), an injection of PGFZa analogue (luprostinol) was given on the second day of estrus. The results (interval to ovulation) were intermediate between those of hCG and controls and were not significantly dif- ferent from either group. The study thus failed to show that ovulation was has— tened with the PGFZoc analogue. In a recent report (348), the analogue alfaprostol did not appear to increase LH levels when given in early estrus. The authors suggested that the action of PGFZa in hastening ovulation may there- fore occur at‘ the follicle rather than at the pituitary. Clearly, much study will be needed on the efficacy and mecha— nism of action of PGFZoc analogues on the stimulation of ovulation. 7.6B. Delaying Ovulation until Release from Treatment The use of progestins as synchronizing agents has been extensively investigated in many species. The rationale is to keep the animals in an artificial progestational state for a prolonged period until the corpora lutea in all animals of the group have regressed. Estrus and ovulation are expect- ed to occur at a predictable time after with- drawal of the progestin. The clinical appli- cation of progestins in mares, including synchronization of ovulation, has been reviewed (1518, 1134). Exogenous progesterone. The original experiment on the effects of exogenous progesterone on the estrous cycle of mares was done by Loy and Swan (996); this study, although limited, was the first to indicate a potential applied use for proges- terone in mares. Fair synchronization of ovulation was obtained in pony mares with a regimen of 19 days of progesterone (50 mg/day), followed by hCG six days after withdrawal. Percentage of mares ovulating was 52% on 7 and 8 days and 71% on 6 to 9 days after withdrawal. A regimen of 20 daily injections of proges— terone (300 mg on Day 1 and 100 mg thereafter) without hCG produced unsatis— factory results (1201). Ovulation was not suppressed in some mares. Other progestins. Two orally adminis- tered synthetic progestins (MAP and MGA) that inhibit ovulation in other species were ineffective in mares at the doses tested (996). Chlormadinone acetate (CAP; 89) and norgestomet (synchromate- B; 249) are other progestins that are being tested for estrous cycle control in mares. In 1975, an initial report was made on the use of an orally administered pro- gestin for regulation of ovarian cyclicity (altrenogest or Regumate; 1745). During 1979-83, several studies were done, and in 1984 the progestin was approved for use in the United States {1523). Altren- ogest is being used during the resurgent phase of the anovulatory season (pg. 163). Endocrinology of the Ovulatory Season 283 TABLE 7.4. Effect of 15 Daily Altrenogest Treatments Early in the Breeding Season End point Treated Control (Number mares) 14 13 End of treatment to: Estrus (days) 3.4 7.2 Ovulation (days) 8.8 13.7 Length estrus (days) 6.4 6.0 Pregnancy rate 79% 85% Significant difference between groups in length of interval from end of treatment to estrus and to ovu— lation. Treatment was started on specific calendar days without regard to day of estrous cycle. Adapted from (1508). Results of a trial involving daily treat— ment for 15 days during the ovulatory sea- son are shown (Table 7.4; 1508). The inter- val from last day of treatment (or the corresponding day in controls) to estrus and to ovulation was reduced and less variable in treated mares. Pregnancy rates were not altered from those of con- trol mares in this and other studies, even when the length of treatment was as long as 60 days (1526). The interval to estrus after cessation of an altrenogest regimen has been reported to be 3 to 6 days (1523, 1526). Mares in estrus at the start of treat- ment ceased to exhibit estrus in 2 or 3 days. There seems to be inadequate infor- mation on the effect of the product on ovu- lation when ovulation is imminent at the start of treatment. The ability of the prod- uct to synchronize ovulation, along with a definition of synchrony, also has not been described adequately. It has been noted that synchronization of the onset of estrus is good, but that the time of ovulation in relation to the onset of estrus is as vari- able as in untreated mares (1203). Intravaginal sponges. The successful adaptation of vaginal sponges impregnat- ed with progestin (altrenogest) as an aid in reproductive management of mares has been reported (1203). All 24 mares retained the sponges, and onset of estrus was well synchronized after withdrawal (3.8 days i1.0 SD). Vaginal irritation occurred, but nevertheless, the pregnancy rate was 71%. Development of the vaginal sponge system has been encouraged because of the elimination of daily treat- ments. A scheme involving repeated suc- cessive synchronizations with a progestin was noted; the mares were mated after each synchronization until pregnancy occurred. Results were encouraging. A report on the use of vaginal sponges impregnated with progesterone is avail- able (413). Inhibition of ovulation in mares with a large follicle. A key consideration in determining the optimal length of treatment and dosage is the ability of progestins to inhibit ovulation when treatment is started during estrus. Unfortunately, there is only limited infor— mation on this point. When 50 mg/day was given intramuscularly to one horse mare and 100 mg/day to another begin- ning on the first day of estrus, both mares continued in estrus and ovulated on day 4 of estrus while on treatment (996). In another study, treatments of 50 mg/day were given to four pony mares beginning on the second day of estrus; all four went out of estrus, but ovulation was inhibited in only two mares (757). In a subsequent study, a dose of 75 mg/day in ponies inhibited estrus and ovulation when treatment began on days 1, 2, or 3 of estrus, but not when it began on days 4 or 9 (one mare/period). Clarifying studies are needed on the effect of various treat- ment regimens on the probability of ovu- lation when the regimens begin during estrus. In this regard, a recent trial indi- cated that altrenogest does not suppress the growth of large follicles in some mares (977). As a result, ovulation occur- red during treatment or earlier than expected after the end of treatment. This finding is in accordance with the propen- sity of some breeds to ovulate during pro- gesterone dominance (diestrous ovula- tions; pg. 223). The addition of PGFZoc after the end of a progestin treatment regimen 284 Chapter 7 would be expected to improve estrous cycle control by regressing corpora lutea that formed during progestin treatment. Progestin and estrogen combination. Loy and associates have developed an approach during the past decade that involves administering a combination of a progestin and estrogen with or without the inclusion of PGFZa in the regimen (995, 1590). This approach was developed because of the problem of development of the follicles during progesterone treat— ment even though ovulation apparently was prevented. Total ovulation preven- tion with progesterone alone has not been adequately studied, considering the abili- ty of mares to ovulate While under pro- gesterone dominance. The recommended treatment in horse mares consists of 150 mg progesterone and 10 mg estradiol—17B given daily for 10 days alone or combined with an injection of PGan on the first and last days. This regimen resulted in ovulation in 15 of 16 mares 9 to 13 days after the last treatment (995). In another study, initiation of the regimen on the first or second day of estrus did not block ovulation in 8 of 13 mares. Thus, injection of PGan on the last day of treatment is advocated. The authors concluded that the pro- gram produced a tighter grouping of ovu- lations than what has been reported for progestins alone; however, a progestin regimen and the combined regimen were not directly compared. The authors com- mented that development of follicles over a Wide range of sizes during progestin treatment results in a wide dispersion in post—treatment ovulations. The combined program seemed to more readily suppress follicular development. In subsequent work (1590), these investigators used a 15- day treatment schedule with a single injection of PGFZOL on the last day. The results supported the earlier conclusion that this regimen provides reasonably precise control of ovulation (Figure 7.32). In two trials, 87% (n=31) and 80% (n=128) ovulated in 8 to 14 days and 9 to 16 days, respectively, after the last treatment. The progesterone-estradiol combination also has been used effectively in postpartum mares (pg. 487,- 253). Studies have been initi— ated on controlled release of steroids, using biodegradable microspheres (262). 7.60. Termination of Luteal Phase with Prostaglandins With this approach, PGan or one of its analogues is given to cause immediate regression of the corpus luteum. The orig- inal reports on the use of PGFZa and its analogues, respectively, for inducing lute- olysis in mares appeared in 1972 (427) and 1973 (66). Since then, many reports and reviews have been published (e.g., 56, 936, 1195, 1096, 931, 1102, 226, 111, 774, 340, 249, 794). Side effects of PGFZa and its ana- logues have been well researched and discussed (56, 936, 1096, 1138); dose and route of administration must be given careful attention. Treatment with PGFZcx is especially useful for individual mares when it is known or suspected that a mature corpus luteum is present. For Synchronization with E 2 + P4 15 _l 0 Number of mares 01 910111213141516 Number of days from last injection to ovulation Figure 7.32. Results of ovulation synchronization by an estradiol-progesterone regimen, showing dis- tribution of intervals from day of last injection to ovulation. Adapted from Taylor et al. (1590). Endocrinology of the Ovulatory Season 285 example, a single injection can be used when breeding was missed for any reason and when it is desirable for the mare to return to estrus. Prostaglandin treatment in mares with a mature corpus luteum results in estrus in 2 to 4 days and ovula- tion in 7 to 12 days. The onset of estrus was more synchronized than day of ovula- tion (66). This is attributable to the vari- ability in the interval from onset of estrus to ovulation (pg.190). As noted earlier, an ovulating hormone, such as hCG, may be useful in the regime to reduce this variability. It is important to note that the corpus luteum is refractory to treat- ment with PGFZoc and its analogs for 4 or 5 days after ovulation (66, 428, 1192, 431). In addition, variation can be expected according to the diameter of the largest follicle at the time of luteolysis (991). If luteolysis occurs when a dominant follicle is at the end of its growing phase, ovula- tion can occur earlier than when the dom- inant follicle is smaller, especially in horse mares with a major follicular wave in early diestrus (pg. 178). However, if the follicle is large but is beginning to under- go atresia or if all follicles are small, the interval to ovulation after induced luteol- ysis could be prolonged. In one study (991), the presence of follicles larger than 85 mm reduced the interval to ovulation to a few days. Because luteolysis does not naturally occur in the presence of a large follicle, it is not surprising that such an artificial situation can result in an unusual endocrinologic balance and may result, for example, in weak estrous behavior (pg. 91). Mares that are already in estrus when a group is treated will not be synchro- nized with those mares that have a mature corpus luteum. For scheduling the time of ovulations in a large group of mares or when the stage of the cycle is not known, sequences of PGan treat- ments have been advocated. The empha- sis has been to develop a regimen of two sequential injections, spaced in such a way that the corpus luteum of most mares will be at a sensitive stage at the time of the second injection. Combining treatments. In many test- ed programs, PGan was combined with other treatments (hCG, GnRH, or pro- gestin). The use of PGFZa with the progestin—estrogen protocol was noted above. Programs involving two injections of PGan are designed to induce luteolysis in all corpora lutea more than four days old at first injection so that all mares will have a corpus luteum of a susceptible age at the second injection. Such programs have been followed by an ovulation induc- er (hCG or GnRH) 5 or 6 days after the second PGFZoc treatment. In the listed examples (Table 7.5), two injections of PGFZa seemed most effective when hCG was given 6 days after each injection. In another study (1720), PGan was given 14 days apart and hCG was given 5 days later; 81% were in estrus on days 14 to 20, but pregnancy rates for mares bred on preplanned days were low (25% to 40%). French workers have studied the use of altrenogest in combination with other agents. Altrenogest-impregnated vaginal sponges were used for eight days with an injection of PGan on the last day (1203, 443). Synchronization of the beginning of estrus was good (i2 SD = 1.8 days), but TABLE 7.5. Examples of Synchronization Protocols % mares ovulating in reference to last hCG injection No. Within During >6 mares Before 3 days 3-6 days days A 48 19% 69% 4% 8% B 23 26% 39% 17% 17% C 21 14% 62% 19% 5% D 39 0% 54% 28% 18% A = days 0 and 14, PGF; days 6 and 20, hCG B = days 0 and 18, PGF; day 24, hCG C = days 0-18, progest; day 24, hCG D = days 0-18, progest; day 7, PGF; day 14, hCG (day 0 = first day of treatment) (A, 1214, 1215; B-D, 758, 759) 286 Chapter 7 synchronization of ovulation was not (i2 SD = 3.3 days). When hCG was given on the seventh day after sponge removal, 70% of the mares ovulated within four days (1203). In a subsequent study, the effects of various factors on the degree of synchronization were examined (443). The treatment regimen was eight days of progestin-impregnated vaginal sponges and PGF2a treatment on the last day. Sponge insertion immediately reduced circulating LH levels, and this was fol- lowed by an increase in LH at sponge removal and a surge in FSH. When used during the estrous cycles in April, the interval to ovulation was four days longer than when used later in the year (14 days compared to 10 or 11 days). Several differences in levels of various hormones were found at various times of the year, but none were linked to the removal-to- ovulation interval, except for lower peak LH in the April group. Such seasonal effects of treatment early in the year are likely related to the less prominent LH surges and longer estrous periods that are associated with the first ovulations of the year (pg. 254). In a subsequent report (1205), several variations to the eight-day progestin regimen were tried. Addition of estra— diol benzoate to the protocol reduced the FSH secretion during treatment and reduced the removal-to—ovulation inter- val. However, ovulation synchronization was not improved. Addition of daily testosterone injections, however, re— duced LH concentrations, but not FSH concentrations, during treatment and improved the degree of synchronization. The improved synchronization was shown by a significantly smaller stan- dard deviation, apparently as a result of fewer early ovulations. Use of the anti— estrogen, tamoxifen, did not alter the results. Breeding without detection of estrus. Ideally, estrus detection should not be necessary in synchronization programs, especially if a reliable method is avail- able for monitoring the reproductive tract (e.g., ultrasonography; 590). Hyland and Bristol (794) evaluated a procedure that involved giving PGFZa on day 0 (first day of treatment) and on day 15 and hCG on day 20. Insemination was done on one of the following schedules: 1) day 21 in the blind, 2) day 21 plus day 23 in the blind, and 3) days 19, 21, 23, and 25 while in estrus (controls). Pregnancy rates were lower (although not significantly; n=30 to 54) for the inseminations in the blind (57% and 59%) than for the program involving estrus detection (72%). A high rate of estrus synchronization was attained (80% within 48 hours after the second prostaglandin injection). Palmer (1203) used a program that involved adminis- tration of a progestin on Days 7 to 21 in mares mated during an initial synchro- nized estrus (also see pg. 283). If a mare was not diagnosed pregnant at the end of the progestin regimen, a new sequence of hCG injections and mating was done on Days 27, 28, and 29. The procedure was repeated until pregnancy was established. The rate of synchro- nization of return ovulations in the non- pregnant mares was given as 63%. Cumulative pregnancy rate after three regimens or cycles was 7 4%. Other programs. In addition to the pro- gestins and prostaglandins, an orally administered potent gonadotropin inhibitor (methallibure) has been tested in mares (507). Estrous and ovulation syn- chronization and conception rates were good. Unfortunately, side effects such as anorexia made the compound undesir- able. Another approach is suggested by the experiments with antisera against equine pituitary extract (pg. 258). The anti— sera caused regression of both the ovula- tory follicle and the corpus luteum when given for a few days. This method there— fore has the potential for synchronization, regardless of cycle stage. Endocrinology of the Ovulatory Season 287 7. 6D. Implementation of Cycle Control Programs Suitability of mares. Compared to other species, certain endocrinologically related phenomena in mares seriously hamper cycle-control efforts. These include: 1) rel— atively long follicular phase, 2) physiologic differences among breeds (especially ponies versus horses), and 3) ability to ovulate even when under the influence of high progesterone levels. The prolonged follicular phase in mares greatly compli- cates control. For example, if an injection of PGan is given when mares are in ran- dom stages of the estrous cycle, more than 50% of the mares likely will not respond— those in estrus or with a corpus luteum less than five days old. Even though a sec- ond injection will improve the amount of control obtained, it is difficult to agree on an optimal interval between injections. Differences in cycle length attributable to breeds (ponies versus horses; pg. 173), sea- son (pg. 174), and mare-to-mare variation (pg. 173) are important obstacles to formu- lating a standard approach. It is also important to note that extrap- olation of research results from one breed to another may lead to difficulties. Many physiologic differences between ponies and horses are noted throughout this book (pg. 230). Of special importance for cycle control, is the more frequent occur- rence of ovulations during diestrus in horses, especially in certain breeds, than in ponies. It appears that many experi- ments, especially those involving ponies, were not challenged by such natural phe- nomena as mid-diestrous ovulations to the degree that may occur in horses. The ability of the mare to ovulate in the pres- ence of high progestin levels (pg. 450) com- plicates attempts to develop optimal pro- gestin doses and regimens. On the positive side, it appears that the long follicular phase in mares may work for, as well as against, the operator. Cycle control with progestins does not appear to decrease pregnancy rates in mares. In cat- tle, however, a reduction in pregnancy rates can occur (1826). The apparent differ- ence between cattle and mares in this regard is attributable to the longer post- treatment estrus in mares. Thus, the ovu- lating follicle may be free of progestational influence for a longer time span in mares than in cattle. Use in the individual mare. Estrous cycle control can be particularly effective with an individual mare, especially when informa- tion on the stage of the estrous cycle is available or can be estimated. The individ- ual mare approach is helpful to the owner of one or two horses. In such circum- stances, estrus may be difficult to detect since a stallion is not usually available. Taking the mare to the stud may mean being without the horse for a prolonged period. If the mare is in estrus, as deter- mined by behavioral or physical signs (especially appearance of cervix), and a large follicle (>30 mm) is present, a single injection of hCG may be all that is required. If on the basis of history (recent estrus) or transrectal examination, a recent ovulation is suspected, a single injection of PGFZoc can be given after a few days delay. The delay is to ensure that the corpus luteum is at least five days old. Because of variability in time of onset of estrus and interval from onset of estrus to ovulation, an injection of hCG six days after the PGFZa injection is recommended. Post- treatment monitoring of follicular devel- opment, wherever practical, should be useful in adjusting the day of hCG treat- ment. If it is known that the mare is in diestrus but beyond Day 4, the injection of PGan can be given immediately. Anabolic steroids. Anabolic steroids (androgenic compounds) are sometimes used in the equine industry. Anabolic steroids have been reported to affect reproductive function in both mares and stallions. Readers confronted with this problem are referred to recent publica- tions (1512, 1522, 191, 1.92, 1501, 1009). 288 Chapter 7 SUMMARY: Temporal Relationships among Circulating Hormones and Ovarian Events ‘ Gonadotropins & inhibin Progesterone EProgesterone, PGF 2a; §&|utea| dynamics ‘ Follicular estro en 9 Estrogen & follicular dynamics Luteal estrogen? (Growth of dom. fol. l-—— Primary fol wave (Growth ofdom. fol. Atresia of sub. fols. Atresia of sub. fols. The follicular patterns are for estrous cycles with only one major fellicular wave—the pri: mary wave that gives origin to the ovulation associated with estrus (dom fol. and sub . £015.: dominant and subordinate follicles, respectively) The circulating hormone profiles-:5 are a representation of means for many estrous cycles. Hormone Surges, espeéially for: #- PGFZa, inhibin, and FSH, occur at different times among cycles and are masked by the. means; in individual animals, hormone surges occur during the time encompassed by the high mean values The circulating estrogen concentrations repreSent the follicular contri: 1 bution plus the apparent luteal contribution; more study IS needed. ‘ s ‘ x ' : l. 10. 11. 12. 13. Endocrinology of the Ovulatory Season 289 HIGHLIGHTS: Endocrinology of they Ovulatory Season Mean circulating LH concentrations are low during midndiestrus, begin to increase a few days before estrus to a maximum 1 or 2 days after ovulation, and then decrease over the next 4 to 6 days. See facing page for schematic hormone profiles. Mean circulating FSH concentrations are reciprocally related to LH concentra" tions except that levels of both gonadotropins increase for a few days during the periovulatory period. Diestrous surges of FSH do not follow a consistent pattern. Circulating progesterone concentrations begin to increase approximately 12 hours postovulation, reach high diestrous values by Days 5 to 7, and begin to decrease on Days 13 or 14 (3 days before estrus). Circulating concentrations of estrogens begin to increase 6 to 8 days before ovula— tion, reach a peak 2 days before ovulation, and then decline. Concentrations of androgens increase in the follicular fluid and blood plasma just - ' before ovulation and may play a role in the accumulation of FSH in the pituitary during estrus. In absence of ovaries, FSH levels are higher than during any stage of the estrous cycle, and LH is higher than during diestrus but lower than during estrus. Progesterone has a negative effect and estradiol a positive effect on circulating LH concentrations. Although results of some studies have been equivocal, others indicate that pro- gesterone has a positive effect and estradiol a negative effect on circulating FSH concentrations. ‘ ‘ A proteinaceous fraction of follicular fluid depresses circulating levels of FSH. This effect is augmented by estradiol. The corpus luteum of diestrus is dependent on a continuing supply of a gonadotropin, probably LH. Prostaglandin F20: is produced by the endometrium and causes luteolysis in the absence of pregnancy; if the uterus is removed, the life span of the corpus luteum is prolonged. Progesterone, estradiol, and oxytocin have roles in the uterine production of PGan. PGan is delivered to the ovaries through a whole-body pathway in mares in con- trast to a local uteroovarian pathway in ruminants. 290 Chapter 7 MILESTONES: Endocrinology of the Ovulatory Season First report that an injection of hCG during estrus hastens ovulation (cited in 993). First study on control of the estrous cycle with exogenous progesterone (996). Characterization of circulating concentrations of progesterone during the estrous cycle (1497). Demonstration that uterine removal prevents regression of the corpus luteum (609). Exogenous PGFZOL shown to be a potent luteolysin (427). Demonstration that the corpus luteum is dependent on a continuing supply of gonadotropins (1270). Characterization of circulating concentrations of LH during the estrous cycle (1768). Characterization of circulating concentrations of estrogens during the estrous cycle (1249). Characterization of circulating concentrations of FSH during the estrous cycle (490). Finding that PGFZOC concentrations in uterine venous drainage increased dur— ing late diestrus (433). Demonstrations of a negative effect of progesterone and a positive effect of estradiol on circulating LH concentrations (555). Role of ovaries in controlling levels of gonadotropins as determined by ovariec- tomy experimentation (540). One of a series of experiments that demonstrated that androgens play a role in the accumulation of FSH in the pituitary during estrus (1607). Demonstration of suppression of FSH and follicles during the estrous cycle by a nonsteroidal fraction of follicular fluid (181). Reports that pulses of LH, FSH, and GnRH in pituitary venous blood (23) and LH and FSH in systemic blood (1602) tend to occur concurrently. ——Cfiapter 8— MATERNAL ASPECTS OF PREGNANCY The various aspects of pregnancy are covered in three consecutive chapters: this chapter (maternal aspects), Chapter 9 (embryology and placentation), and Chapter 10 (endocrinology). Those aspects of the terminal portion of preg- nancy that may be related to the mecha- nisms of parturition are deferred to Chapter 11. The present chapter includes oogenesis, mating, sperm and ova trans- port, interactions between the uterus and embryo, changes in the ovaries and uterus, and pregnancy diagnosis. The last section reviews the current status of equine biotechnology involving gametes (sperm and ova) and embryos. 8.1. Oogenesis The term germ cells refers collectively to the gametes (ova in the mare, sperm in the stallion) and the cells from which the gametes are derived. Oogenesis is the process of development of the ovum. It begins with the oogonia, which originate from primordial germ cells in the embryo, and ends with the formation of secondary oocytes. Research on oogenesis in the mare has been stimulated by the desire to culture and fertilize oocytes in vitro. Studies on oogenesis have been done in earnest in mares only during the past few years. The state of oogenesis at birth and the development of the oocytes and folli— cles between birth and puberty are open research areas. The details of oogenesis across species can be found in textbooks and reviews (479, 245). Those wishing to study oogenesis in detail should first review the mechanisms of mitosis and meiosis, as given in embryology and genetic textbooks. An introductory overview of the continuity of life in the horse through the perpetuity of germ cells appears in Figure 8.1. Structure of oocytes. The fine structure of equine follicular oocytes is similar to that of other mammals (546). Approximate diameter of mounted oocytes was 125 um, excluding the zona pellucida and corona radiata. The zona pellucida, located between the oocyte and corona radiata, was 7 to 10 mm thick. Lipid droplets are large and dense in equine oocytes. In the living state, the vitellus of the horse ovum has a black appearance and contains many closely packed fatty globules (Figure 8.2,A). In transmitted light, the opacity of the globules obscures the nucle- us. In this respect, horse ova resemble swine ova more closely than those of other farm species. Primary oocytes are distinguishable by the absence of a polar body and the presence of a germinal vesi- cle. The germinal vesicle is a nucleus arrested in prophase I of meiosis; it has a clearly visible n‘ucleolus, finely granu- lated nucleoplasm, and a distinct nuclear membrane. Secondary oocytes are most readily recognized by the presence of a polar body. Secondary oocytes are in meiosis II, and sometimes the chromo- some complement (1N) is organized as a metaphase plate. The remaining half of the chromosome complement has been discarded in the polar body. The struc- tural changes following penetration of the oocyte by a sperm are described in the next chapter (pg. 347). 292 Chapter 8 SUMMARY: Continuity of Life in the Horse x B. Primordial germ cells Similar life cycle for stallion maturation division Ovulation (puberty to senescence) oocyte ‘ maturation division (reductional) FIGURE 8.1. Continuity of life in the horse through perpetuity of germ plasm. All somatic (body) cells and almost all germ cells die, but life is passed in unison from stallion (one sperm) and mare (one ovum) to an individual of the next generation beginning with a fertilized ovum. The letter designations in the fol- lowing account of this marvelous system refer to those on the figure. Countless divisions to form body of mare D. Primary oocyte SfllElzl E. Primordial follicle ~ - A. Fertilized ovum. The fertilized ovum contains all the genetic information from the stallion and the mare. The genetic information is housed in chromosomes of the cell nucleus. In horses, the number of chromosomes is 64 (32 from the sire and 32 from dam). That is, each gamete (sperm or ovum) contains one-half (1N) of the number of chromosomes in a fertil— ized ovum (2N). B. Primary germ cells. At some point, the cleaving cells of the fertilized ovum differentiate into primordial germ cells and somatic cells. In farm animals, pre- sumably including horses, the primordial germ cells, the forerunners of the gametes, are believed to first become detectable in the endoderm of the yolk sac (203). They migrate to the tissue that eventually differentiates into gonads, probably by the end of the embryo stage (Day 40). C. Oogonia. The primordial cells multi- ply, in the fetal gonads by mitosis. This results in massive numbers of oogonia (mare) or spermatogonia (stallion). D. Primary oocyte. Some of the oogonia enter into the first stage of meiosis in the fetal ovaries, beginning at approximately Day 80 of pregnancy. The resulting cells are called primary oocytes and are arrest— ed in the first stage of meiosis (prophase I).'The oogonia remain in the arrested state until atresia or until stimulated in the preovulatory follicle. Stimulation is a function of the ovulatory LH surge in the adult. E. Primordial follicle. At about mid- pregnancy, some of the primary oocytes in the fetal ovary become associated with surrounding cells (forerunners of the granulosa), and the envelope of sur- rounding cells is called a primordial fol- licle. At birth, the ovaries contain thou- sands of primary oocytes in arrested meiosis and enclosed in primordial folli— cles. The pool of primordial follicles at birth serves as the reservoir of gametes for the length of the mare’s reproduc- tive life. ‘ Maternal Aspects of Pregnancy 293 F. Mature follicle. The primordial folli- cles develop sequentially “into primary follicles (one distinct granulosa layer), secondary follicles (more than one granu— losa layer), and antral (fluidfilled) folli- cles. A mature preovulatory follicle devel- ops during each estrous cycle. During the LH surge associated with ovulation, the primary oocyte of the preovulatory follicle is activated and meiosis is resumed. Just after the peak circulating levels of LH, based on studies in nonequine species, the nuclear membrane of the arrested primary oocyte disintegrates. This pro- cess is called germinal vesicle breakdown and signals the resumption of meiosis. G. W. During the first maturation or reduction division of the oocyte, the number of chromosomes goes from 64 (diploid; 2N) to 32 (haploid; 1N), and the resulting cell is called the sec- ondary oocyte. The remaining 32 chromo- somes are discarded in the first polar body (black dot) which is retained Within the zona pellucida of the secondary oocyte. The secondary oocyte is arrested during the second meiotic division in the metaphase stage (metaphase II). H. Ovum. The equine secondary oocyte is discharged at ovulation and now is called an ovum. Meiosis resumes when the ovum is penetrated by a sperm. Upon resumption of meiosis, another polar body is produced and the resulting female nucleus (32 chro— mosomes) and the male nucleus (32 chro- mosomes) expand (pronuclei) and then merge (64 chromosomes). The resulting structure is the fertilized ovum, completing a cycle in the perpetuity of life. The events associated With fertilization are depicted in Figure 9.1 (pg. 347). 294 Chapter 8 w Mwammw\\ “w” \N y M M» w» FIGURE 8.2. Seven ova from oviducts of a non-bred mare on the second day of diestrus. (a) New ovum from current estrous cycle. (b-g) Ova in various stages of degeneration. From (1541). Preparation of equine chromosomes for electron microscopy using gold labeling 1086) and the characterization of the X- chromosome inactivation have been described (1342). Nature of meiotic arrest. The mecha- nisms involved in the prolonged (years) maintenance of the primary oocyte in an arrested stage at the beginning of meiosis have been studied in nonequine species (reviews: 245, 1058, 479). Indications are that maturation-inhibiting substances are involved and that inhibition is blocked by a substance produced by the granulosa cells when stimulated by gonadotropins. The meiotic arrest ceases when a primary oocyte is removed from the follicle and cultured in vitro. Stage of ovulated oocyte. For many years, the stage of oogenesis at the time of ovulation in mares was thought to dif- fer from that of most other mammals. In a description of a few recently ovulated (oocytes in the 1940s, it was concluded that since polar bodies were not found, the cells were primary oocytes (675). Others (1670) reported in the 1960s, how- ever, that newly ovulated oocytes in the mare possessed one polar body, indicating that the first meiotic division was com- pleted before ovulation and a secondary oocyte was ovulated. These authors con- cluded that sperm entry stimulates the formation of an additional polar body as it does in most mammals; it was suggest- ed that the earlier workers may have been studying ova in early degenerative changes and were therefore unable to find the polar body. Subsequent workers also reported finding follicular oocytes that were in the secondary stage (1746, 403). In a more recent study (887), eight oocytes were collected from preovulatory follicles 36 hours after an injection of hCG. All oocytes (except one that was not deter- minable) had a first polar body, demon— strating that the oocyte just before Maternal Aspects of Pregnancy 295 expected ovulation was a secondary oocyte. In conclusion, the equine oocytes, like those of other mammals, are discharged as secondary oocytes and not as primary oocytes (887, 1213). It took over 40 years of sporadic study in this species to move from the assumption that the ovulated gamete is a primary oocyte to the firm conclusion that it is a secondary oocyte. This is an example of the slow progress toward knowledge that is inevitable when funds are not available for a concerted thrust. Cumulus-oocyte complex. The oocyte and its cumulus investment is often called the cumulus-oocyte complex. In one study, the cumulus-oocyte complex averaged 2.5 mm in diameter and size was unrelated to size of follicle (smallest follicle used: 5 mm; 1173). In addition to the maturation of the oocyte during the LH surge, the granulosa cells (cumu- lus cell investment) undergo change. The cumulus cells initially are in tight apposi- tion and later become loose; the two extremes are referred to as compact and expanded. Studies in nonequine species indicate that compact cumulus cells are tightly adherent to the zona pellucida and undergo expansion in response to gonadotropins and follicular steroids (cited in 245). A recent abstract (1789) on in vitro maturation of equine oocytes indi- cated that the addition of FSH or eCG to the culture medium increased cumulus cell expansion; LH was not effective. Studies in cattle (116) indicate that during expansion the granulosa cells produce hyaluronic acid, a carbohydrate belonging to the glycosaminoglycan (GAG) family. Hyaluronic acid may be involved in the expansion or loosening of the cumulus and is believed to play a role in preparing sperm for fertilization. Recent study indi- cates that GAG increases in the tubular genitalia of mares during estrus (1693), as has been described for other species. 296 Chapter 8 8.2. Natural Mating Mating practices vary considerably among farms and breeds, and no attempt will be made to describe mating tech— niques in detail. Readers are referred to other sources (338, 1333, 1362). The most striking aspect of equine mating proce- dures, compared with those of other farm species, is the high degree of managerial preparation and assistance used on some farms. Such activities may include various forms of mare restraint (twitch, knee straps, hindleg hobbles, blindfolds, tranquilizers), protective gear (felt boots for stallions, neck padding for mares), and hygienic preparation (washing of the perineal area, wrapping the tail, washing the penis). During mounting, the atten- dant may maneuver the mare’s tail and the stallion’s penis to assist entry of the penis into the vulva. After many years of tradition, at least one of these practices, washing the penis, has been questioned (237); systematic washing caused the nor- mal flora to be replaced with pathogens. Ejaculation is recognized by a character- istic flagging of the tail or by a urethral pulse which may be felt on the underside of the penis (1362). 8.2A. Number of Mares Mated per Stallion per Season Examination of productivity records of the Hanoverian breed in Germany indi- cated that foaling rate diminished when more than 80 mares were served by natu— ral mating per stallion per season (1084). The decrease was progressive, extending from a foaling rate of approximately 55% when 80 mares were bred to 36% when 170 to 179 mares were bred. These authors cited studies which indicated that a mean of about three services are used per mare per ovulatory period. On this basis, 240 natural services per year were an approximate maximum. This fig— ure agrees with an earlier report on ejac- ulation frequencies and semen character- istics (1577). A goal on breeding farms is to reduce the number of services per ovula- tory period and thereby increase the number of mares that can be mated natu- rally to a stallion per season. This prob- lem is especially severe during the pro— longed estrus associated with the year’s first ovulation. 8.23. Optimal Time Optimal mating or inseminating criteria may be the most fundamental need for efficient reproductive manage- ment. Yet there have been few critical studies on optimal mating regimens; reg- imens are based primarily on tradition and opinion. Most reports were based on field data or were limited by circum- stances that did not permit rigid, random assignments to various test groups—confounding was inevitable. The long and variable interval from onset of estrus to ovulation is an obstacle in mating programs that are designed to minimize both mating frequency and the length of the interval from mating to ovulation. Criteria for predicting the time of ovulation are not adequate (pg. 192). As a result, methods have been devised for hormonal induction of ovulation to reduce the number of matings (pg. 279). Consideration must be given to fertile life of both ova and sperm as a basis for development of mating programs. Factors determining the optimal interval between mating and ovulation are the viable life of ovum, viable life of sperm, and sperm capacitation requirements. The Viable life of sperm governs the maximum length of the interval from mating to ovulation that is consistent with good fertility. The viable life of the ovum and the requirements for sperm capacitation are determining factors in the efficiency of postovulatory mating. The possible dele- terious effects of aged gametes must be considered. Studies in other species indi- cate that embryos that result from sperm or ova approaching the end of their fertile life may undergo a high rate of embryo death or teratologic changes. Apparently, there have been no direct and critical studies on the life span of either Sperm or ova in the mare’s genital tract. Optimum interval between matings. It is generally recommended that the interval between matings should not be more than 2 or 3 days (381). In earlier stud- ies (before 1970), pregnancy rates for mares mated 1, 2, 3, or 4 times during estrus were 40%, 52%, 48%, and 44%, respectively (85); no relationship was found between the number of times a mare was mated per estrus (range of means on 11 farms: 1.1 to 2.3) and the mean number of estrous periods before pregnancy occurred (792). In this regard, a pregnancy rate of 53% (434 mares) was obtained by mating only once during estrus; mating two or more times resulted in a pregnancy rate of 56% (723 mares; 1638). In contrast, in a more recent study (1720), mares inseminated frequently had higher pregnancy rates than mares insem- inated only once; it was concluded that the single-insemination method cannot be rec- ommended in the absence of a reliable method for predicting day of ovulation. In another trial (1718), artificial insemi- nations were done every other day throughout estrus, beginning on the first or second day of estrus; mares that became pregnant were inseminated more frequently (P<0.05) than those that did not (3.4 versus 2.8). By the same token, estrus was longer for mares that became pregnant. A similar insemination protocol was used during the spring transitional period in 64 mares; mares were insemi- nated as many as 34 times because of the long estrus that often is associated with the first ovulation of the year. First cycle pregnancy rates were positively corre- lated With the duration of estrus (i.e., number of inseminations). Repeated inseminations apparently were not detrimental to pregnancy establish- ment, indicating that chronic inflamma- tory processes did not develop. The Maternal Aspects of Pregnancy 297 authors noted that one mare in the tran- sitional period was inseminated 27 times and one 34 times—both became pregnant. The results of earlier studies (no advantage in mating more than once per estrus) and those of the more recent stud— ies (increased pregnancy rates with more than one insemination per estrus) seem contradictory. It may be important that the earlier work involved natural matings and the more recent work involved artifi- cial insemination. For example, repeated artificial inseminations would increase the reservoir of sperm available at the time of ovulation. It is especially difficult to reconcile the divergent results without specific information on the relationships between day of insemination or mating and day of ovulation. Critically designed and objective experimentation will be needed to develop recommendations for optimal mating programs. Optimum time when only one insemi- nation is used. It has been concluded (128) that the survival time of sperm in the mare’s genital tract is 5 or 6 days. Pregnancy rates were good in Thorough- breds mated as early as 8 or 9 days before the end of estrus (Table 8.1); 90% of the ovulations occurred on the last two days of estrus. A report from Yugoslavia (841), on the other hand, indicated a rapid drop in pregnancy rates as the number of days before ovulation increased (Table 8.1). In a recent study (1824), there was no signifi- cant difference among days for a single artificial insemination done 1 to 6 days before ovulation (Table 8.1). Mares inseminated with fresh semen the day before ovulation had a nonsignificantly higher pregnancy rate. When data were combined, insemination 1 to 3 days before ovulation resulted in a significantly higher pregnancy rate than insemina- tion 24 days before ovulation (76% ver- sus 45%). Nevertheless, the 45% rate for insemination—to-ovulation intervals of 24 days is substantial. Four of nine mares became pregnant when insemi- nated 6 to 8 days before ovulation; the 298 Chapter 8 TABLE 8.1. Effect of Time of a Single Breeding on Percentage of Pregnancies Breed (reference) Day of ‘breeding in‘ relation to estrus or ovulation Day of estrus (teasing 3x/week) 1 2 3 4 5 6 7 Thorough- % 63% 65% 64% 56% 39% 28% 20% breds (290) No. 38 160 239 113 46 14 5 No. days from mating to end of estrus >9 9 8 7 6 5 4 3 2 1 0 Thorough- % 0% 50% 56% 50% 63% 63% 60% 58% 52% 61% 50% breds (128) No. 5 8 18 19 41 63 67 27 18 6 Thorough- % . . . . . . 30% 20% 31% 56% 66% 56% 51% 42% 37% breds (1638) N0. . . . . . . 8 16 30 73 335 199 134 14 No. days from mating to ovulation —6 —5 —4 —3 —2 —1 0 +1 +2 Bosnian— % . . . . . . 0% 33% 54% 92% 55% 25% 0% mountain (841) N0. . . . . . . 2 11 67 98 24 5 ------- (1155) % . . . 10% 40% 52% 65% 60% 60% 54% No. . . . 30 20 124 164 256 13 Ponies (1824) % 44% 63% 33% 60% 65% 89% 52% 6% No. 9 8 12 51 9 94 70 longest interval in mares that became pregnant was seven days. These results support the conclusion that equine sperm in the mare reproductive tract sometimes have a long survival time—as long as one week. In contrast, sperm in other large domestic species retain their fertilizing capacity in the female tract for only 1 or 2 days (1531). Long life of sperm in the mare’s reproductive tract is compatible with a long estrus (e.g., 7 days) with ovu- lation toward the end and occasionally after the end of estrus. Despite the long sperm survival time in at least some circumstances, the above rates indicate that a prolonged (24 day) interval from insemination to ovulation does decrease the pregnancy rate. However, further study using more mares will be needed to adequately test the hypothesis that natural mating or artificial insemination with fresh semen one day before ovulation is more effective than mating two days before ovulation. The practical problem, of course, is to clinically differentiate Day -1 from Day -2. If hCG is used, mating could be done the day after hCG treatment since most mares ovulate two days after hCG treatment (pg. 279). Practitioners have indicated that the optimal mating-to-ovulation interval is stallion dependent. In the recent study (1824), there was no indication that the reduced pregnancy rate for insemination— to-ovulation intervals of 24 days was attributable to certain stallions. However, when data were partitioned on a per stal— lion basis, the number of observations was small, and the possibility that sperm from some individual stallions had a longer life was not resolvable. In contrast to studies with fresh semen, studies with frozen/thawed semen have shown that insemination one day before ovulation results in better pregnancy rates than insemination two days before ovulation (pg. 335; review: 1824). Efficiency of postovulatory mating. Presumably, efficiency of postovulatory insemination is dependent upon the life span of the ovum and the requirements for sperm capacitation. It is well estab— lished that pregnancy can occur in mares inseminated after ovulation, using either natural mating or artificial insemination with fresh or frozen/thawed semen (review: 1824). One practitioner used a single natural mating on the day of ovula— tion and reported a pregnancy rate of 217/355 (61%; 172). In a recent study (1824), pregnancy rate for postovulatory insemination on Day 0 was 24 percentage points lower than for preovulatory insem— ination on Days -3 to —1 (52% versus 76%; Table 8.1). These results are consistent with those of other workers who obtained a rate of 11 of 34 (32%) for mares insemi- nated 0 to 24 hours after ovulation, com- pared to 18 of 30 (60%) for 72 to 0 hours before ovulation (1204). Pregnancy rates for six-hour intervals from ovulation to insemination on Day 0 decreased progres- sively, with a significant decrease occur- ring between the 0-t0-6 hour group and the 18-to-24 hour group (Figure 8.3). These results are consistent with those of previous controlled studies of postovula- tory insemination (1204, 855, 912); however, small mare numbers were used in the previous studies. Insemination on Day 1 resulted in an extremely low pregnancy rate (6%), and all pregnancies were from insemination during the first six-hour period of Day 1 (hours 24 to 30; 1824). If ovulation detection is done every two days, 50% of the mares on the detected day of ovulation would be, on the average, at Day 0 and 50% at Day 1. That is, insemination on the day of detected ovu- lation with examinations done every two days would be expected to produce a Maternal Aspects of Pregnancy 299 Postovulatory insemination (11/14) 80 (13/20)? No. pregnancies/ No. inseminated g; 60 (D E 3' 40 I: (B C O) o a 20 (0/10) (0/12) (0/9) C)No. embryonic losses 0 3 3 3 4 1 6-12 18-24 30-36 42-48 Interval from ovulation to insemination (hours) FIGURE 8.3. Effect of postovulatory insemination on pregnancy rates and embryo-loss rates. Difference (P<0.05) in pregnancy rates among the four six—hour intervals on Day 0 (0-6, 6-12, 12-18, and 18-24 hours). First significant decrease (P<0.05) in pregnancy rates was between hours 0—6 and 18-24. No significant differences in embryo-loss rate among intervals. Adapted from (1824). result represented by the average for the Day 0 and Day 1 groups (average of 52% and 6% = 29%). Results of the above-cited studies indi- cated that equine ova retain maximal Via- bility for approximately 12 hours after ovulation, in common with the other large domestic species (1531). There are no criti- cal data on the time required for capacita- tion of sperm in horses. Results of the recent study (1824} indicated that postovu- latory insemination was more effective for sperm from one stallion than from another, but further study is required. In this regard, it has been reported that the development of sheep ova in vitro is dif— ferentially affected by the ram from which sperm were collected (545). The embryos from postovulatory insemination. Embryonic vesicles were detected earlier and mean diameter on day of first detection was greater for mares inseminated before ovulation than 300 Chapter 8 for mares inseminated after ovulation (Figure 8.4; 1824). These end points can be assumed to be interrelated since larger vesicles are detected earlier. The smaller difference at Day 11 was attributed to failure to detect the smaller vesicles at Day 11 in the group inseminated on Day 0, contrasted with detection of almost all vesicles on Day 11 in the group insemi- nated before ovulation. Growth rates of the vesicles over Days 12 and 14 were similar between the preovulatory and Day 0 groups. The differences in vesicle diameters between groups were based on the use of the day of ovulation (Day 0) as a refer— ence for defining the day of pregnancy. Actual age of the conceptus on a given postovulatory day, however, was probably as much as 24 hours younger for the Day 0 group than for the preovulatory group; in the Day 0 group, capacitated sperm were not immediately available for fertilization when ovulation occurred. The difference in diameter between vesicles in the two groups was equivalent to approxi- mately one day’s growth and can be attributed, at least in part, to the delay between ovulation and insemination and the requirements for sperm capacitation in the Day 0 group. Embryo loss in associ- ation with postovulatory mating is dis- cussed elsewhere (pg. 537). Time of mating versus sex of foal. Chinese workers have published consider- able data indicating that time of mating relative to ovulation has a profound effect on gender of foals. This belief apparently originated in ancient times and has been studied by several groups of Chinese workers in modern times (review: 1299). In a recent study (1299, 1300), mares were given a single insemination 24 hours before ovulation or within four hours after ovulation. For mares inseminated before ovulation (n=294), 88% of the foals were female, whereas for mares insemi- nated after ovulation (n=570), 79% of the foals were male. The authors concluded that Y-bearing sperm travel faster but Day of insemination & embryo diameter 25 M O —L 01 Diameter (mm) 3 Number of days after ovulation FIGURE 8.4. Effect of day of insemination on diameter of embryonic vesicles. Significant differ- ence between groups inseminated before versus after ovulation. Number of observations is given in parentheses. Adapted from (1824). have a shorter life span. The birth of a large proportion of fillies when mares are inseminated before ovulation is not com- patible with the approximately 50:50 gen- der ratio obtained on breeding farms in other countries, even though most mat- ings are done before ovulation. In a sur- vey of a large Thoroughbred farm in Canada, for example, the sex ratio was 52% male to 48% female (n=1704; 752); this ratio does not differ significantly from 50:50. In a limited experiment in 65 pony mares (600), 44% inseminated before ovulation and 59% inseminated after ovulation had female fetuses, fail— ing to confirm the Chinese results. Nevertheless, the Chinese data have been consistent among studies and have involved large numbers. Curiously, there are reports that artificial insemination in humans just before or at the time of ovulation results in a predominance of males, whereas insemination two or more days before ovulation results in a predom- inance of females (review: 950); these results are in the same direction as reported for horses in China, but the sub- ject in humans is controversial. Determining Parentage. Equine blood- typing is an accurate method (see 1566 for probability calculations) and has been used to identify individuals and establish parentage for more than 30 years (cited in 1566). Blood-typing may be required in association with artificial insemination or embryo transfer; the spe- cific rules vary among registries. Owners and registries may question paternity of a foal and require blood-typing when the mare was bred to more than one stallion, especially if the interval between change of stallions was less than 45 days. The effect of length of interval between use of different stallions on parentage was stud— ied in 108 mares (238). The interval between use of different stallions and the percentage of mares pregnant to the first stallion were as follows: 1 to 7 days, 33%; 12 to 26 days, 4%; 29 to 45 days, 0%. 8.20. Deposition and Transport of Sperm in Mare Genitalia Site of deposition. Presumably, the equine ejaculate is deposited directly into the uterus. However, the only direct indi- cation of uterine deposition is based on the ultrasonic finding of large fluid pock— ets in the uterus immediately after natu— ral mating (614). The cervix is quite expandable during late estrus; a hand sometimes can be inserted through it to palpate the endometrium (644). The effect of the extent of relaxation of the cervix on the proportion of the ejaculate deposited into the uterus has not been studied. It is unknown whether there is a relationship between the fertilization rate and the proportion of an ejaculate deposited at various sites. Hypotheses on site of sperm deposition and pregnancy rate and the interrelationship of cervix and penis during copulation merit direct testing in mares. Sperm transport. Sperm transport has received limited study in mares but has Maternal Aspects of Pregnancy 301 received much attention in other farm species. The following brief account of sperm transport in nonequine species is based on a review (692). Sperm transport failures are believed to account for a major portion of fertilization failures. Sperm can be transported efficiently only during estrus or after estrogen injection in ovariectomized animals. Transport to the oviduct occurs within a few minutes for a minor portion of the deposited sperm, but these early arrivers probably do not fertilize the ova. Smooth muscle contraction, ciliary action, fluid currents of the reproductive tract, and flagellar activity of sperm are the primary propel- lors. Sperm transport in nonequine species can be improved by adding to the semen or administering certain com- pounds such as PGonc, oxytocin, or estra- diol. Most sperm are lost from the repro- ductive tract within a few hours, probably through external discharge. In mares, up to 2% of sperm were recovered 12 hours after inseminating 30 ml of fresh semen (1231). More sperm were in the oviducts in mares inseminat- ed during estrus than during diestrus, which agrees with studies in other species. Backflow through the cervix probably occurs in mares and contributes to sperm elimination (122); large numbers were found in the vagina four hours after insemination into the uterine body. Results of a recent study (120) indicated that sperm are present in the equine oviduct within two hours after insemina— tion; the number increased by four hours and decreased by six hours. The decreased numbers at six hours may have been due to gradual elimination of sperm from the oviduct and cessation of further transport from the uterus to the oviducts. Sperm reservoir. It is believed that the cervical crypts serve as a reservoir for sperm in those species (ewe, cow, goat) in which sperm are deposited into the cervix (692). However, in the pig, in which a por- tion of the semen is believed to be deposited into the uterus, the oviduct at 302 Chapter 8 the uterotubal junction is believed to act as a reservoir based on prolonged main— tenance of high sperm numbers (1705). In mares, deep edematous longitudinal folds are present at the uterotubal junc- tion during estrus (243). It was suggested that the deep furrows may constitute the pathway to the ampullary region and act as storage sites, similar to what has been reported for pigs. Much of the fold- ing and edema disappear after ovulation. The uterotubal reservoir is thought to be the main site of capacitation and selec— tion of sperm in mares and pigs; in the other farm animals, these functions occur during transport through the cervix and uterus. The uterotubal junc- tion in mares also may act as a filter to prevent excessive sperm numbers from reaching the ampulla. Passage of sperm from an oviduct into the peritoneal cavi- ty or transabdominal migration between oviducts apparently has not been inves- tigated in mares and could be the subject of a specific research project. In other species, such as cattle, transperitoneal migration of sperm has been demon- strated. 8.3. Transport of Ova in Oviducts One of the enigmas in reproductive physiology is the paradoxical ability of the isthmus of the oviduct to transport sperm toward the ovary and the site of fertilization while transporting the fertil- ized ovum in the opposite direction. Transport of ova and sperm involves at least four primary factors (214): 1) pro- grammed contractions of smooth muscle layers, 2) rate and direction of the beat of cilia, 3) luminal fluid currents, and 4) sperm motility. The oviduct is a very complex tubular system. Because fore— most authorities in this research area acknowledge that our understanding of gamete transport is in its inception, no attempt will be made to summarize cur- rent work in the nonequine species. 8.3A. Time of Entry into Uterus Fertilization of the ovum occurs in the ampulla of the oviduct in mares (1541 ), just as it does in other species. The time of entry of the fertilized ovum into the uterus in nonequine farm animals is approximately Days 2 to 4 (785). Trans- port through the ampulla is relatively rapid, but arrest occurs at the junction of the ampula and isthmus for approxi- mately two days. The ova then enter the isthmus where they remain until enter- ing the uterus under the regulatory influ- ence of the uterotubal junction (665). In association with the development of a technique for recovery of ova, observa- tions were made on the time of entry of fertilized equine ova into the uterus (1170). Ova were recovered from the uterus on Day 6 after ovulation in 47% of 36 uterine flushings, but none were recovered from nine flushings on Day 5. Similar results involving oviductal or uterine flushing have been reported (197, 562, 198, 675), although recently embryos were recov- ered on Day 5 in 2 of 8 mares (745). It can be concluded that fertilized ova in mares usually enter the uterus on Day 6. At the time of its arrival in the uterus, the con— ceptus is usually a morula and occasion- ally an early blastocyst (pg. 350). Insertion of rabbit ova into the uterus of mares two days before recovery attempts at Day 5 apparently hastened the transport of the fertilized equine ovum into the uterus (1170). However, a recent study (562) found that placing fluid medium alone into the uterus on Day 4 hastened oviduct transport. It was also noted that the Day 14 pregnancy rate was reduced in mares that received uterine infusion, raising questions about the advisability of postovulatory intrauterine treatments in bred mares. Administra- tion of PGan, however, did not increase the Day 5 embryo recovery rate by uter- ine flushing (745). 8.3B. Retention of Unfertilized Ova in Oviducts An unusual phenomenon in mares is the retention of unfertilized ova in the oviducts. This important discovery was made in 1966 in South Africa (1670) and has attracted attention among specialists in oviductal function (683). In the original work (1670, 1667), as many as 10 ova per mare in all stages of cytolysis were found in the oviducts. The ova were retained for as long as seven months or more and eventually degenerated. Many subsequent studies have con- firmed the phenomenon of retention of unfertilized ova (listed in 1641). Retention of ova in the oviducts of bred and nonbred pony mares was demonstrated (1541) by the following results: 1) the recovery of ova as late as 18 days after the end of estrus, 2) the recovery of ova from both the ovulatory and nonovulatory sides, and 3) a large mean number of ova recov- ered per mare (means: 3.8 for 17 nonbred mares and 1.9 for 32 bred mares). Similarly, in an extensive slaughterhouse study (1172), an average of four ova were found per mare during the season of ovar- ian activity. The retention of ova occurred primarily in the middle one—third of the oviducts in both bred and nonbred mares (1541) or in the region of the junction between ampulla and isthmus (520). micture of retained ova. The degen- erative stages in retained ova reportedly (1670) involved condensation of cytoplasm into a compact mass, followed by pas- sage of yolk material through the cell membrane into the perivitelline space. The degenerative stages, based on com- parisons among retained ova (1670), do not seem to agree with those of the degenerative stages of ova of known ages (201). It was concluded that estimating the age of a retained ovum by its stage of degeneration is unreliable. In another study (199), the cell membrane of old ova was broken down and debris was dis- persed throughout the zonal cavity. The Maternal Aspects of Pregnancy 303 degree of fragmentation of the Vitellus ranged from a single large cytoplasmic mass with little debris in the perivitelline space to complete fragmentation of cyto- plasm resembling a morula. In another study (473), freshly ovulated ova were dis- tinguished from retained ova by the pres- ence of a variable number of cumulus cells and a distinct perivitelline space. Many retained ova consisted only of a zona pellucida enclosing clusters of gran— ules. Comparisons of a freshly ovulated ovum and degenerating retained ova are shown in Figure 8.2. Parthenogenesis. If one is unaware of the ova-retention phenomenon, the frag- mentation of old retained ova can be con- fused with normal cleavage. In this regard, parthenogenetic cleavage (sponta- neous) was reported to occur in 9% of unfertilized horse ova (1670). Although such spontaneous cleavage has been reported for many species, it has been pointed out that certain cases of degener- ative fragmentation can be mistaken for parthenogenetic development (1070). More recently, it was reported that partheno- genetic division of unfertilized horse oocytes appears to be rare both in vitro and in vivo (1851). Mechanisms. Several suggestions have been made and a few hypotheses have been tested on the mechanism causing dif- ferential or selective retention (unfertil- ized ova) or passage (fertilized ova). The following questions should be considered: 1. Is the mechanism local? The first and as yet unresolved question concerns local versus bilateral or systemic mecha- nisms. There are indications that some old ova pass into the uterus along with the fertilized ovum (1541). Significantly fewer old ova were recovered from the oviducts in mated mares than in nonmat- ed mares. In addition, more old ova were recovered from the uterine one-third of the oviducts in mated mares than in non- mated mares, providing additional indi- cations of movement of some old ova toward the uterus in mated mares. It was 304 Chapter 8 confirmed in a uterine flushing experi— ment on Day 6 (1801) that some unfertil- ized ova do enter the uterus in mated mares; 26 unfertilized degenerative ova were recovered along with 62 embryos. Diameter of all old ova was about 150 um and all ova had an intact zona pellucida. The difference between sides (ovulatory and nonovulatory) in number of old ova in the oviducts was not significant (1541); this provides rationale for the hypothesis that the mechanism of passage of fertil- ized ova is not entirely unilateral, and this hypothesis needs to be tested. 2. Are ova trapped by intraoviductal masses? The granulosa cells near the time of ovulation apparently secrete a basophilic filamentous mucoid substance which coats the follicular antrum (1667). The substance enters the oviduct with the ovum and embeds the ovum and corona radiata cells in a mucoid sticky mass. Thus, the recently ovulated ovum was described as encased in a large irregular gelatinous mass of follicular origin; the ovum became separated from the mass on the second day. Globular gelatinous masses were found in 76% of 424 oviducts, but based on histologic study, the masses were thought to consist of desquamated tubal epithelium (1181). The masses within the oviduct have been examined by light and scanning electron microscopy (1641). They were found in 16 of 24 oviducts and were grayish—white and easily recognized under a dissecting scope. It has been noted that the masses can be seen by the naked eye when oviducts are held against a light source (520). Some were threadlike (1 to 1.2 mm by 3.5 to 7.5 mm), and some were globu- lar (1 to 1.5 mm diameter; 1641). All of the masses were in the ampullary portion, and most were near the junction with the isthmus. Some (44%) of the masses were attached to the mucosa. They consisted of bundles of fibers, and it was recom— mended that they be called fibrous masses. There is disagreement on the origin of oviductal masses. Regardless of origin, it is not known Whether the mass- es play a role in retaining unfertilized ova in the oviducts, but it was concluded (1181) that ova were retained more frequently in oviducts that contained these globular masses. On the other hand, retained ova are sometimes located distal to the mass- es (520). Late entry. Oviductal masses were reported to be more common in mares that were seven years old or older (59%) than in mares less than seven years (24%; 1863). Occasionally, large masses occupied the entire lumen and appeared to distend the lumen. It was the author’s opinion that the masses could occlude the lumen and interfere with embryo transport. The masses were thought to be nonoviductal in origin; con- trary to other reports, the masses were not attached to the oviductal epithelium, and there was no indication of desqua- mated tubal cells. 3. Is there a local effect of side of ovu- lation or presence of the corpus luteum? Oviductal embryo transport is not depen— dent, in an absolute sense, on side of ovu- lation; transfers of oviductal embryos to the contralateral side have resulted in uterine pregnancies (1255, 197). 4. Do the degenerating ova truly repre— sent retention? It has been suggested that the excess ova originate from the rupture of small follicles near the ovulation fossa due to the increasing pressure of the large preovulatory follicle (376). By using marked ova, however, direct evidence was obtained that the excess ova result from oviductal retention (200). Furthermore, fertilized ova treated to become arrested at the 2- or 4—cell stage were retained (197), indicating that additional cleavage is needed before the embryo is recognized by the retention mechanism. 5. Is retention due to differences in ova surface characteristics? The possibility that trapping results from morphologic surface differences between fertilized and nonfertilized ova has been examined (199) but not substantiated. An untested possi- bility is that the softening of the zona pel- lucida is greater for unfertilized ova, causing them to be less responsive to pro- pelling forces (785). 6. Are humoral agents produced by the fertilized ova? The following experimen- tal results in nonequine species indicate that the fertilized ovum may be capable of controlling its own passage through the oviduct by producing a regulatory agent: 1) The contraction of the rabbit oviduct is greatest in the region of the ovum (214); 2) The oviductal ovum in the pig and in certain rodents contains enzymes normally associated with ste- roidogenesis (406); and 3) Rabbit and rat blastocysts contain measurable quanti- ties of steroids (progestins) as early as Day 5 (406). The following indirect, but weak, indi- cations from research in horses are com- patible with the postulate that the oviductal embryo produces a smooth muscle stimulant: 1) The longer stay and greater development of the equine con— ceptus in the oviduct (pg. 302), compared with that of other species, may favor the production of a humoral agent while the conceptus is still in the oviduct; and 2) In vitro studies have shown that the equine conceptus can produce steroids as early as Day 8 (pg. 66), and further study may demonstrate even earlier steroido- genic capabilities. The hypothesis that the oviductal embryo, but not the unfer- tilized ovum, produces a local stimulant of contractile activity of the oviduct mus- cle should be tested. Perhaps the sub- stance produced by the conceptus for oviductal passage is the same as the sub- stance that stimulates myometrial con- tractions in association with embryo mobility (discussed in next section). Prostaglandins are good candidates because they are potent stimulants of smooth muscle, and the production of PGF20L and PGE2 by the intrauterine con- ceptus has been demonstrated (pg. 441). Late entry. Prostaglandin E2 has been implicated in transport of equine oviduc- tal embryos (1875). Continuous infusion of Maternal Aspects of Pregnancy 305 PGE2 into the oviducts on Days 3 and 4 resulted in recovery of embryos from the uterus on Day 4, two days sooner than expected (treated, 6 of 11 in uterus; con— trols, O of 11). The authors cited unpub- lished data that PGE2 receptors are pre- sent in the oviducts, and uterine embryos inserted into the oviducts on Day 2 were transported into the uterus by Day 4. The solution to the perplexing question of differential ova transport in the mare has important comparative ramifications and emphasizes, once again, the useful- ness of the mare as a research model in reproductive biology. In this regard, a recent report indicated that fertilized ova of hamsters arrive in the uterus earlier than unfertilized ova (1184), and trapping of unfertilized ova has been discovered in a species of bats (1315). This example illus- trates that a dramatic mechanism in one species can lead to discovery of a similar, sometimes more subtle, mechanism in other species (comparative research). 8.4 The Uterus and Physical Embryo-Uterine Interactions The phenomena of dynamic physical embryo-uterine interactions include embryo mobility (traversing the full length of the uterus many times each day), fixation (cessation of mobility), and orientation (rotation of the vesicle so that the embryonic pole is located opposite the mesometrial attachment). These phenom— ena have been reviewed (590, 587). Uterine contractions, tone, and secretions likely play roles in these interactions. This sec- tion describes the occurrence of these phenomena on Days 10 to 20. 8.4A. Embryo Mobility Transuterine migration has long been known to occur in mares because the con- ceptus attaches in the uterine horn oppo- site to the side of ovulation about 50% of the time (1838; review: 575). In 1983, 306 Chapter 8 however, it was discovered in horse mares, through ultrasonic monitoring, that transuterine migration is not a sim- ple one—way passage (581). Instead, the embryonic vesicle bypasses its eventual site of attachment 10 to 20 times per day while traversing the full length of each uterine horn and the uterine body (Figure 8.5). Mobility of the embryonic vesicle has been characterized for pony mares (946), horse mares (584), and jennies (195). The characteristics do not seem to differ among the two types of mares and the two species, except in regard to the day of cessation of mobility (pg. 309). Time of occurrence. Mobility already is occurring when the embryonic vesicle is first detected by transrectal ultrasonogra— phy on Days 9 or 10. The nature of mobil- ity from the time the blastocyst enters Embryo mobility FIGURE 8.5. Example of sequential location changes for a Day 14 conceptus. The numbers are the number of minutes the vesicle spent in each uterine segment. Sometimes the vesicle passed directly from one uterine horn to the other, whereas at other times it traversed the full length of the uterine body. From (584). the uterus (Day 6) to the day the concep— tus first reaches the uterine body is unknown. Standardized mobility trials were devised (584) to quantitate the extent of mobility; the vesicle is located every five minutes for two hours and is assigned to one of nine estimated loca- tions (three approximately equal seg- ments of each uterine horn and the uter— ine body). The mobility on Days 9 and 10 is limited; the vesicle spends more time (>60%) in the uterine body (946, 584). Mobility increases over Days 9 and 10 to reach a maximum on Day 11 or 12 and moblity is then maintained to approxi- mately Day 15 in ponies (Figure 8.6) and Day 16 in horses and jennies. During this maximum mobility phase, the vesicle spends more time in the uterine horns than in the uterine body. Entry of a vesi- cle into a uterine horn from the body appears to occur randomly. No significant preference was detected in the following statistical comparisons: 1) right versus left horns, 2) the horn ipsilateral to the corpus luteum versus the contralateral horn, 3) the horn that the vesicle had entered previously versus the opposite horn, and 4) the horn of eventual site of fixation versus the opposite horn (946, 195). Nature of movements. The mean rate of movement of the embryonic vesicle during the mobility phase was estimated to be 3.4 mm/minute, using the cervix or uterine cysts as fixed points of reference (584). Movement rate is highly variable, however, and rapid point-to-point move- ment is seen occasionally. For example, in one mare the vesicle moved from a point adjacent to the cervix to the caudal por- tion of a horn in approximately two min- utes. In another, the time taken to move from the tip of a horn to the caudal seg— ment was only a few seconds. Major movements of the vesicle give the appear— ance of a gently flowing sphere immersed in a slow—moving stream of water. This is illusionary because ultrasonic observa- tions indicate that the uterine lumen nor— mally does not contain detectable quanti- Embryo mobility (“=7) '0 12 c bC, “\ b0 9 Segment<——> ‘1 / Segment ‘, Location change (number) 9 10 11 12 13 14 Number of days from ovulation ties of free fluid (614). When the vesicle is observed continuously, to-and-fro move- ments, encompassing several seconds, are seen. These are prominent when a large vesicle (e.g., Day 14 vesicle) is monitored while the uterus is Viewed in a longitudi- nal plane, as is seen in the uterine body when using a linear-array ultrasound transducer. The to-and-fro movements also can be seen in the horns by rotating the probe so that a longitudinal View is obtained. They are superimposed on the major embryo location changes that occur between portions of the uterus. Location changes within a uterine horn are more likely to proceed progressively when the vesicle is moving in the caudal direction, whereas location changes within the body are more likely to be progressive when the vesicle is moving cranially (947, 587). The reason for this dichotomy in progres- sive movement is unknown. Perhaps less intraluminal resistance is offered by the horns when the vesicle moves caudally and Vice versa for the uterine body. Alternatively, there may be an inequality between horns and body in the extent of uterine contractions moving in one direc- tion compared to the other. Maternal Aspects of Pregnancy 307 FIGURE 8.6. Mean number of times the embryonic vesicle changed locations over two hours on various days of early preg- nancy. Data are from two-hour trials in which location was determined every five minutes in each mare on each day. The uter— ine segments were the body and three approximately equal por— tions of each horn (seven seg- ments). Mobility on Days 9 and 10 was limited but increased between Days 9 and 11 and reached a plateau of maximum mobility on Days 11 through 14. Mobility was not detected in most mares (5 of 7) on Day 15 and in the remaining two mares on Day 16. Within each line, means without a common super- script are significantly different. Adapted from (.946). Mechanisms involved in mobility. Factors that likely favor mobility of the conceptus include its spherical shape, the acellular capsule that envelops it and increases its rigidity, and the longitudinal endometrial folds. Uterine contractions provide the propulsive force (Figure 8.7). A single intravenous injection of clen- buterol, a B-sympathomimetic agent that suppresses uterine contractility, reduced the number of location changes (947). Expansion and compression of larger vesicles on Days 13 and 14 were observed when the vesicle was in the uterine body or was Viewed longitudinally in the horn (587). These periodic compressions lasted 5 to 14 seconds, and were accompanied by the to-and-fro movements. They were distinguishable from the vesicle compres- sion that was caused by pushing the transducer against the uterus or by move- ment of the intestines. Continuous longi- tudinal observations indicate that uterine movements, which give the endometrium an active flowing appearance, are present at all stages of the estrous cycle and during early pregnancy. However, con- tractile activity is greatest during the phase of embryo mobility (pg. 313). 308 Chapter 8 FIGURE 8.7. Movement of a Day 14 vesicle (EV) over a uterine cyst. The number in the lower right corner of each image is the number of minutes from the beginning of the sequence. At the initial determination (min: 0), the vesicle was in the caudal portion of a horn. The cyst is shown (upper central image) on the ventral aspect of the uterine body 50 mm from the corpus-cornual junction. The relationship of the vesicle to the cyst at each sequential determination at one minute intervals is shown. This sequence demonstrated that sometimes considerable propulsive force is involved in embryo mobility—a force capable of compressing the vesicle when an imped- iment is reached. Adapted from (584). Simulated embryonic vesicles were prepared from the fingertips of rubber surgical gloves filled with water (587). The simulated vesicles were comparable in diameter to a Day 12 embryo. The simu— lated vesicles were mobile, but their rate of movement was significantly less than that of viable conceptuses, indicating that distention due to the embryonic vesicle is not in itself the stimulus for contractions. Furthermore, the simulated vesicles spent most of the time in the uterine body on the day of insertion, in a similar man- ner to that described previously for Day 9 and 10 embryonic vesicles. This finding raises the possibility that in normal equine pregnancy the large Day 12 to 14 embryonic vesicle may provide some active stimulus that increases the magni- tude of uterine contractions, increases the extent of mobility, and increases the number of entries of the vesicle into the uterine horns. The conceptus does pro- duce estrogens (pg. 66), but a test of the hypothesis that estradiol plays a role in embryo mobility was not supported; exogenous estradiol did not increase the extent of mobility on Day 10 or Day 12 (194). However, mobility of the embryonic vesicle decreased in mares without a source of luteal or exogenous proges- terone (850). The control of embryo mobili- ty and uterine contractions are probably interrelated (pg. 313). It is not known whether selective movement of an embryo into the uterus from an oviduct involves the same factors or substances that stim- ulate intrauterine mobility. 8.4B. Fixation The day of fixation has been defined as the first day that no location changes of the embryonic vesicle were detected dur- ing a two-hour mobility trial, or when mobility trials were not done, the first day that the vesicle was consistently in the same location over many consecutive days (580). Day of occurrence. In ponies, many studies have found that the mean day of fixation was Day 15 (review: 195). In a comparison of nonlactating ponies and horses (590), the vesicle fixed on a mean of Days 15 and 16, respectively, and the diameter of the vesicle on the day of fixa- tion was equivalent to one day’s greater growth in the horses (Figure 8.8). In a comparison of jennies and ponies (195), the mean day of fixation was approximately one day later in jennies (Day 15.6) than in ponies (Day 14.7). The embryonic vesi- cle was first detected on Day 10 in more ponies (8 of 9) than jennies (3 of 9), and the diameter of the vesicle tended (P<0.08) to be smaller in jennies. In sev- eral experiments involving daily exami- nation of hundreds of nonlactating pony mares until Day 40, the latest day of fixa- tion for singleton embryos has been Day 17, and movement of the embryonic vesi- cle from one horn to another has not been Maternal Aspects of Pregnancy 309 Day of fixation 03 0 Percent mares N O —l D 13 14 15 16 17 18 Number of days from ovulation FIGURE 8.8. The day of fixation was defined as the first day that the embryonic vesicle was in the same uterine segment during every daily examination. The difference between ponies and horses was sig- nificant for mean day of fixation and for diameter of embryonic vesicle on the day of fixation. The mean day of fixation was approximately one day later in horses (Day 16) than in ponies (Day 15), and the vesicle of horses was equivalent to one day larger on the day of fixation (19.5 i0.7 versus 23.3 i1.3 mm). Adapted from tabulated data (590). detected in any mare after the day of fixa- tion, except in association with impend- ing embryo loss. These findings do not substantiate a previous report (132), based on transrectal palpation, of frequent movement of the entire conceptus between horns after Day 20. Mechanisms. It has been postulated that fixation is a function of increasing size of the embryonic vesicle, combined with increasing intraluminal resistance to mobility caused by the development of uterine tone (580). When these two factors (embryo diameter and uterine tone) reach a critical point, mobility ceases. There are no indications that the conceptus slows before fixation; extent of mobility was not different between one day and two days before fixation (947). The temporal relationships between vesicle diameter, uterine tone, and the 310 Chapter 8 day of fixation in nonlactating horse mares are shown (Figure 8.9; 587). Both vesicle growth and uterine tone increased over Days 11 to 16, and the mean day of fixation was Day 15.8. Failure of the vesicle to expand when viewed in a cross-sectional plane of the uterine horn between Days 16 and 21 was thought to be caused by the high degree of uterine tone reached on Day 16 and the resulting oblong expansion in the uterine lumen (pg. 366). Because increas— ing uterine turgidity probably plays a role in fixation, the mechanisms that control turgidity probably control the time of fixation. Summary of fixation mechanism. The following indicators are consistent with the postulate that fixation is a function of increasing size of the embryonic vesi- cle in relation to decreasing diameter and increasing tone of the uterine horns: 1. Uterine contractile activity contin- ues beyond the day of fixation (pg. 313), indicating that fixation is not caused by cessation of contractions; 2. The embryonic vesicle fixes a day later in horses and jennies than in ponies, attributable to similar size of con- ceptus in horses and ponies (pg. 413) and a Uterine tone & fixation (n=10) O.) 01 Uterine tone 0) O M 01 Uterine tone (coded) M 0 Embryonic vesicle diameter (mm) d 01 _s_ ,I’l' Mean 'day of fixation _L O 11 14 12 13 15 16 17 18 Number of days from ovulation Embryonic vesicle 'I' I I larger uterus in horses. In ponies and jennies, the uterus is probably of similar size but the vesicle is smaller in jennies than in ponies; 3. Within experiments, the vesicles that became fixed earlier tended to be the largest in diameter (585, 587); 4. Fixation in postpartum mares occurs more frequently in the formerly nongravid horn (40, 503, 650) which would be expected to be smaller; 5. Uterine turgidity and fixation have a close temporal relationship; and 6. Fixation almost always occurs at the flexure in the caudal portion of one of the uterine horns where there is proba- bly greater impediment to continued mobility when conceptus diameter and uterine tone reach a crucial point (Figure 1.7, pg. 10). Effect of reproductive status. In the first edition (575), a tabulation was made of incidences of transuterine migration given in earlier studies. There was a slight preference for attachment in the right horn, but the report With the largest data mass indicated a preference for attachment in the left horn. A subse- quent study (268) found attachment more likely on the left in lactating mares and - lsd FIGURE 8.9. These data demon- strate the temporal associations among growth of the embryonic vesicle, the development of uterine tone, and the occurrence of fixation. The lsd bars are the magnitudes of the least significant differences. Adapted from ( 587). 19 20 21 on the right in nonlactating mares. This finding has been confirmed (579, 581) and, in addition, right-side attachment was greatest in maiden mares (Table 8.2). Perhaps the intraluminal resistance to mobility is greater for the right horn in maiden mares and nonlactating (barren) mares; the difference in resistance between left and right horns may be less exaggerated in barren mares than in maidens because of the distending effects of previous pregnancies. In postpartum mares, the embryo became fixed more often in the left uter- ine horn. This is likely related to the finding that embryo attachment in post- partum mares is more frequent in the formerly nongravid horn (503, 40, 650). The formerly nongravid horn was smaller for the first 21 days (1068) or for more than 35 days (650) after parturition. Therefore, the flexure of the nongravid horn would be a greater impediment to vesicle mobility than that of the formerly gravid horn. No difference was found in the time the vesicle spent in the gravid versus nongravid horns during the mobility phase (584, 650), indicating that selection of the horn in which fixation occurred was not a function of the amount of time the vesicle spent in a horn before Day 15. Comparisons were made recently on the effect of reproductive status on vesi- cle diameter, uterine horn diameter, and day of fixation (650). The conceptus grew at a similar rate in postpartum and non— parturient mares. However, the day of fixation did not differ between the reproductive statuses (means: 15.3 and 15.0 days). Although the uterine horns were larger in diameter in the postpar- tum mares on the day of fixation (means: 33 and 28 mm), uterine tone was also significantly greater (mean scores: 3.1 and 2.6). Perhaps the greater uterine tone in postpartum mares allowed fixa- tion to occur at the appropriate time, despite the greater diameter of uterine horns. Maternal Aspects of Pregnancy 311 TABLE 8.2. Effect of Reproductive Status on the Side of Fixation (left or right horn) Side of Side of ovulation fixation Reproduc- No. tive status mares L R L R Lactating 421 47% 53% 60% * 40% Barren 268 45% 55% 41% * 59% Maiden 104 62% * 38% 33% * 67% All mares 793 50% 50% 50% 50% Percentages that differ significantly are indicated by an asterisk. Fixation occurred with greater fre- quency in the right horn in barren and maiden mares; the difference was most exaggerated in the maidens. Adapted from (579). Importance and roles of embryo mobil- ity and fixation. Discovery of the phenomena of embryo mobility and fixa— tion has provided rationale for hypothe- ses on the causes of the following per— plexing phenomena: 1) differential effects of reproductive status on side of fixation, 2) preferential fixation in post- partum mares in the formerly nongravid horn, 3) occurrence of fixation almost always in the caudal portion of one of the uterine horns, 4) lack of agreement between side of ovulation and side of fixation (581), 5) greater incidence of unilateral than bilateral fixation in mares with twins, especially when the vesicles are of unequal size (pg. 549), and 6) ability of a relatively small conceptus to block the uterine luteolytic mecha- nism in a species with a relatively large uterus. In regard to Point 6, it has been proposed (581) that mobility of the con- ceptus favors physiologic exchange between embryo and the maternal envi- ronment. One of these physiologic exchanges involves the ability of the con- ceptus to block the endometrial induc— tion of luteolysis. The role of embryo mobility in this pivotal mechanism is discussed in Chapter 10 (pg. 439). 312 Chapter 8 8.4C. Orientation Orientation refers to the rotation of the embryonic vesicle so that the embryonic pole assumes a certain position in rela— tion to the mesometrial attachment. The position differs among species. In equids the embryo proper assumes a ventral position opposite to the mesometrial attachment. The mechanisms involved in orientation of the embryonic vesicle with— in the uterine lumen of various species are poorly understood and have not been resolved. Uterine and conceptus anatomy. In the mare, it is unlikely that orientation occurs before embryo mobility ceases. In this regard, study with simulated embry- onic vesicles indicated that the vesicle is rotated or rolled during intrauterine loca- tion changes (600). This was observed in simulated vesicles in which an area of their walls was thicker and therefore ultrasonically identifiable. The embryo proper is first identifiable by ultrasound between Days 19 and 21 (review: 590). The embryo proper lies initially in the ventral hemisphere of the vesicle (as seen on the scanner screen), indicating that orientation occurs before Day 19. The embryo proper is subsequently lifted into the dorsal hemisphere of the vesicle by development of the allantois. These obser- vations indicate that orientation occurs between the day of fixation (Day 16) and the earliest day of ultrasonic identifica- tion of the embryo proper (Day 19). On the day of fixation, the conceptus is centrally located in a cross-sectional View of the uterine horn due to the uniform thickness of the uterine wall (590). Beginning on approximately Day 17, the embryonic vesicle begins to lose its spher- ical form with the result that its cross- sectional ultrasound image becomes oblong, triangular, or irregular in outline (Figure 8.10; 580). With the triangular forms, the apex tends to occur in the dor- sal region of the uterine lumen. However, the shape of the conceptus does not remain static, and its outline is seen to change frequently during periods of con- tinuous observations With the scanner. Some of these shape changes can be induced by external pressures on the uterine wall from movements of the abdominal viscera or pressure applied by the ultrasound transducer. Close observa- tion, however, reveals that most are caused by myometrial contractions which appear to exert a kneading or massage- like action on the fixed vesicle. Coupled with this, a disproportional change in the thickness of the uterine wall occurs between Days 16 and 21; the dorsal wall, especially on each side of the midline, thickens considerably. In contrast, the ventral wall becomes thinner and smoother in cross-sectional outline, form- ing a ventral dome—like bulge for the FIGURE 8.10. Hypertrophy of dorsal uterine wall between Days 1'7 and 22. Arrows delineate periphery of uterine horn. Note the thicker encroaching dorsal endometrial folds and the result- ing guitar—pick shape at Day 22. The structure is primarily yolk sac, but the allantoic sac is emerging and is begin- ning to lift the embryo proper from the floor of the vesicle. From (590}. expanding vesicle (Figure 8.10). Shape changes are further discussed in Chapter 9 (pg. 366). Postulated mechanism. It has been postulated that orientation is caused by the combination of the disproportional encroachment of the dorsal uterine wall and the massaging action of the uterus (580, 587). As noted below, uterine contrac- tility continues after fixation. These phys- ical relationships force the thickest part of the conceptus wall, the embryonic pole, into a ventral position. This hypothesis is consistent with the six instances of natu— ral disorientation that have been reported (587, 590). Five of these were associated with natural embryo reduction of unilat- erally fixed twins where the surviving embryonic vesicle was normal in size and appearance but was positioned so that the umbilical cord attached to the ven- tral, rather than dorsal, hemisphere of the allantoic sac. Thus, the presence of two vesicles interfered with the orienta- tion process. In the remaining mare, dis- orientation seemed to be associated with poor uterine tone and the resulting elon- gation of the conceptus. Proper orienta- tion did not occur in the absence of luteal or exogenous progesterone (850). Sticki- ness of the uterine secretions and relative turgidity of the sinus terminalis region also are thought to play a role in mainte- nance of orientation (472). Understanding the phenomenon of orientation is impor- tant in comprehending the developing anatomy of the conceptus and the nature of natural embryo reduction in mares with twins (pg. 552). 8.4D. Uterine Contractions Uterine contractions and the associat- ed to-and-fro movements and compres- sion and expansion of the embryonic vesi- cle can be visualized when a uterine segment (horn or body) is Viewed longitu- dinally by ultrasound (587). In longitudi- nal View, the contractions can sometimes be observed as undulations on the ventral Maternal Aspects of Pregnancy 313 aspect of the uterus. Occasionally, the waves traverse the length of the portion of the uterus being viewed. When a major contraction passes over a large embryonic vesicle (15 mm, Day 14), the vesicle some- times partly collapses (Figure 8.7); however, the small vesicles (3 to 9 mm) do not exhibit the compression and expansion phenomenon. The characteris- tics of uterine contractions during concep- tus mobility have not been described ade- quately. Problems in studying the wave patterns ultrasonically include the fre- quent inability to maintain the transduc- er in constant plane for an adequate time and the difficulty in distinguishing between true uterine contractions and contractions of closely apposed intestine. The latter problem has been partly over- come by doing the contractility studies when the urinary bladder is expanded so that intestinal viscera do not impinge on the ventral wall of the uterine body (646). Control of uterine contractions. The mechanisms involved in uterine contrac- tility during early pregnancy are not known. It has been suggested that the mobile embryonic vesicle causes its own mobility by stimulating uterine contrac- tions (587). The stimulant could be an estrogen since the conceptus at the stage of maximal mobility produces estrogens (pg. 66). However, treatment of mares with systemic injections of estradiol did not result in an earlier increase in embryo mobility or increased uterine activity scores (194). Similarly, in a study in sea- sonally anovulatory mares, the addition of estradiol to a progesterone regimen did not alter activity scores from those obtained by progesterone alone (349). Attention should be directed toward other potential embryo-produced myometrial stimulants (e.g., PGFZa, PGEz), or other methods should be devised for testing the estrogen hypothesis. A role for proges- terone in uterine contractility was indi- cated by an increase in activity scores after 14 days of treatment in anestrous mares (349). Furthermore, mobility of the 314 Chapter 8 embryonic vesicle, and probably uterine contractility, decreased in mares Without a source of luteal or exogenous proges- terone (850). Temporal relationships between con- tractions and embryo—uterine inter- actions. Uterine contractility during early pregnancy has been quantitated ultrason- ically by a derived scoring system in pony mares (646, 350, 650) and jennies (350). Comparisons were made between early pregnancy and the corresponding days of the estrous cycle; contractile activity dur- ing the estrous cycle is discussed else- where (pg. 215). Contractility in pregnant mares and jennies increased at the time of expected increase in embryo mobility (Figure 8.11). Maximum activity in non- pregnant animals was reached at the expected time of luteolysis (Days 14 to 18), whereas in pregnant animals maxi— mum activity occurred 4 days earlier (Days 10 to 14). Activity remained at maximum levels through the day of fixa- tion (350, 650) or decreased to an interme- diate level on the day of fixation (646). These and other studies (587, 194) provide temporal support for the postulate that fixation is not caused by cessation of con- tractions and that the continuation of contractions at the maximal or only part- ly reduced level for a day or two after fix- ation plays a role in orientation of the embryonic vesicle after fixation. 8.4E. Uterine Tone Midventral Views of the reproductive tract of pregnant mares 10 to 50 days after the end of estrus are shown in Figure 9.8 (pg. 354). These photographs show the external appearance of the uterus, as well as the ventrally directed embryonic bulge, for various stages. Description of tone changes. A remark- able change, clearly palpable and visible, occurs in the equine uterus early in preg- nancy, including the nongravid horn. Uterine tone and the compressed thick- ness of the uterine wall increase greatly Uterine contractility Uterine activity (score) Nonpregnant (n=8) 0 4 8 12 16 20 Number of days from ovulation FIGURE 8.11. Changes in uterine activity scores as determined by ultrasonography. A star repre- sents a difference between the two groups (P<0.05) on the indicated day. Adapted from (350). by days 14 to 17. In many mares, the increased tone is so pronounced that on transrectal palpation the uterus that was previously quite flaccid takes on the con- sistency of a sausage or rope. The amount of tone change, however, varies consider— ably among mares, and it may be unclear in some whether a change in tone has occurred. In one study (1625), 7 of 52 preg— nant mares failed to develop increased tone and 4 of the 7 had low progesterone values. The dramatic increase in uterine tone was first described half a century ago (381, 383). It was reported that pregnancy could be anticipated by the extent of uter— ine tone at Day 15 before the vesicle was detectable. An account (1664) of palpable uterine changes during the estrous cycle is given elsewhere (pg. 209). A detectable uterine thickening (tone) occurs after ovu— lation, but after Day 12 a decrease occurs in nonpregnant mares, paralleling the progesterone decline. In pregnant mares, a marked increase occurs after Day 12 and reaches a plateau at approximately Day 25 (1664, 650, 697). After the embryonic bulge becomes visible or palpable, the tone is maintained in the uninvolved por- tions of the gravid horn and throughout the nongravid horn and uterine body. The form and consistency of the cervix also change early in pregnancy (1499). The cervix is constricted, elongated, and firm on palpation at 17 to 21 days. The uterine body and cervix feel like a firm narrow structure traversing a portion of the pelvic cavity. In a recent study (650), a transient increase in uterine tone with a peak at Days 5 or 6 was detected in both postpar— tum and nonparturient pregnant mares. A subsequent study (648) was done in the blind (operator unaware of reproductive status or day of ovulation). The results confirmed this observation and found no difference between pregnant and non- pregnant mares. The transient increase in tone is likely related to a secondary estrogen surge in early diestrus (pg. 242) rather than to entry of the blastocyst into the uterus. Cause of turgidity. The substance responsible for, or contributing to, the Effect of steroids on uterine tone (n=5/group) P = Progesterone (100 mg) 1E2: 1 mg Estradiol 5E2 = 5 mg Estradiol C = Nonpregnant control on Isd Uterine tone (score) N 10 15 20 l— Treatment period—l 25 Number of days from ovulation Maternal Aspects of Pregnancy 315 extensive uterine tone in early pregnancy when the uterus has been primed with progesterone may be estradiol. Scores for uterine tone, taken without knowledge of treatment group, approached those of early pregnancy when diestrous mares were treated with a combination of the two steroids beginning at Day 10 (Figure 8.12; 697). The combination was most effective when the dose of estradiol was low. Furthermore, in seasonally anovula- tory mares with minimal follicular activi- ty, pretreatment with progesterone pro- duced a degree of uterine tone equivalent to that of diestrus. In addition, exogenous progesterone maintained diestrous uter- ine tone after luteolysis. However, proges- terone, followed by progesterone plus estradiol, produced greater uterine tone than either hormone alone or when the combination was given without previous progesterone priming. In an earlier study (178), increased uterine tone was recorded during combined progesterone and estra- diol treatment without progesterone priming; however, this experiment may have involved examiner bias since the Pre nant "*K 9 FIGURE 8.12. Changes in uter- ine tone in mares treated on Days 10 through 29 with combi- nations of progesterone and estradiol. Tone was scored from 1 (flaccid, as during estrus) to 4 (maximum, as in early pregnan- cy). The tone of early pregnancy was mimicked most closely by administration of progesterone plus the low dose (1 mg) of estra- diol. The lsd bar is the magni- tude of the least significant dif- ference. Adapted from (697). 30 35 316 Chapter 8 scoring was not done in the blind. In the absence of an objective technique, uterine tone studies can most effectively be con- ducted by an operator who is not aware of treatment groups, day of ovulation, or even the hypothesis under test. In another study (194), exogenous estra- diol did not increase the extent of mobili- ty of the embryonic vesicle but did hasten the day of fixation (Figure 8.13). Fixation occurred on or before Day 14 in more mares in the estradiol-treated group. However, data for uterine tone were equivocal and further study is needed. In an initial experiment (648), both uterine horns were ligated on Day 11 so that the embryonic vesicle was restricted to the uterine body and caudal portion of the horns (n=5 treated and 5 controls). Uterine turgidity over Days 12 to 17 was maintained at diestrus levels in the ligat- ed horns (conceptus confined to uterine body), but increased, as expected, in the Day of fixation Control / (n=22) Number of observations 11 12 13 14 15 16 17 18 Number of days from ovulation FIGURE 8.13. Effect of estradiol treatment on day of fixation of the conceptus. Significantly more fixa- tions occurred on Day 14 or earlier in the estradiol- treated group than in the control group. Adapted from (194). nonligated horns. Similarly, scores for uterine echotexture were lower in the lig- ated horns, suggesting less exposure to estrogens. This study supported the hypothesis that the traveling conceptus distributes a substance that causes increased tone. The substance is most likely an estrogen, since the uterine echo- texture of exposed horns, but not ligated horns, showed ultrasonic indications of increased edema; edema is characteristic of the endometrium during estrus (pg. 208). The above described findings and the demonstrations that the conceptus pro- duces estrogens at the appropriate time (pg. 66) indicate that the estrogen source for the increased turgidity is the concep— tus and that the estrogens are locally dis- tributed. Some estrogens also could origi— nate from increasing follicular activity in the face of continuing progesterone out— put or from estradiol production by the corpus luteum (pg. 242). Progesterone is essential for uterine contractions and therefore embryo mobility and is also needed for uterine turgidity. The expand- ing embryonic vesicle did not become fixed and orientated when the luteal pro- gesterone source was removed and exoge— nous progesterone withheld (850). Tone versus size. Diameter of uterine horns was determined by suturing an echogenic bead to the serosal surface of the horns and ultrasonically measuring horn diameter at the bead (647). The results are shown in Figure 8.14. The diameter was greatest during estrus and then gradually decreased. The decreasing diameter temporally corresponded to reported increasing uterine tone, indicat— ing that increase in tone is due to tonic contraction of the myometrium which results in decrease in diameter. This rela- tionship has been confirmed (650). Perhaps uterine tone can be quantitated for research purposes indirectly by mea- suring changes in horn diameter. Uterine horn diameter .i i ii. ‘I “1 Nonpregnant [H (n=15) r \." Diameter (mm) M O) N h Pregnant (n=6) N M M O 0 5 10 15 20 25 30 Number of days from ovulation FIGURE 8.14. Cross-sectional diameters at middle of uterine horns as determined by ultrasonic mea- surements. A bead was sutured to the outer surface of the horns so that measurements were taken in the same place each day. The interaction between day and reproductive status was significant, due primarily to a divergence in diameters beginning on approximately Day 15. Adapted from (64 7). 8.4F. Endometrial Histology The histology of the uterus during the embryo stage has been a neglected research area in mares, contrasted to the many studies on placentation during the fetal stage. Workers in Ireland (860) have done an initial study on endometrial his- tology. Interestingly, the height of the surface epithelium and diameter of uter- ine glands were significantly greater on Days 2 to 5 in pregnant mares than in nonbred and bred-nonpregnant mares. The increased height did not appear to be related to the effects of breeding. Significantly more lymphocytes and eosinophils were found in pregnant mares on Days 6 to 9, suggesting an immunologic response to the embryo. Clearly, confirmation and extension of this research area is indicated. Maternal Aspects of Pregnancy 317 8.4G. Uterine Secretions During early pregnancy the uterine environment is not only physically, but biochemically, dynamic. Various uterine secretions are produced that presumably are essential to the conceptus and have the following proposed functions (508): 1) enable the sperm to thrive and ascend the tubular genitalia, 2) provide a system for metabolic exchange of nutrients and waste products between the embryonic vesicle and the endometrium, 3) main- tain the proper physical-chemical envi- ronment (e.g., osmolarity), 4) fulfill immunologic and antibacterial require- ments, and 5) provide lubrication for embryo mobility and orientation and, later, for adhesiveness to aid in mainte- nance of conceptus position and orienta- tion. The proteins have been most exten- sively studied. Most are of blood serum origin, but some may also be synthesized by the endometrium. In some species, pregnancy specific proteins have been identified. Uterine proteins have been shown to serve as enzymes and as car- riers of molecules of hormones, vitamins, and minerals (cited in 1051). Studies by Sharp and associates on uterine secretory patterns during early pregnancy have been reviewed (1428). A uteroferrin-like protein is present in uterine fluid during only the luteal phase of the estrous cycle but is maintained in pregnant mares; this protein has been partially characterized (1051) and may function in iron ,transport to the concep- tus (1843). Total recoverable luminal pro- teins tended to peak during mid-diestrus in nonpregnant mares. In pregnant mares, however, total proteins tended to be depressed during Days 8 to 16 with an increase on Days 18 to 20 (Figure 8.15; 1843). The authors suggested the decrease on Days 8 to 16 may have resulted from increased metabolic needs of the concep- 318 Chapter 8 Content in uterine flushings 100 Total acid phosphatase 80 Pregnant (n=3 to 5) \ 60 40 Activity (pm Pi/ H) 20 Weight (mg/flushing) 8 10 12 14 16 18 20 Number of days from ovulation FIGURE 8.15. Acid phosphatase and recoverable uterine proteins in pregnant and nonpregnant mares. The interaction of day by reproductive sta- tus was significant (P<0.005) for acid phosphatase and tended to be significant (P<0.1) for total pro- teins. Adapted from tabulated data in Zavy et al. (1843). tus. Acid phosphatase in the uterine flushings of pregnant mares continued to rise after Day 14, whereas the levels declined in nonpregnant mares (Figure 8.15). The role of acid phosphatase is unknown. Increases in carbohydrates and enzymes in the uterine lumen of early pregnant mares have also been reported (1842). In this regard, carbohydrate metabolism was detected in individual Day 7 to Day 10 horse embryos (1329, 1328); the contribution of the pentose- phosphate pathway to glucose metabol- ism increased over Days 4 to 12 (258). The authors stated that this finding is consistent with the onset of steroid pro- duction by the generation of nucleic acids. Administration of estrogens and pro- gesterone stimulated the accumulation of enzymes (681), and long-term proges- terone treatment enhanced the luminal concentrations of proteins and acid phos- phatase (743). Uterine estrogen concen- trations in pregnant mares increased locally in the uterine lumen in propor— tion to the size of the conceptus, but con- centrations did not increase in the peripheral circulation (pg. 66). Increased estrogens may have local functions in uterine secretory activity, blood flow, and water balance (1843) and also may play a role in embryo-uterine physical interactions (increasing uterine tone which, in turn, leads to decreasing diam- eter of the uterine horns and embryo fix— ation). Uterine secretions also may be important in providing a lubricated sur- face for embryo mobility and orientation. In this regard, it was recently suggested that sticky uterine secretions help maintain orientation (472). SUMMARY: Embryo-Uterine Interactions Maternal Aspects of Pregnancy 319 Fixation § Orien— A. Maximum ‘ ; tation mobility Luteal progesterone Cross—sectionai diameter of conceptus Uterine contractions Estrogen from conceptus <————-————-—- Relative change ——————-> Uterine tone Diameter of uterine horns 10 11 12 13 14 15 1617 18 Number of days from ovulation 9 FIGURE 8.16. Interrelationships among events in early pregnancy. A. Maximum intrauterine mobili- ty of the embryonic vesicle occurs on Days 11 to 15 (ponies) or 16 (horses) and is characterized by movement of the vesi- cle throughout the length of the uterus 10 to 20 times per day. Fixation (cessation of mobility) is followed ‘by orientation of the vesicle so that the embryo proper is locat- ed opposite the mesometrial attachment. B. A continuing progesterone source is necessary for each of these events. Absence of progesterone results in a decrease in mobility and in the lack of fixation and orientation. C. During mobility the conceptus increases in diameter. The cross-sectional diameter of the conceptus ceases to increase shortly after fixation, presum- ably because of increasing tone and decreasing diameter of the uterus. During the static phase of cross—sectional expansion (Days 16 to 26), there is a com- pensatory increase in conceptus length to accommodate the increasing growth of the conceptus. D. Maximum mobility of the embryonic vesicle results from increasing uterine contractility. Progesterone is a stimulant of uterine contractions but not to the extent that occurs during the maximum mobility phase. It is postulated that the conceptus produces an additional sub- stance that provides the additional stim- ulus for contractility. E. Beginning on Day 12, the conceptus produces estrogens in increasing amounts in preportion to its increasing diameter. The mobile conceptus delivers estrogens to all parts of the uterus. The estrogens, in turn, increase uterine turgidity but apparently are not responsi~ ble for the increased contractility. F and G. With increasing uterine tone, the diameter of the tubular uterus decreases. The decreasing diameter of the uterine horns and the increasing diame- ter of the embryonic vesicle reach a point Where vesicle mobility can no longer occur. As a result of a continuation in contractions after fixation, the fixed vesi- cle rotates and becomes orientated With the embryo proper in the ventral position. 320 Chapter 8 8.5. The Embryonic Bulge A localized enlargement due to the growing conceptus becomes discernible by direct observation through an abdominal incision on the 14th day after the end of estrus or shortly thereafter (Figure 9.8, pg. 354). It is noteworthy, however, that mares vary considerably in this regard. The embryonic bulge (vesicular bulge or gestational sac) may be detected by trans- rectal palpation by Days 16 to 19 and, according to one report (581), as early as Day 15. Early pregnancy diagnosis (before Day 20) was first described more than 50 years ago (381, 383); the embryonic bulge was detected as early as Day 16 and consistently by Day 23. The vesicle was described as being the size of a ban- tam’s egg at Day 16 and the size of a golf ball at Day 20. It was noted, however, that detection of the vesicle at such early stages was easier in maiden or barren mares. In a herd of nonlactating horse mares, the embryonic bulge was first detected by transrectal palpation in 40 mares on Days 15 to 24 (mean: Day 17); the diagnosis and location (left versus right horn) were confirmed by ultrasonography (581). During develop- ment of a technique for manual elimina- tion of one member of a twin set (583), it was noted that embryonic vesicles can sometimes be felt as early as Days 12 to 14 during the mobility phase; the horns at this time are more flaccid, and the turgid vesicle can be felt as the finger and thumb move across it. A popping sensa— tion associated with rupture can some- times be detected during digital compres- sion of a selected vesicle (pg. 556). The bulge palpated early in bovine pregnancy (beginning at approximately Day 30) is due to the amniotic vesicle; in mares, on the other hand, it is due to the allanto- chorionic vesicle, i.e., the entire concep- tus. Because of the spherical shape of the equine conceptus, the bulge is palpable much sooner in mares than in cattle. The early embryonic bulge is located in the caudal portion of one uterine horn and primarily distends the ventral aspect of the uterine wall, producing a dome— like, thin-walled, ventral enlargement. Dimensions of the embryonic bulge and the nongravid portions of the uterine horns in pony mares were tabulated in the first edition (575). The measurements were made through a midventral laparo- tomy. The conceptus grows progressively from its initial location at the base of the horn toward the tip of the horn, filling the horn much like an expanding balloon. The length of the contained bulge becomes greater than the width by approximately Day 24, and the conceptus then becomes increasingly oblong as it invades the gravid horn. If the horn is incised, the vesicle assumes a more rounded form (Figure 9.8, pg. 354), indicat- ing the extent to which the shape of the contained vesicle is molded by the horn. Approximately one-half of the gravid horn is filled by Day 40, and only the tip of the horn (approximately 3 cm) is empty at Day 50 (575). Recent study by ultrasound (648) found that the uterine body, gravid horn, and nongravid horn filled on Days 54 _+_2, 61 i3, and 70 i3, respectively. The filling process is dynam- ic, however, and displacement of the allantoic fluid, but presumably not the allantochorionic membrane, occurs periodically from a uterine segment or an entire horn (pg. 411). The turgidity of the nongravid portions of the gravid horn seems to continue until the horn is invaded by the vesicle. It appears that relaxation and accommodation of the uterine wall occurs only when the uter- ine wall is contacted by the expanding conceptus. Such an observation raises questions about the nature of local control of the vesicle over the tone of the uterine wall. Perhaps the vesicle produces a substance that locally relaxes the turgid wall so that vesicle expansion can be accommodated. 8.6 Estrous Behavior During Pregnancy The phenomenon of occasional estrous behavior in pregnant mares has been known for more than 50 years (1395, 1098). Incidences of 5.4% (1395), 10% (1664), and 9.6% (1625.) have been reported. In the lat— ter study, the phenomenon was recorded 18 to 21 days after mating. An association between the expression of estrus during pregnancy and low progesterone levels also was reported (1625). All of 52 preg— nant mares, including five with estrous behavior, had follicles >20 mm 18 to 21 days after mating, and the authors con- cluded that the presence or absence of large follicles did not seem to be implicat- ed in the expression of estrus. However, the concentrations of plasma proges- terone were lower in the mares showing estrous behavior but did not drop below 2 ng/ml. Full estrus or intromission was not observed in any of 12 pony mares exposed to a stallion for 20 minutes per day on five days per month of pregnancy (103). Progesterone may be sufficient to cause the absence of estrous behavior during diestrus or perhaps may have a positive effect on nonestrus signs (pg. 98). Estrus-inhibiting progestins (e.g., 50c-pregnanes) may be responsible for nonestrus signs later in pregnancy. The pregnanes may account for the absence of estrus despite the high estrogen and low progesterone levels that are characteristic of the last half of pregnancy. An interesting aspect of the relation- ship between estrous behavior in preg— nant mares and the gender of the concep- tus was reported recently (698). The incidence of a female conceptus (12 of 12) was significantly greater than the inci- dence of a male conceptus in mares that exhibited estrous behavior. The authors noted that the finding was novel and requires confirmation. Most (65%) of the estrus detections were on Days 12 to 20, but the study was not designed to deter- Maternal Aspects of Pregnancy 321 mine incidence or whether the phe— nomenon is more likely to occur at a cer- tain time during pregnancy. Estrous behavior usually did not appear to be as intense as the behavior exhibited during the estrous cycle. The cause of the estrous behavior in mares with a female concep- tus is not known. The early conceptus produces estrogen (pg. 66), and an experi- ment is needed to determine if estrogen productivity is greater for female concep- tuses. 8.7. Maternal Ovaries Much mystery surrounds the physio- logic importance of some of the anatomi- cal changes of the equine reproductive tract during pregnancy; this is especially true for the ovaries. In this section we will see, for example, that the ovary may (or may not) go through extreme activity involving the development of large folli- cles and many corpora lutea. Yet, very early in pregnancy, the highly active ovaries can be surgically removed without loss of pregnancy (pg. 437). The anatomical changes undergone by the ovaries of the pregnant mare were described by workers in California (324) as part of the pioneer- ing series of studies on endometrial cups, eCG, and the ovaries. Some of the confusion about ovarian changes could have been avoided if subsequent workers had read the California reports more carefully. Quantitation of various ovarian compo- nents during pregnancy in 65 pony mares was tabulated in‘the first edition (575) and will not be repeated, here. The table suggested several generalizations. As indicated by the standard deviations, there was considerable variation for all components. An inspection of means for many of the follicular end points gave the impression of increasing magnitudes up to Day 60, followed by a decrease. The increasing ovarian weight was largely attributable to the growth of follicles. 322 Chapter 8 In addition, changes in minced extra- luteal tissue weight suggested increased development of supporting tissues (vascu- lature and connective tissue). It should be noted, however, that this end point included the walls of cut follicles, as well as some very small follicles left intact in the mincing procedure. The formation of new corpora lutea beginning at approximately Day 40 con- tributes to the increasing ovarian weight. Between Days 40 and 60, some of the new corpora lutea are in the form of corpora hemorrhagica or hemorrhagic follicles that weigh as much as 54 g (575). After Day 60, decreasing ovarian weight can be attributed primarily to decreasing num- bers of large follicles. The small ovarian weights (<30 g) beginning at approxi- mately Day 190 reflect the near absence of follicles larger than 10 mm and the regression of both the primary corpus luteum and those that form after Day 40. 8. 7A. Follicular Dynamics In a recent ultrasound study, nonpreg- nant and pregnant mares did not differ in follicular profiles until the preovulatory Follicular profiles 36 Nonpregnant (n=20) . 30 —-0— Pregnant (n=40) r r r‘ 0* ¢. N .5 Diameter (mm) as 12 12 Number of days from ovulation 18 24 Second largest follicle growth spurt in the nonpregnant mares (Figure 8.17, 590). In pregnant mares at this time (e.g., Day 20), a growth spurt of a selected follicle apparently did not occur. Presumably, this was the result of the absence of an LH surge in the preg- nant mares. The mean diameter of the largest follicle reached a plateau; the plateau, however, represented different follicles at various times. Evaluations of follicular dynamics in pregnant ponies also have been done by transrectal palpa- tion or examination of excised ovaries (1503, 1504). The resulting data are used here to depict follicular changes during pregnancy. Other reports substantiate these findings (27, 129). Means and regres- sion curves that best characterized follic- ular data are shown in Figure 8.18. The development of medium (10 to 20 mm) and large follicles (>20 mm) and diameter of the largest follicle increased markedly during early pregnancy. Note that follicu- lar numbers increased considerably in early pregnancy (during approximately Days 10 to 40 or 60) and then decreased to very low values by Days 180 to 200. In a necropsy experiment (1503), there were fewer large follicles 18 days after the end Largest follicle FIGURE 8.17. Diameter of the largest and second—largest follicles in pregnant and nonpregnant mares. Data were discontinued on the day that the first mare in each group ovulated (nonpregnant, Day 17; pregnant, Day 36). The stars indicate a significant difference for diameter of largest follicle between groups on Days 15, 16, and 17. The follicular profiles of nonpregnant and pregnant mares did not differ until the preovulato— ry growth spurt in nonpregnant mares. Adapted from (590). 30 36 Follicular profiles (n=16) 00. Size groups 2 E E 1 3 z 0 30 Diameter (mm) M O —l D 20 60 100 140 180 200 Days of pregnancy FIGURE 8.18. Means and regression curves that best characterized the means for follicular changes during pregnancy in ponies based on transrectal palpation. Adapted from (1504). of estrus in pregnant mares than in non- pregnant mares. There were, however, more follicles larger than 10 mm at Day 30 (10.0) than at Days 10 (2.3) or 18 (3.0), again demonstrating growth of follicles early in pregnancy. The increasing num- bers of follicles in early pregnant mares precedes the detectability of circulating eCG (590, 27, 129, 1503, 1504). The diameter of the largest follicle was significantly greater at Day 60 than at any other day. Follicular profiles, beginning on approximately Day 20, represent an aver- age of follicle diameters from many mares. Growth spurts of individual folli- cles would be masked if they occurred on different days among mares (590). In a preliminary inspection of follicular pro- files for individual follicles, growth and regression of large follicles were similar to what occurs during the spring transi— Maternal Aspects of Pregnancy 323 tional period (Figure 8.19; 184). Other pre- liminary observations have suggested also that periodic surges of follicular activity are superimposed on the overall pattern of increasing numbers of follicles during early pregnancy. This possibility is considered elsewhere (pg. 449) in connec- tion with a discussion of the occurrence of surges of FSH. Clearly, follicular dynam- ics in pregnant mares have not been resolved adequately and should be evalu— ated with the aid of monitoring of individ- ual follicles by ultrasonography. Season (month) affects follicular devel- opment during early pregnancy, as well as during the estrous cycle (pg. 174). In conjunction with an experiment on the effects of FSH suppression by treatment with the proteinaceous fraction of follicu- lar fluid, ovarian follicles were monitored ultrasonically on Days 14 to 42 (182). Individual follicles Diameter (mm) 51015 20 25 30 35 40 45 50 Number of days from ovulation FIGURE 8.19. Diameter changes of individual large follicles during pregnancy in two mares. The third large follicle ovulated in each mare, presum- ably as a result of eCG production. From (184). 324 Chapter 8 Mares were mated in the summer (June) or in the fall (September to October). There was greater follicular activity in the summer control mares than in the fall control mares. This agrees with an earlier report (30) of greater ovarian and follicu- lar size and greater numbers of secondary ovulations during the first four months of pregnancy in mares mated early in the breeding season, compared to mares mated after July 11. It also has been reported (603) that mares that become pregnant during the anovulatory season following GnRH-induced ovulation have reduced follicular activity during the ensuing pregnancy (pg. 168). 8. 7B. Corpora Lutea The primary corpus luteum of preg- nant mares is the structure that forms at the site of the ovulation that results in conception. Secondary corpora lutea and accessory corpora lutea are those that form from ovulated and unovulated folli- cles, respectively, While the mare is under progesterone dominance (diestrus and pregnancy; 599). Secondary and accessory luteal glands that form during the pro- duction of eCG will be referred to collec— tively as supplementary corpora lutea. That is, secondary corpora lutea result from ovulation while the mare is under progesterone dominance, whether or not the ovulation can be attributed to eCG. Primary Corpus Luteum. The history of our knowledge of the life span of the primary corpus luteum in mares is of spe- cial interest and provides an example of the self-perpetuation of faulty informa- tion until it becomes dogma (Figure 8.20). This phenomenon is neither unusual in biologic research nor peculiar to it. As depicted in the figure, it was believed for many years that the primary corpus luteum of pregnancy regresses at about the end of the first month and is then replaced by other corpora lutea. The con- cept of a short—lived primary corpus luteum was not supported by results of an experiment in 1971 (1503). The primary corpus luteum (marked with India ink) was maintained in ponies until approxi— mately 160 to 180 days after the end of estrus when it regressed along with the supplementary corpora lutea. Mares were necropsied at 24 to 220 days. The weight of the primary corpus luteum was not sig- nificantly different for 24, 30, 40, 50, and 60 days, but there was a gradual decrease (linear regression) in weight from 60 days (4.3 g) to 220 days (0.8 g). The primary corpus luteum was pink suggestive of continued function in all pregnant mares, except for the one killed on 220 days. All luteal structures (primary and supple- mentary) were in an advanced stage of regression in O of 6, 3 of 4, and 4 of 4 mares killed on 150, 180, and 210 days, respectively. Within mares, all luteal structures (excluding corpora hemorrhag- ica) appeared similar in color. It seems likely, therefore, that the primary and all of the supplementary corpora lutea reached an advanced stage of regression (small, hard, brown to orange-brown, dif- ficult to separate from surrounding tis— sue) at approximately the same time (by 180 to 220 days). After the formation of supplementary corpora lutea, the pri- mary corpus luteum could be positively identified only by the ink mark. This sim— ilarity may have contributed to the per- petuation of the belief that the primary corpus luteum regressed about the time the supplementary corpora lutea began to form. In conjunction with another experi- ment, which also involved marking with India ink, the primary corpus luteum at 140 days was large (mean weight: 2.8 g) and its appearance and color at that time were similar to those of supplementary corpora lutea (1504). In another study (1521), it was found that marked pri- mary corpora lutea were not regressed at 100 days in 3 of 3 horse mares and that the corpora lutea were still capable of in vitro production of progesterone. The primary corpus luteum secreted large quantities of progesterone into the ovari- an venous system up to at least 80 days, the last day studied (1506). More recently, ultrasonic studies (186) have demonstrated that the primary corpus luteum not only fails to regress but undergoes a resurgence in growth and activity at about the time eCG begins to appear in the circulation (Day 35). The cross-sectional area of the ultra- sonic image of the primary corpus luteum was significantly greater on Days 39 to 42 than on Day 33. The increase in cross— sectional area paralleled an increase in circulating concentrations of proges- Maternal Aspects of Pregnancy 325 terone. The resurgence phenomenon is further discussed in Chapter 10 (pg. 443). Supplementary corpora lutea. The supplementary luteal glands that devel- op during eCG production are closely associated with follicular dynamics. These curious structures begin to form at approximately Day 40, develop into functional luteal glands, many of which are comparable to the primary corpus luteum, and regress by approximately Day 180. A series of photographs of both primary and supplementary corpora lutea from pregnant ponies is shown in Figure 8.21. 1931: Days 1 to 40 are characterized by 1°CL ORIGINAL and Days 40 to 150 by 2°CL (324) OBSERVATION 1937: 1°CL regresses at about end of 1st MISSTATEMENT month (877) . 1947: "According to (1372) E : PROGRESSIVE : ENTRENCHMENT 1948: "It has been established that (678) For totaIIy 1°CL has a life incomprehensible of some 40 days reasons : _ . I SELF- 19503 19705" 5 PERPETUATION TEXTBOOKS AND REVIEWS : (95, 1132, 1155, 1361, 1332) 5 At about the At about 35 to 35th day 40 days Between days 40 to 50 DOGMA I (- FIGURE 8.20. Apparent literature chronology which led to the misconception that the pri- mary corpus luteum regresses early in pregnancy. The numbers in parentheses refer to lit- erature references. 326 Chapter 8 Gross appearance of supplementary corpora lutea varies considerably, reflect- ing a divergence in morphogenesis. Between approximately Days 40 and 70, some are extremely large (e.g., 50 g) and appear much like blood clots (corpora hemorrhagica or hemorrhagic follicles; pg. 202). The blood clots later recede, and with the development of the luteal cells, the structures take on the appearance of mature corpora lutea. Some of the sec- ondary corpora lutea (those resulting from ovulation) thus go through develop- mental stages similar to those of some primary corpora lutea. Some supplemen— tary corpora lutea are relatively solid, (milllltlll”HM!11le“11H”Willi!Hilliililli‘iillfllll; 1 2 3 at} 5 6 FIGURE 8.21. Appearance of primary corpus luteum (arrow) and supplementary corpora lutea in ponies. Primary corpora lutea were marked with an injection of India ink on Day 3. The ovaries have been cut midsagittally and opened like a book. Some of the supplementary corpora lutea on Days 40, 50, and 60 are corpora hemorrhagica. Fully developed supplementary corpora lutea may be solid and easily confused with the primary corpus luteum, or they may contain a central cavity lined with white tissue or filled with an organized fibrinous clot. whereas others contain central cavities of various diameters. The cavities may con- tain a clear, yellow lymph-like fluid or a plasma-like clot, and some may be lined with a white fibrous material. Other cavi- ties contain fibrinous reddish or brown clots. In one study (1503), 68% of the sup- plementary corpora lutea (n=55) were spherical and, of these, 45% had a promi- nent central cavity while 55% were rela— tively solid. The remaining 32% were irregular or gourdlike in shape, with a ' tract or neck leading to the ovulation fossa. The presence of tracts leading to the fossa indicated, though not conclu- sively, that approximately a third of cor- pora lutea developed from ovulated folli- cles (secondary corpora lutea) and the remainder from luteinization of unrup— tured follicles (accessory corpora lutea). Supplementary corpora lutea contain— ing large blood clots (corpora hemorrhag- ica) were found on Days 40, 50, and 60 but not later (1503). Two of four corpora hemorrhagica had an ovulatory papilla. A recently ovulated ovum was found in the corresponding oviduct in both instances (575). From available data on the shape of corpora lutea, periodic transrectal examinations, and presence of recently ovulated ova, it is clear that ovulation does occur during eCG production but ovulation apparently accounts for only a minority of the supplementary corpora lutea. The corpora lutea that form early during high eCG production are more likely to result from ovulation. (1503), based on the following: 1) Corpora hemor- rhagica with ovulatory papillae were found before Day 70, but seldom there- after; 2) Recently ovulated ova have been found in the oviducts of mares necropsied before Day 60 but not thereafter (1503); and 3) Significant increase in numbers of ova in the oviducts as pregnancy advances has not been found. Further- more, in one study (1503), the last detected ovulation was on an average of Day 74, yet the number of supplementary corpora lutea increased from 2.8 per mare at Day Maternal Aspects of Pregnancy 327 70 to 10.2 per mare at Day 140. Recent study (1021) of slaughterhouse specimens confirmed that initially most corpora lutea form from ovulations (secondary corpora lutea) and then from luteiniza— tion of follicles (accessory corpora lutea); classification was based on presence or absence of an ovulatory process. The number of corpora lutea increases as pregnancy advances up to Days 180 to 200, at which point all corpora lutea regress. The numbers of supplementary corpora lutea and follicles >10 mm found in one study are shown in Figure 8.22. Inspection of the figure indicates that the number of follicles >10 mm decreased and number of corpora lutea increased between approximately 40 and 120 days; a significant negative correlation (r=—0.63) occurred within mares between these two end points (1503). There was no difference among days in the sum of num- ber of follicles and supplementary corpora lutea. These comparisons indicate that the formation of supplementary corpora lutea accounts for the decrease in the number of follicles >10 mm. Number of ovarian structures 10 Corpora lutea .’ G) / I O) .5 Number per mare N 40-59 80-99 120-149 Days of pregnancy 180-209 FIGURE 8.22. Changes in number of corpora lutea (primary and supplementary) and number of folli- cles >10 mm during pregnancy in pony mares. Adapted from tabulated data ( 5 75). 328 Chapter 8 SUMMARY: Ovarian Dynamics Follicles Follicles 20 60 80 Regression of primary & supplementary CL . 100 120 140 160 180 200 \ Days of pregnancy Figure 8.23. A schematic overview of changes in the number of follicles and supplementary corpora lutea during pregnancy (Days 20 to 210). The changes in the number of large follicles (>10 mm) are based on Figure 8.18 (palpation data) and Figure 8.22 (necropsy data). This information is reasonably dependable. The number of small follicles (2 to 10 mm) is based on necropsy data (575) but is less reliable due to small numbers of ani- male. The following concept emerges as a working model: the total number of gross- ly Visible follicles (>2 mm) remains fairly constant throughout Days 10 to 210, but changes occur during this time in the pro— portion of small to large follicles. The number of large follicles increases from Day 10 to approximately Day 50 and then gradually decreases to very low values by Days 140 to 160. The number of small fol- licles is lowest on Days 30 to 40. The increasing number of large follicles thus can be accounted for by growth, as evi- denced by a decrease in the number of small follicles. The decrease in the number of large follicles after Day 50 can be attributed to the formation of supplementary corpora lutea. Initially (Days 40 to 70), some of the conversions to corpora lutea result from ovulation (secondary corpora lutea) and some result from luteinizationi of‘ anovulatory follicles (accessory corpora \ lutea) After Day 70, the majority of cor- _ pora lutea form from anovulatory follicles Between Days 160 and 180, all corpora lutea, including the primary corpus - luteum, begin to regress. This simplified concept should not obscure the complexity Within mares and lack of uniformity among mares in the dynamics of follicular and luteal morpho- - genesis. It is not unusual, for example, to find mares 60 to 160 days pregnant with no secondary or accessory corpora lutea. It appears, therefore, that neither the \ tremendous follicular development nor the formation of supplementary corpora - lutea is entirely essential for pregnancy. This nonessentiality is a reflection of the wide variation or imprecise centrol occur- ring among mares. Also obscured by the smooth, hypothetical lines are the rhyth— * mic or irregular fluctuations that occur within mares. The availability of ultra- sound scanners for monitoring individual large follicles should soon lead to clarifi- cation of rhythmicity in folliculogenesis ‘ during pregnancy. 8.8. Gestation Length Several factors have been examined for their influence on the length of gestation. There is confusion about the possible effects of some factors; a review (1344) can be used for locating the various conflict- ing reports. One of the problems may be that some workers use day of breeding as the beginning point, while others use ovu- lation. These two points can differ by as much as a week (pg. 297). Environmental factors. Various envi— ronmental sources of variation in gesta— tion length were studied in Arabian mares in California (773). Gestation length for mares bred in winter (338 days) and spring (342 days) were significantly longer than for mares bred in summer (331 days) and fall (329 days). Well-fed mares had a gestation four days shorter than mares on a maintenance ration. Nutritional and seasonal effects did not interact; that is, the seasonal effect was independent of nutrition. It was concluded that season of breeding accounted for 44% of the varia— tion in gestation length, While the level of nutrition accounted for 5%. In another study (626), the length of pregnancy was not different between mares receiving 100% versus 120% of the recommended requirements for digestible energy during the last three months of pregnancy. Feeding mares to obesity during gestation did not have a significant effect on length of gestation, any measured aspect of par- turition, or on size of the foal (919). However, mares in thin condition had a nine-day longer gestation (733). In another notable study (1344), records were examined for 522 Thoroughbred mares in Australia, using day of ovula- tion as the reference point. Gestation length was longer for mares mated early in the breeding season. In another study with Thoroughbreds, mares bred in February to April (N. Hemisphere) had gestation lengths five days longer than mares bred in June (752). The foals born early in the year (January to March) had Maternal Aspects of Pregnancy 329 lighter birth weights than those born later (April to June), despite the slightly longer gestation length (753). In a recent study in Arabian mares in Egypt (469), month of foaling significantly altered ges- tation length; gestation was longer for Winter foalings. It appears that mares are able to make a limited adjustment in ges- tation length such that foals tend to be born in the spring. Such an adjustment, although not marked, may have some survival advantage for the species. An influence of photoperiod length on gesta- tion length has been demonstrated by Texas workers (755). Light treatment began on December 1 and mares foaled primarily in March and April. Mares with a 16-hour fixed photoperiod had a reduced mean gestation length (10 days shorter than controls). Size of the new- born foal was not significantly affected. Breed. Breed is another factor said to affect the length of gestation. Although lists giving gestation lengths for various breeds are available, it is usually not clear Whether the differences are a mean- ingful reflection of true breed differences. Means of 322 to 345 days have been reported for various breeds. The expected date of parturition for Thoroughbred farms is often conveniently calculated as 11 months, or 333 to 336 days, from last service; reported means for the service-to- birth interval are usually 338 to 340 days, with ranges for individual observa- tions of 310 to 374 days (1361). For exam- ple, gestation length in a Thoroughbred study was 335 i6 days (mean iSD) with a range of 326 to 343 days. Other factors. Dam (1344) and sire (1338) had a significant effect on gestation length. Foal gender had a significant effect in some studies but not in others. In various studies, Thoroughbred colts were carried 1.7 days (1344), 2.5 days (sig- nificant, 752), and 7 days (P<0.07; 919) longer than fillies; another study (773) did not find an interaction between season and foal gender on gestation length. Age of mare did not alter length of gestation 330 Chapter 8 in an extensive study in Thoroughbreds (752); this result agrees with the results of some studies, but other studies have reported longer gestation for older mares (review: 752). Year of breeding also signifi- cantly influenced gestation length, but the causes could not be determined (752); this finding underscores the confounding that can be expected in attempting to compare gestation lengths among breeds when the breeds are not part of the same herd. Gestation length also was signifi— cantly reduced in mares carrying twins (823). Many factors can affect gestation length so that number of days since ovu— lation serves only as an estimate of the expected day of parturition. Short gestations. Foals born two weeks or more prior to 340 days are usually con- sidered premature (1059). Those born three weeks early show signs of underde- velopment and may need special care to survive. The birth of a normal vigorous foal three or more weeks early indicates a need for re-examination of the breeding records (1059). Reports on the charac- teristics and guidelines on assessment of equine prematurity or dysmaturity are available (767, 1358; for references, see 1359). Prolonged gestations. Apparent pro— longed gestation has been infrequently reported (1659). Vandeplassche (1682, 1681) reviewed prolonged gestation in mares and estimated that the incidence of exces- sively prolonged gestation (370 to 387 days) is probably about 1%. The author reported the rare occurrence of embryonic arrest of 25 to 35 days (diapause) begin- ning between 16 and 35 days and noted that such a phenomenon is common in some other species (e.g., mink, marsupi- als). The observations preceded the avail— ability of ultrasound scanners. Scanners may provide the technology needed to determine whether embryonic diapause exists in mares, as opposed to errors in breeding dates. In daily examinations of hundreds of mares at 11 to 40 days, no indication of diapause was noted (600). 8.9. Pregnancy Diagnosis A brief outline of pregnancy detection is given in Table 8.4. The table provides an overview and indicates where detailed information may be obtained in this text or elsewhere. In addition, a discussion of specific points can be found in research reports on comparisons of laboratory tests (1039) and on comparisons of transrectal palpation and immunologic tests (1232). The subject of pregnancy diagnosis will not be discussed in detail here since reviews are readily available and include information on the technique of transrec- tal examination of the equine reproduc— tive tract by palpation (95, 1333, 1362, 1848, 1334) and ultrasonography (590, 1514, 1069). Transrectal approach. Early detection of the embryonic bulge by palpation was described earlier (pg. 320). Considerable discussion has been published (28, 810, 899, 1717) on whether pregnancy diagnosis by transrectal palpation may be harmful to pregnancy. Published reports, however, have not documented that manual ovari- an examinations or pregnancy diagnoses, as done on breeding farms by profession— als, are harmful to the establishment or continuation of pregnancy. One group of workers (1717) found that transrectal pal- pations during estrus had a detrimental effect on pregnancy rates, although daily palpations during early pregnancy caused no pregnancy loss. The studies, however, were not designed to evaluate the effects of manual examinations as routinely practiced in the field since inexperienced palpators (students) were used without limitations on palpation time. A revolu- tionary approach to pregnancy diagnosis was introduced in the past decade (1208) and involves transrectal ultrasound scan- ners (reviews: 590, 1514, 1069). These instruments can accurately display the embryo in real-time images by Day 11. Appearance of mucosa. The Visual con- dition of the mucous membranes of cervix and vagina has been described (383) as an aid to pregnancy diagnosis. As early as Maternal Aspects of Pregnancy 331 TABLE 8.4. Methods of Detecting Pregnancy Days after ovulation Basis Method Comments 11 to term Real time Transrectal The preferred method when available images ultrasonography 16 to 24 Failure to exhibit Estrous determination A few pregnant mares will show estrous the behavioral, signs (pg. 321) or nonpregnant mares physical, or may fail to show estrus due to silent hormonal signs estrus or pseudopregnancy (pg. 228) of estrus Vaginal and cervical Descriptions of mucous membranes and appearance cervix are given for estrus and diestrus (pg. 211) and for pregnancy (pg. 330) Uterine and cervical Turgid tone (pg. 314) is good presumptive tone indicator before bulge, but does not differentiate from pseudopregnancy (pg. 228) Progesterone assay High values (>1ng/ml) are presumptive indicators, but do not differentiate from pseudopregnancy or cycles of unusual length; values can be quite low in an occasional pregnant mare (1220, 1409) 16 and 17 Absence of Single injection of False positives in mares with pseudopreg- estrus in estrogen with nancy or early embryonic death (prolong- response to observations ed life of corpus luteum). 90% efficiency estrogen for estrus in limited trials (pg. 332) 19 to 60 Vesicular bulge Rectal palpation Size, location, and other characteristics given in Section 8.5 (pg. 320) 28 to term Changes in Mucin test Positive results as early as Day 20 and is stained vaginal especially effective after Day 80; false mucus positives from pseudopregnancy; special precautions needed to differentiate from anestrus (pg. 332) 45 to 90 eCG in blood Immunologic and Commercial kits and dipsticks available biologic tests in some countries (819, 1337, 1704; pg. 72) Very accurate, but false diagnoses can result from low eCG production or maintenance of endometrial cups after abortion (pg. 450) 45 to term Estrogens Immunoassays Conjugated or total estrogens measured in urine, blood, or milk 60 to term Uterine contents Transrectal palpation Ballottement and palpation of fetus 90 to term Fetal heart beat Doppler ultrasound Fetal pulse detected between Days 90 and 240 (535, 1104) 150 to term Estrogens in Chemical (Cuboni) Cuboni test involves adding chemicals to urine and biologic tests urine and observing fluorescence; reliable after Day 150 (345) 332 Chapter 8 Day 24 in some mares, the vagina and cervix become very pale and blanched and assume a pearly appearance. Small blood vessels and capillaries are prominent, forming a network over the surface. These disappear as pregnancy advances, and the mucous membranes become more blanched. The folds of the cervix become sealed together by a thick, tacky mucoid secretion so that the external os seems to be obliterated. The vaginal speculum does not slip in readily, and the vaginal walls do not balloon as in the diestrous mare; separation of the walls occurs slowly as though they are peeling apart. The mucosa has a very dry appearance, and its secretion is sticky or even gumlike (according to the stage of pregnancy); the amount and stickiness of the secretion in the vagina increases with advancing pregnancy. Descriptions of the appear- ance of vaginal and cervical mucous membranes and fluids for estrus and diestrus are given elsewhere (pg. 211). Mucin test. A pregnancy test that uti- lizes changes in cervical-vaginal mucus was developed by Japanese workers in the 1920s and 1930s. The method, called the mucin test or Kurosawa method, is described here briefly since it is apparent— ly not well known in other countries. The test is reportedly applicable over a long period of pregnancy (383). The technique involves spreading a sample of mucin from the cervical os onto a glass slide. The mucous smear is fixed in alcohol, dried, and stained with methylene blue or hematoxylin. Stains from pregnant mares reportedly are thick and dark and contain globules of mucus and epithelial cells when viewed under a microscope. The smear from nonpregnant mares is thin and pale and does not contain globules of mucus. Nishikawa (1153) gives a detailed account of the characteristics of cervical- vaginal mucus during various reproduc- tive states; he notes that the Kurosawa method must be used with caution during the anestrous season. At that time, the smears contain mucous globules, but dif- ferentiation can be made between anestrus and pregnancy by the absence of epithelial cells in the anestrous condition. The reliability of the test in the pseudo- pregnant condition (pg. 228) has not been determined. False positives are likely, however; four mares gave positive tests after death of the conceptus (383). The Japanese have considerable experience with such techniques, and their reports may be examined by those interested in this area (1108, 1153). Estrogen injection test. Another unusual procedure for diagnosis of preg- nancy in mares, the estrogen injection test, has been described (1319, 1495). The technique is based on the principle that the effect of the injected estrogen depends on the functional status of the corpus luteum. For example, 2.5 to 5.0 mg of diethylstilbestrol was used as a test dose in one study (1495). Originally, the test (1319) was based on a report (1153) that an estrogen injection induced estrus in non— pregnant mares without a functional cor- pus luteum (Day 16 and 17 after last ser- vice) while estrus failed to occur when the corpus luteum was present (Days 2 to 12 in nonpregnant and pregnant mares, and Days 16 and 17 in pregnant mares). Early pregnancy factor. A factor (early pregnancy factor) has been detected in the serum shortly after fertilization; its use as an early indicator of pregnancy is being investigated in several species (903, 1179), including mares (246). The use of a pregnancy-specific protein for serological detection of very early pregnancy in the horse has been investigated (938). Samples from 16 mated mares during the first three weeks after mating were com- pared to those of nonmated mares. Eleven mares became pregnant (indicated by ultrasonic scanning at Day 90) and the protein was detected in ten (90%). The protein was first detected on Day 6 and was detectable until the end of the experi- ment. The protein was also detected tran- siently in 2 of 5 mated mares that were not pregnant, but these false positives could have been due to pregnancy loss. The protein was not detected in 14 non- mated mares. This technique could become valuable in studies of early embryonic loss before ultrasonic detectability of a conceptus. Hormonal tests. Several approaches utilizing eCG have been investigated, and simplified commercial kits, including dip- sticks and enzyme immunoassay kits, are available (pg. 72,- 1704, 1814, 1701, 973). The use of various immunologic methods for pregnancy detection through eCG has been described (193, 1525). Immunologic diagnosis by agglutination of latex parti- cles is another innovation (387, 530). Radioreceptor assay is an accurate quan- titative approach (997, 500). Pregnancy diagnosis by radioimmunoassay of estro- gens in the urine, serum, plasma, feces, or milk also has been described (pg. 427,- 1489, 487, 1612, 846, 1109, 1490). For the estro— gen approach, blood serum or plasma appears to be preferred, and reliable detection of pregnancy can be done after TABLE 8.5. Acceptability of Artificial Insemination in the United States in 1990 Not acceptable Suffolk Thoroughbredl< Acceptable but number ofolffspring limited Peruvian Paso (45/yr) Welsh Pony (12/yr) Acceflablgif done at fiplace Qf_collecti0n; usualllimglies use of fresh semen Appaloosa Pony of Americas Arabian Quarter Horse Belgian Draft Horse Standardbred Missouri Fox Tennessee Trotting Horse Walking Horse Paint Horse Acceptable; usually semen may be transported Clydesdale Lipizzan Connemara Pony Morgan Friesian Paso Fino Hackney Saddlebred Hanoverian Frozen semen accepted Clydesdale Peruvian Paso Hackney Saddlebred Paso Fino * Acceptable if accompanied by natural service Maternal Aspects of Pregnancy 333 Day 60 (1489). Assay of progesterone in blood or milk also is being investigated for pregnancy diagnostic purposes (930, 1220, 1409}. Pregnancy diagnosis and contraception in feral mares. In feral horses, pregnancy diagnosis can be done from fecal samples by means of steroid conjugates (891, 998, 892, 890, 1200); no false diagnoses after Day 120 were recorded in one study (1490). The development of contraceptives for feral populations is currently receiving atten- tion. Approaches being tested include delivery of estrogens and progesterone in silastic implants (1278, 1275, 1277), remotely delivered immunocontraception (antibod- ies against porcine zona pellucida; 889, 890, 968, 1456), and microencapsulated delivery of testosterone propionate (1648). 8.10 Equine Biotechnology: Gametes and Embryos 8.10A. Artificial Insemination Artificial insemination will not be dis- cussed in detail. Information on the tech- niques of artificial insemination around the world with raw, extended, or frozen semen in horses are available in the fol- lowing reports: Australia (437), China (1620), England (55), France (1370), Germany (1082), Japan (1154), Netherlands (388), Poland (1620), Sweden (668), and United States (1832, 1692). Information can be found on collecting, handling, storing, and evaluating semen (1832, 401, 868), fac- tors affecting quality and quantity per ejaculate (1620, 1259), practical aspects of cooling and transporting semen (916), fer- tility evaluation of the stallion (1260, 1371), and determining stallionzmare ratios for natural and artificial insemination pro- grams (952). Acceptance of artificial insemination. Results of a 1990 survey of breed reg- istries in the United States in regard to their policies on artificial insemina- tion are shown (Table 8.5). Registries were asked whether they accepted foals 334 Chapter 8 produced by artificial insemination and whether any special rules were used. The listing of breeds in Table 8.5 according to narrowly defined categories is intended to provide an overview of the current situa— tion in one country. Those planning to use artificial insemination should, of course, first consult directly with the appropriate registry; most of the registries have spe— cial rules. Inspection of Table 8.5 suggests that the equine industry in the United States is a long way from reaping the benefits of artificial insemination that have been realized for other farm species. Nine of the 22 surveyed registries accept foals produced by artificial insemination but only if the insemination is done at the place of collection. This precludes the transportation of semen and usually requires the immediate use of raw or extended semen. This approach is used by Standardbred and Quarter Horse farms in the United States (159). A notable exception is the Thoroughbred Registry which continues to allow only natural service, except that a portion of the ejacu- late can be manually inserted in associa— tion with the natural breeding. Although severe restrictions on the use of artificial insemination have continued among the major breeds, there have been notable advances toward acceptance since the survey for the first edition (14 years between the 1976 and 1990 surveys; 575). At least 5 of the 22 surveyed registries now permit the use of frozen semen. One registry (American Saddlebred Horse Association) has permitted the use of arti— ficial insemination since 1946 but consid- erably broadened the rules in 1990; trans- portation of cooled and frozen semen can now be done, although the stallion must be living on January 1 of the year in which the semen is used. The American Morgan Horse Association allowed artifi— cial insemination in 1979 and shipping of semen in 1985 (1318). The registry expects that these rule changes will decrease average stud fees but will increase total revenue of stallion owners and will increase the numbers, geographic distri- bution, and, most importantly, quality of the breed. Raw and cooled semen. Insemination with unprocessed semen results in preg— nancy rates comparable to those of natu- ral service (55, 1082). In this regard, one group of workers (1716) concluded that artificial insemination provides a higher pregnancy rate than natural service, and another group (1198) concluded that artifi— cial insemination with unextended semen resulted in pregnancy rates similar to those obtained with cattle. A manage- ment routine used on some Standardbred and Quarter Horse farms in America and Australia involves the collection of semen from stallions on alternate days (55). The semen may be diluted with extender and used on many mares (e.g., 17 or more; 1257). This procedure permits increased safety to all participants (horses and handlers) and encourages semen evalua- tion. The advantages of transporting and storing semen are, however, lost to these breeds. Cooling of extended semen can prolong fertile life span of sperm (review: 1832). Commercial containers are available for cooling, storing, and ship- ping stallion semen. The Semen reaches a final temperature of 5°C. This approach allows overnight storage or shipping. The cold semen can be inseminated without prior warming. One author noted that the maximum interval from collection to use of raw, extended, and extended-cold semen is 1 hour, 12 hours, and 2 to 3 days, respectively (1620). For informa- tion on semen extenders, consult recent reviews (1832, 868). Frozen semen. Freezing of stallion semen has been successful, although reported results of pregnancy rates have been variable (e.g., 29%, 32%, and 56%; 1832). In a recent report, packaging meth— ods were compared for frozen semen (984); pregnancy rates were 46% and 55% (50% overall, n=46). Embryo-loss rate did not seem to be elevated over the expected. In a study in Czechoslovakia (1124), a preg- nancy rate of 56% was achieved (n=959 mares for an average of 1.4 cycles during 1981 to 1985), and apparently differences were found among stallions. Because of limited numbers and the nature of the horse industry, field evaluation of the merits of this technology to the industry are not available. This contrasts with the computerized population-wide evalua- tions that are constantly generated in the cattle industry. One author (1620) estimat- ed that at least 1,116 mares were insemi— nated with frozen semen in 1987 in Europe and the United States, whereas about 32,000 were done in 1985 in three provinces in China. These differences probably reflect the differing economic set ups that are peculiar to the horse indus- try in the two locations. Sperm from frozen semen have a much reduced life span in the mare’s reproductive tract, but good pregnancy rates can be obtained if insemination is near the time of ovula- tion. Pregnancy rates were highest in mares inseminated with frozen semen within 12 hours of ovulation, whether or not the insemination preceded ovulation (1198). More widespread acceptance of the use of frozen semen by breed registries would likely stimulate research activity in this area. The concern of the breed registries in the United States centers on the impact that widespread artificial insemination with frozen semen might have on the economic structure of the industry—a structure based on a narrow mare—to-stallion ratio. It will be interest- ing to see which direction the industry will take in years to come. 8.1 OB. Assisted Fertilization Assisted equine fertilization tech- niques include the following: 1. Placing an unfertilized oocyte from a donor into the oviduct of a bred recipient (gamete intrafallopian transfer; GIFT); 2. Collecting mature oocytes (sec- ondary oocytes) from preovulatory folli- Maternal Aspects of Pregnancy 335 cles or from the oviduct of a donor, fertil- izing the ovum in vitro, and transferring the resulting embryos into a recipient; and 3. Maturing primary oocytes from small follicles in tissue culture and trans— ferring the unfertilized mature oocyte as in Point 1 or fertilizing the mature ovum in vitro as in Point 2. Research on assisted fertilization is just beginning in mares but has been under study for many years in other farm species and primates. Large-scale in vitro programs have developed in the human field during the last decade (458). The third annual report of the U. S. Registry on in vitro fertilization and embryo trans- fer in humans in 1988 gave the following results (1073): 12% (2,243 pregnancies) live delivery rate after transfer of in vitro fertilized ova and 21% for the GIFT approach. Even when done under the most ideal conditions, such procedures have not been highly successful. Because survival per embryo is low, 3 or 4 embryos may be transplanted per recipi- ent (458); multiple pregnancy then becomes a major problem. Some of the motivations for developing assisted fertilization procedures in mares include the following: 1. To establish pregnancies in mares that would be otherwise infertile; 2. To develop means for rapid multipli- cation of certain genetic lines; 3. To study the biology of oogenesis, fertilization, and early embryo develop- ment; . 4. To provide material for other technologic advances, such as microma- nipulation or genetic engineering; 5. To provide oocytes for in vitro fertility tests for stallions; 6. To develop a tool for research areas (e.g., twinning mechanisms, effects of senescence on oocytes and fertility, embryo-loss mechanisms); and 7. To be the first, in historical perspec- tive, to develop a given technique. This incentive is seldom, if ever, given but may 336 Chapter 8 be the greatest motivator of all since it involves a positive aspect of competitive- ness. Collection of mature oocytes. Follicular oocytes of mares have been collected from large follicles by aspiration after exposing the ovary through a flank incision (1710), passing the needle through the flank after stabilizing the ovary with a hand placed in the rectum (282, 1213) or through a colpotomy (740, 739), or by using an ultra- sonically guided needle through the flank (1212, 887). The oocyte may be matured in vivo by an injection of hCG followed by aspiration in 20 to 36 hours (1212, 887, 1213, 740, 282). A recommended interval from hCG treatment to attempts at oocyte retrieval is 34 hours (206); this is likely to be 10 hours before ovulation in 80% of the mares. Aspiration of preovulatory follicles 36 hours after hCG administration result- ed in an initial decrease in progesterone output by the resulting corpus luteum (282). However, this finding was not con— firmed in a subsequent study (1740); the authors suggested that flushing the folli— cle with large volumes of saline in the previous study may have removed some granulosa cells. It appears that follicular fluid can be aspirated at any time after giving hCG to a mare with a follicle 235 without interfering with luteinization. It has also been noted that 2 of 7 mares that did not yield an oocyte upon aspiration of the preovulatory follicle became pregnant despite removal of the follicular fluid (1212). In 2 mares, follicles aspirated on Day 26 of pregnancy yielded oocytes 36 hours after hCG injection; both were in metaphase II. Collection and culture of immature oocytes. Nuclear maturation (timing and chromosomal features) of in vitro cultured equine oocytes resembles that of other domestic species (888). Primary oocytes have been collected from small follicles of slaughterhouse ovaries by aspiration (403) or by scraping the exposed inner wall of the follicle (395, 1174). In one study (1174), a mean of eight oocytes (89% of follicles) was collected per pair of ovaries by the scraping technique, compared to a mean of three for aspiration (34% of follicles). In no case was more than one oocyte obtained from a follicle. One study used cultured oocytes from follicles >5 mm (403). The following conclusions were made: 1) 62% of the oocytes resume meio- sis, 2) germinal vesicle breakdown can occur within 12 hours, and 8) secondary oocytes can form within 48 hours. In a subsequent study (888), similar results were obtained, except it was concluded that a secondary oocyte (metaphase II) was reached by 24 hours. In a similar study (1711), the germinal vesicle was pre— sent in 70% of the oocytes at the start of culture, but metaphase I and metaphase II (secondary oocyte) formed in 20 to 24 hours and 40 hours, respectively. The time required for formation of a secondary oocyte after the termination of arrest in the primary oocyte is apparently similar in vivo and in vitro (e.g., 36 to 48 hours). The effect of type of culture medium on the in vitro maturation of equine oocytes has been reported (1790). A recent abstract (1789) indicated that maturation of equine oocytes was not enhanced by the addition of LH or FSI-I to the culture medium. However, eCG increased the number of matured oocytes but also increased the number of degenerating oocytes. Gamete intrafallopian transfer. The GIFT procedure has been reported for mares (1067, 1062). The technique involved retrieving the oocyte from the preovulato- ry follicle of hCG-treated mares and transferring the oocyte to the oviduct of a recipient that was inseminated before or just after transfer. Fifteen oocytes were transferred into mated recipients that had their oocyte removed by aspiration of the preovulatory follicle. Ten oocytes were recovered 48 hours later, and three were fertilized. Each fertilized oocyte was then transferred to a recipient; one recipient became pregnant, one aborted, and one did not become pregnant. Thus, 1 of 15 (7%) transferred oocytes led to a viable fetus. In another trial by these workers, 15 oocytes and equine sperm were trans- ferred to rabbit oviducts; 9 oocytes were recovered but none were fertilized. The GIFT approach produced poor results, and unless a mare is extremely valuable, it does not seem to be practical given the current status of the technology. In addition, no mention was made of blood- typing to assure parentage; presumably the pregnancies could have originated from ovulation of an undetected second follicle in the mated recipients. In vitro fertilization. In the past year, several laboratories have reported on var- ious aspects of in vitro fertilization in mares. An important aspect of in vitro fertilization, which will not be reviewed here, involves preparation of sperm, espe- cially to satisfy the needs for capacitation and the acrosome reaction (215, 1692, 1695, 1383, 1851). Techniques in horses for prepa- ration of media, preparation of sperm, the use of hamster zona—free test ova, collec- tion and preparation of ova, and evaluat- ing and fertilizing ova were recently discussed (215). In vitro fertilization of in viva-matured oocytes (collected from preovulatory follicles from hCG-treated mares) had limited success. Structures resembling two pronuclei were noted in one of 26 oocytes, and a condensed sperm head was noted in another oocyte that was in metaphase II. In another study (1850), oocytes were matured in vitro and fertilized in vivo. Ovaries were obtained at a slaughterhouse. The usual recovery rate was 10 to 20 cumulus-oocyte com- plexes per pair of ovaries. In vitro matured oocytes (n=29) were transferred to four bred recipients; the uteri were flushed on Day 8, and seven blastocysts were recovered. In another study (206), oocytes were collected from the oviducts or by aspiration of a preovulatory follicle 34 to 45 hours after hCG treatment. The oocytes were fertilized in vitro and 6 of 84 underwent cleavage. In a more recent report (395), 21 of 143 (15%) oocytes matured in vitro became fertilized Maternal Aspects of Pregnancy 337 in vitro. An oocyte was considered fertil- ized when a decondensing sperm head, one pronucleus with a sperm tail, or two pronuclei were found in the ooplasm. Most (86%) of the fertilizations had two pronuclei. Successful fertilization was obtained from groups of ooctyes that were obtained from follicles that ranged in diameter from 5 to 6, 5 to 9, 5 to 10, 8 to 15, 8 to 18, and 8 to 21 mm, demonstrat- ing that even small follicles (5 to 6 mm) yielded oocytes that were fertilizable. A recent report (1851) has been published on in vitro fertilization of horse oocytes with emphasis on methods of prepara- tion of sperm. Another report (1175) on in vitro fertilization is currently available only in abstract form. Most recently (1207, 1217), one foal was born following transfer of eight in vitro fertilized embryos into the oviducts of eight recipi- ent mares. In vitro culture of equine embryos is discussed in Chapter 9 (pg. 350). In summary, successful assisted fertilization attempts in horses have been reported for in vivo fertilization of transferred ova, in vitro fertilization of in vivo matured ova, in vivo fertilization of in vitro matured ova, and in vitro fer— tilization of in vitro matured ova; suc- cess rates have been low. 8.100. Embryo Transfer Transfer of embryos from mare to mare has been done since 1972 (65). Reports involving this technology mush— roomed in the 1980s. Many reviews and instructions have been published (e.g., 47, 1063, 1401, 1403), and a recent compilation of research reports is available (54). In addition, many short courses and workshops are being conducted. Recent reports relate to collection of older embryos (Days 10 to 16; 1485), viability of embryos (1295), embryo development after intrafollicular oocyte transfer (738), and other technique modifications and improvements (438, 735, 244). The mechan- ics of the technology will not be repeated 338 Chapter 8 here. This discussion will be limited to items that may be of interest to reproduc- tive biologists. Research uses of embryo transfer. Some of the ways the technology has been used as a research tool are as follows: 1. Determining the roles of the oviducts versus the uterus in embryo sur- vival and loss (1255); 2. Studying the pathogenesis of subfer- tility in old versus young mares (147); 3. Studying the nature of endometrial cup formation, production of eCG, and maternal-fetal immunity by the use of transfers between species (e.g., donkey in horse, zebra in donkey; 59); 4. Examining the role of progesterone in pregnancy maintenance (748, 1066); 5. Studying reproductive biology of mules (86); and 6. Studying the effect of uterine capaci- ty on fetal development (928, 1621). Acceptance of embryo transfer by the industry. Most breed registries have developed strict policies in regard to regis— tering foals born by embryo transfer. Some of the restrictions involve advance notification, presence of a registry repre- sentative, copies of transfer logs, blood- typing, and a limit of one foal or one twin set per donor per year. This latter restric- tion denies to the horse industry much of the advantage of embryo transfer (propa- gation of desirable genetic material) that has been enjoyed by the cattle industry for many years. The opposite approach used by the two animal industries center on economic traditions that need not be dis— cussed here. Considering the restrictions and reluctance to accept artificial insemi- nation, it is remarkable that embryo transfer has achieved its current level of acceptance in the United States. Only 6 of 21 registries in Table 8.6 do not permit embryo transfer. At the other extreme, three registries have already accepted the use of frozen embryos. Over half of the registries allow embryo transfer, but with a limit of 1 to 4 foals per donor per year. Applied uses of embryo transfer. Some examples of applied or potential uses of the technology are as follows: 1. Breeding and preserving exotic or threatened species (e.g., Przewalski’s horse; 925, 1579, 496); 2. Obtaining pregnancies from older or subfertile mares; 3. Placing younger mares into produc— tion a year earlier (1542, 812, 1398); 4) Allowing a mare athlete to produce a foal while remaining in competition (1418, 1823); 5. Allowing a late foaling mare to pro- duce an embryo and still remain open for an earlier start the following year; 6. Producing uniform experimental animals; and 7. Serving as an integral part of other forms of biotechnology (e.g., in vitro fertil- ization, cloning, sexing, semen evalua- tion). Identical horse twins are being pro- duced by micromanipulation, and bisecting techniques have been discussed (1061, 63, 1125, 1493, 1787); success rates have been 10% to 20%. ' TABLE 8.6. Acceptability of Embryo Transfer in the United States in 1990 Not acce ted Connemara Pony Standardbred Friesian Suffolk Missouri Fox Trotting Thoroughbred mm Belgian Draft Horse Tennessee Walking Clydesdale Horse Morgan Accep_ted but number ofifoals limited Appaloosa (1/yr) Paint Horse (1/yr) Arabian (1/yr) Peruvian Paso (2/yr) Hackney (2/yr) Quarter Horse (1/yr) Hanoverian (1/yr) Saddlebred (2/yr) Lipizzan (10/1ife time) Welsh Pony (4/yr) Paso Fino (2/yr) Frozen embryos accepted Clydesdale Welsh Pony Lipizzan Some aspects of the technique. As in other species, the initial embryo collec- tion techniques involved cannulation of the oviducts or uterus through a mid- ventral laparotomy (67, 201). These approaches have been replaced by embryo collection through transcervical uterine flushing and embryo insertion transcervi- cally or through a standing flank incision (1063); the techniques were modified from procedures developed for cattle. The proce- dures are simpler in mares than in cattle because: 1) the cervix is more easily pene- trated, 2) the uterine horns are not tapered and coiled as in cattle, 3) the embryonic vesicle is large and more readi- ly located, and 4) synchrony between donor and recipient is less restrictive. In regard to synchrony, records accumulated over several years have indicated that ovu- lation of donors three days before to two days after the recipient (six-day spread) resulted in satisfactory pregnancy rates (1063). The synchrony problem has been further reduced by the use of exogenous hormones in ovarian—intact (1063, 1236, 1280) or ovariectomized recipients (747, 1066, 734, 748, 741). There are disadvantages to the use of ovariectomized recipients which must be weighed against the advantages (see panel discussion in 54). With ovariec- tomized recipients, steroid treatment can begin on the day of donor ovulation or up to a few days after donor ovulation. Most transfers are done between Day 6 and Day 9 (1063). Earlier attempts may precede the arrival of the embryo in the uterus, and later attempts result in a vesicle that is less able to withstand transfer. It would be helpful to develop transfer technology to allow transfer at Day 11 or to develop ultrasound technol- ogy that will consistently allow detection of Day 8 or Day 9 embryonic vesicles. When either of these goals for technologic improvement are met, the operator will be able to determine whether a potential donor has an embryo for transfer without going through the flushing procedure. Maternal Aspects of Pregnancy 339 Multiple embryo collections. A disad- vantage in equine, as apposed to bovine, embryo transfer is that mares superovu— late less effectively (pg. 260,- 1817). In a study in horse mares during the ovula- tory season, the embryo collection rate per ovulation for induced multiple ovula- tors (mean ovulations per mare: 4.6) was similar to that of single ovulators (0.6 and 0.7 embryos per ovulation, respectively; 1818). The embryo collection rate per donor was therefore higher (2.9 versus 0.7). However, the transfer success rate per embryo from induced multiple ovula- tors (11 of 21) was lower than for recipi— ents that received an embryo from single ovulators (7 of 8). Results indicated that some Day 7 embryos from induced multi- ple ovulators were impaired; however, the number of pregnant recipients per donor was increased because of the greater number of embryos available for transfer. Mares that frequently double ovulate spontaneously have an advantage as donors; the embryo recovery rate and recipient pregnancy rate per ovulation was equivalent between single and spon- taneous double ovulators (1063, 1511), con- trasting with the results for induced mul- tiple ovulators. 8.10D. Preservation and Transport of Embryos Usually embryo transfer is done within an hour of collection. Techniques are being developed, however, to preserve embryos so that transfer programs will have more flexibility and long distance shipping and storage will be practical. Culture systems that maintain Viability for at least 12 to 24 hours also are needed for biotechnology procedures (e.g., sexing, bisecting, and in vitro fertilization). Culture systems. Several in vivo and in vitro culture systems have been used for equine embryos, especially to facilitate transportation. Rabbit oviducts were used successfully to store embryos for 40 to 49 340 Chapter 8 hours for transportation from England to Poland (71). Storage in various culture media has been used successfully for preservation for 12 hours (303) and up to 24 hours (cited in 337). Satisfactory results also have been reported for 24- hour storage using a bovine fetal mono- layer system (1778, 1779). Cooling. Containers built for transport of equine semen are being adapted for cooling and transport of embryos (1421). In a recent field trial (337), commercial trans— portation of embryos stored at 5°C result— ed in pregnancy rates that were similar to those for fresh embryos; most of the embryos were stored for less than 12 hours, and the range was 5 to 24 hours. Pregnancy rates at Day 50 were not sig— nificantly different between transported and control embryos (18 of 24 and 17 of 26, respectively). In an earlier study (284) by the same laboratory using the same culture media and cooling system, a preg— nancy rate of 55% (11 of 20) was obtained for embryos stored for 24 hours. It is con- cluded that equine embryos can be suc— cessfully stored at 5°C for 24 hours (286; Late entry, 1859) to 36 hours (1022) and should make commercial transportation feasible. Freezing. Methods are being developed for cryopreservation of equine embryos (1327). The principles are similar for all living cells (1415). Water must be removed before intracellular freezing to prevent formation of large damaging ice crystals. If too much water is removed, however, the results also will be negative. Thousands of calves have been born from frozen embryos. In humans, the survival rate for the embryo—freezing process is about 80% and the number of babies orig— inating from frozen-stored embryos worldwide is probably close to 500 (458). However, results have been poor for equine embryos above 0.25 mm in diame- ter (363, 1494, 1800, 1587, 1517, 1494). Pregnancies were established from 4 of 8 embryos 30.2 mm in diameter and from only 2 of 24 larger embryos. Embryos 250 um were enclosed only in a capsule. Electron microscopy has been used to study the details of the cap- sule (518). It first appeared as a thin uni- form layer between the zona pellucida and trophoblast of Day 6 embryos. By Embryology and Placentation 353 Day 8, the zona pellucida was shed and the capsule was only 1 mm thick. In histo— logically fixed embryos ZDay 11, the cap- sule was 3 um thick and had a homoge— neous finely stippled appearance. Photomicrographs of the capsule are shown (Figure 9.7). In addition, an appar- ent capsule in association with a Day 11 embryonic vesicle is shown in Figure 9.19 (pg. 367). Capsule structure. The capsule consists of glycoprotein molecules arranged in a collagen-like network. It is a tough elastic material that sometimes remains intact after accidental rupture of the blastocyst during collection (1485). Conceptuses col- lected on Day 14 sometimes had an intact capsule with the yolk sac collapsed within it (198). Apparently when the conceptuses reach 18 to 20 mm, the capsule is more firmly attached (cited in 196). Perhaps the capsule contributes to the popping sensation that can be felt during manual elimination of one member of a twin set (583). Reviews or reports on the capsule FIGURE 9.7. Upper left: Collapsed capsule after shrink- age of a blastocyst during tissue cultureflpflgflght: Close-up of the thin capsule surrounding a 2 mm blastocyst (Days 9 to 10). There has been some shrinkage of the blastocyst as indicated by the clear zone between the cap- sule and blastocyst wall. Lower fl: Blastocyst (Days 10 to 11; 5 mm). The capsule is obvious because of blastocyst shrinkage, as indicated by the wide clear space between the blastocyst wall and the thin capsule. Lower pigm: Ruptured yolk-sac vesicle at Days 16 to 17 (intact diame- ter, 16 to 22 mm). The damaged capsule is detached from the vesicle (white lines). Adapted from ( 198). Photographs courtesy of K. Betteridge and R. Bériault. 354 Chapter 9 consider physiochemical and staining properties (196), possible role of enzyme dissolution (399), biochemical composition (1182), and study by magnetic resonance imaging (1168). Capsule origin. The origin of the cap- sule may involve both the trophoblast and endometrium (review: 196). Uterine involvement is indicated by the observa- tion that in vitro cultured precapsular embryos do not form a capsule (1061, 1748). The capsule is difficult to demonstrate in vitro (196), although one group reported its formation in vitro while the conceptus was still a morula (1493). Immunologic studies have also implicated the uterus (234; review: 196). Trophoblastic involve- ment is indicated by the microvillus attachment between the capsule and tro- phoblastic cells (198, 518). Zona-free bi— sected blastocysts transferred at Day 7 had an apparently normal capsule when examined one week later (1061). This result indicated that the zona pellucida was not a necessary structure in capsule formation. Others (1493) have suggested, however, that the zona may act as a mold for the outer surface of the forming cap- sule. Capsule function. The specific functions of the capsule are unknown, but several possibilities have been raised. It may have a protective role in an otherwise adverse environment (e.g., in association with postpartum pregnancies). The fine structure suggests that it would exclude Viruses and bacteria, but like the zona, it would probably admit macromole- cules (518). Other suggestions (1493) are that it may shield the antigens of the con- ceptus from recognition by the maternal immune system or that it may act as a fil— ter in embryo-maternal exchange. Although the capsule is thin (e.g., 3 um), it is quite strong and can be assumed to be an important structure in conceptus development. It is the outer protective wrapping around a comparatively deli- cate package—a package that is subject- ed to considerable pressures by uterine contractions during embryo mobility (pg. 312) and orientation (pg. 305). So much so, that the Day 13 or 14 vesicle may undergo periodic compressions (e.g., every 5 to 14 seconds). During compression the width of the spherical vesicle can become twice as great as its height (584, 587). The resiliency and elasticity of the capsule may allow these natural distortions with- out damage to the yolk-sac wall. In addi- tion, the capsule may provide needed strength for the physical orientation of the conceptus under the massaging action of uterine contractions (580). It is reasonable, therefore, that a fundamental role of the capsule is in embryo-uterine physical interactions, but this remains to be tested. PHOTOGRAPHIC PLATES The series of plates on the following eight pages depict midventral views of the gravid uterus (Figure 9.8), conceptual drawings of the origin of placental mem- branes (Figure 9.9 and 9.10), the embry- onic vesicle and embryo proper (Figures 9.11 and 9.12), the fetal conceptus (Figures 9.13 and 9.14), and videoendo- scopic views of the early fetus (Figure 9.15). These figures will be referenced in subsequent sections. The legends for Figures 9.11 to 9.15 are at the end of the series (pg. 363). FIGURE 9.8. Facing page. Midventral views of reproductive tract on Days 3 to 50 (A—D). Mares were placed in dorsal recumbency with caudal ele- vation, a midventral laparotomy was done, and intestines and urinary bladder were retracted (575). Photographs were taken immediately after exposure of the reproductive organs to avoid post- exposure and handling changes. Note the tubular form of the uterus that developed by Day 16 (com- pare Days 3 and 16). The vesicular bulge (Days 30 and 50) is in the caudal portion of a horn, and the bulge is directed ventrally. The nongravid por- tions of the uterus continue to maintain turgidi- ty. Note large ovary at Day 50 (arrows). The exposed conceptus (dorsal excision of uterus) is shown on Days 14 and 18 (E, F); the horns short- ened after removal. The exposed vesicle was spherical at Day 14 but flattened at Day 18. 355 Embryology and Placentation B. Day 16 Day 50 D . Day 18 F FIGURE 9.8. Days 3 to 50. Legend on facing page. E. Day 14 356 Chapter 9 DAY12 YOLKSAC DAY9 BLASTOCYST l 0 Trophoblas Developing Inner cell mass yolk sac Blastocoele Developing Embryonic dISC mesoderm DAY19 Bilaminar omphalopleure . Sinus terminalis Trilaminar omphalopleure Splanch- _ . .~; nopleure: W EnFOd‘grm Trophoblast / , mgggdglrfn ‘Chorion Somatic mesoderm PExocoelom - rimitive gut Exocoelom Amniotic fold Choggnri . n Embryo ° oamnio Amniotic sac DAY 25 Developing chorionic girdle Developing allantois Somatopleure - of amnion Hindgut Chorion Allantochorion FIGURE 9.9. Depiction of germ layer origin of the placental membranes for Days 9 to 25. Embryology and Placentation 357 Girdle cells invading endometrium Uterine gland Microcotyledon Opening of gland into uterine lumen Allantochorion 3"“ fl ‘9‘ g: r" Nongravid horn 3 "-"' ECTODERM "H. ENDODERM 0‘0 MESODERM - YOLK SAC ‘ ALLANTOIC SAC 21-21-35 UTERUS » acfi’ . 1:52.012?“ GraVId ‘13:? ("b horn 3.; -’-‘- FIGURE 9.10. Continuation for Days 30 to 80. Close-up ofmicroplacentomes is for Day 150. Chapter 9 358 Day 24 D E. Day 30 F. Day 30 FIGURE 9.11. Days 21 to 30. Legend on page 363. 359 Embryology and Placentation chorionic girdle embryo sinus terminalis genital tubercle tail limb Vitelline artery pontine flexure VV = Vitelline vein allantoic sac b0 = bilaminar omphalopleure yolk sac s l f st t va 5 e gt 2. l 9 d n a 1 l 9 S e r m F r pm. m cg p a )7 FIGURE 9.12. Day 36. Legend on page 363. 360 Chapter 9 E. Day 5 FIGURE 9 13. Days 40 to 60. L gend on page 363. 361 Embryology and Placentation B. Day 60 Day 100 14. 13 and 9. o 9 1gures KEY for F 1C sac allanto as e r how 1 up 00 Pa .ch mp .mm «m0 hm en mi nm 33 RH ab :2 P0 C ab ZuEC ial cups endometr fetus yolk sac E. Day 270 FIGURE 9.14. Days 60 to 270. Legend on page 363. Chapter 9 362 FIGURE 9.15. Day 69. Legend on facing page. FIGURE 9.11. Days 21 to 30. A. Day 21. Spherical conceptus (26 mm; oblique View) showing the yolk-sac vasculature. B. Day 24. Yolk—sac vesicle as Viewed from the embryonic pole. C. Day 24. Embryo proper immediately after removal from the uterus (length: 6 mm). D. Day 24. Embryo proper and allantois. The yolk sac and amnion were removed. The belt (arrow) around circumference of the vas- cularized allantois indicates the area of fold- ing to form a cup around the embryo proper. E. Day 30. Embryonic vesicle. The nonvascu- lar bilaminar omphalopleure has become a rel- atively small area. The allantois is more highly vascularized than the yolk sac. The avascular- ized chorion between allantois and yolk sac is the location of the chorionic girdle (arrow). F. Day 30. Embryo proper. The triangular- shaped pontine flexure is prominent and is useful for estimating age. FIGURE 9.12. Day 36. Single specimen. A. The partially submerged embryonic vesi- cle is spherical and flattened (diameter, 80 mm). The allantoic sac is dominant but the the yolk sac still contained circulating blood. The embryo has been forced closer to the abembryonic pole. The chorionic girdle is a distinct band (width: 9 mm). The missing portions of the girdle indicate attachment to the endometrium had begun. B. Opposite side of vesicle showing the bilaminar omphalopleure and the sinus termi- nalis. C. Embryo proper (15 mm) and amnion before removal of placental membranes . D. Embryo proper after removal of amnion. The pontine flexure is almost closed (compare to Figure 9.11F). E. Left limb was removed to expose the prominent genital tubercle (forerunner of penis and clitoris). FIGURE 9.13. Days 40 to 60. A. Day 40. The uterus was excised and allantoic fluid aspirated. The exposed allanto- chorionic membrane. is shown clinging to the endometrium. L B. Day 44. Videoendoscopic View from with- in the allantoic sac showing the in situ rela- tionships (compare with A). C. Day 44. Videoendoscopic View of in situ endometrial cups. D. Day 50. Before the uterine wall was Embryology and Placentation 363 reflected, the bilaminar omphalopleure was located near the mesometrial attachment in the center of the horseshoe-shaped ring of endometrial cups. E. Day 50. Conceptus after evacuating and opening the allantois to expose the amnion, fetus, and yolk sac. F. Day 60. Uterus was evacuated and evert— ed to expose the horseshoe-shaped band of cups at the caudal portion of one uterine horn. Note the variations in shape. Same specimen as in E. FIGURE 9.14. Days 60 to 270. A. Day 60. The yolk-sac remnant extends for approximately 5 cm along the length of the umbilical cord. The amniotic cavity encloses the cord for a considerable distance. Fetal head is no longer between front limbs. B. Day 60. Videoendoscopic View from with- in the allantoic sac showing the in situ attach- ment of the umbilical cord to the inner surface of the allantochorion. C. Day 80. The allantochorion was severed and drained, and the membranes were left attached to the area of the endometrial cups. The umbilical cord is attached within the ring of cups. A honeylike material is present in the depression of the cups. D. Day 100. Outline of amnion is delineated by arrows. Note the amniotic and the allanto- chorionic portion of the umbilical cord and the tortuous vessels in the amnion. E. Day 270. Inner surface of allantochorion. Note the pedunculated allantochorionic pouch (below) and the nonpedunculated pouch (above). The pouches contain remnants of the endometrial cups. FIGURE 9.15. Day 69. Time series of Videoendoscopic images of a fetus, encompass- ing approximately four minutes. From (601 ). A. The fetal—amniotic unit being forcibly expelled from the right uterine horn. B. Front View of fetus lying completely in the uterine body. C.-F. Entry of fetal-amniotic unit into the par- tially closed left uterine horn by means of vig- orous limb and whole body movements. In the last View, only the hind limbs and hooves (arrow) are visible. 364 Chapter 9 9.6. The Yolk-Sac Stage (Days 11 to 21) The external appearance of the turgid Day 16 gravid uterus and exposed embry- onic vesicles for Days 14 and 18 are shown (Figure 9.8). The relative changes in size of the embryonic vesicle in the yolk-sac stage can be appreciated by the ultrasono- grams for Days 10 to 21 (Figure 9.16). Before describing the intrauterine devel— opment of the conceptus, the concept of germ layers will be introduced. The origin of the layers of the placental membranes (ectoderm, endoderm, and mesoderm), is traced diagrammatically (Figures 9.9 and 9.10). The beauty of the methodical pro- gression from blastocyst to the various placental membranes can be appreciated from the diagrams. For clarity, an open space is shown between the tissues that originated from the three germ layers; however, there is contact and sometimes fusion between the layers. Endodermal encirclement. Encircle- ment of the blastocoele by an inner lining of endodermal cells completes the con- version of a blastocyst to a bilaminar FIGURE 9.16. Ultrasonograms of the embryonic vesicle over Days 10 to 45. On Days 10 to 16 the vesicle is spherical and then becomes irregular as shown for Days 18 to 24. All of the placental fluid (black) on Days 10 to 18 is yolk-sac fluid. The embryo proper is visible at Day 21 as a white dot on the lower hemisphere of the vesicle. The allantoic sac is clearly visible as a separate fluid pocket beneath the embryo proper on Day 24. The allantoic sac becomes relatively larger until the membrane separating the two sacs converge dorsally to form the umbilical cord. The cord is distinct at Day 40 (end of embryo stage). The cord gradually elongates, and the fetus reaches the ventral aspect of the allantoic sac by Day 48. Each image encompasses approxi- mately a 50 X 50 mm section. From (590). (two-walled) yolk sac (Figure 9.9). Some continue to use the term blastocyst even after encirclement by endoderm; here, the term yolk sac will be used. The day of completion of encirclement by endoderm has not been determined, indicating the need for systematic histologic studies of the early conceptus. For this text, the day of encirclement will be considered arbi- trarily as Day 11 for the sake of didactic partitioning of the conceptus into develop- mental periods. For completeness, some of what follows will be based on studies in other species as given in embryology text- books, but the need for specific study in the equine species is emphasized. The yolk-sac lumen is directly continuous with the primitive—gut lumen (forerunner of digestive system of the embryo) and, therefore, whatever the yolk sac absorbs from the uterus becomes available to the embryo proper. The cells of the internal or endodermal lining of the yolk sac are cuboidal, whereas the cells of the trophoblast are columnar with the char- acteristics of absorptive cells. Mesodermal invasion. A third layer, the mesoderm, begins to invade between the trophoblast (ectoderm) and endoderm of the yolk-sac wall from the embryonic disc (developing embryo proper; Figures 9.9 and 9.17). The time of initial mesoderm invasion and its progression has not been established but probably begins by Day 14. Sequential histologic studies are needed to clarify the rate and nature of mesodermal penetration and develop- ment in equine embryonic vesicles. The mesoderm forms blood islands which enlarge and coalesce to form a continuous network in the yolk-sac wall. This net— work connects with similar channels in the embryo proper. Thus a vitelline- embryo circulatory system is established, complete with an increasingly powerful heart. The yolk sac does not contain stored food material as in birds, but with ‘ vascularization, it becomes an efficient organ for purveying nutritive material to the rapidly developing embryo. The lead- Embryology and Placentation 365 Figure 9.17. Dorsal View of embryonic disc 14 days after end of estrus. ing edge of vascularized mesoderm is demarcated by a prominent collecting vein called the sinus terminalis (Figures 9.10 and 9.11,A). The yolk-sac wall between the sinus terminalis and the embryo proper is three-layered (ectoderm, mesoderm, and endoderm) and is there- fore called the trilaminar omphalopleure. The remaining distal wall is two—layered (ectoderm and endoderm) and is called the bilaminar omphalopleure. The bilami- nar omphalopleure with its distinct periphery (sinus terminalis) is an impor- tant structure for anatomists because it is grossly recognizable throughout gesta- tion; it marks the pole opposite to the developing embryo proper (Figures 9.10 and 9.11,A,E) and later marks the site of umbilical attachment of the fetus (Figures 9.10 and 9.13,D). Histologic appearance of the three-layered yolk—sac 366 Chapter 9 wall is shown and described in Figure 9.18. Some aspects of the ultrastructure of the yolk-sac embryonic vesicle have been reported (518). The trophoblastic cells were columnar and later became tall columnar. The increasing absorptive function of the trilaminar omphalopleure is suggested by the changes in the tro- phoblastic cells, which are in contact with uterine milk, and by development of the yolk—sac vascular system. Shape changes. The in situ equine embryonic vesicle maintains a distinct spherical form until the cessation of embryo mobility (Day 16; Figures 9.16 and 9.19). Before fixation, the vesicle is turgid, as indicated by its ability to main- tain a spherical shape even when removed from the uterine lumen (Figure 9.8). After fixation, the vesicle becomes irregular with a tendency toward an in situ guitar-pick shape when viewed in a cross-sectional plane of the uterine horn (Figures 9.16). When removed, the vesicle is flat and no longer has a turgid spheri- cal form (Figure 9.8). The shape changes are believed to be associated with rota— tion of the embryonic vesicle so that the embryo proper becomes located ventrally, opposite to the mesometrial attachment (pg. 312). The change in shape can be accounted for by the accommodation of the expanding vesicle to the turgid uter- ine horn. The vesicle does not increase in cross—sectional diameter over Days 16 to 26, apparently because the expanding vesicle during this time begins to elon- gate slightly in response to the continued FIGURE 9.18. Histologic sections from a Day 18 conceptus. A. Cross section through the embryo and adjacent membranes, showing the continuity of the mesoderm of the somites (s) with the mesoderm that surrounds the exocoelom (ex). The somatopleure is ventral to the exocoelom and the splanchnopleure of the yolk sac is dorsal. B—C. The three layers of the trilaminar omphalopleure are shown from bottom to top: trophoblast, meso- derm, and endoderm. Note the islets of developing blood cells in the mesoderm (B) and the early blood cells in the lumen of vessels (C). The cells of the internal or endodermal lining of the yolk sac are cuboidal, whereas the cells of the trophoblast are columnar with the characteristics of absorptive cells. FIGURE 9.19. Day 11 embryonic vesicle. Note the spherical shape and the embryonic disc (four o’clock). The halo-like appearance on the right is likely due to the capsule; the vesicle has apparently contracted away from the capsule on the right side but remains attached on the other side. The rough surface of the vesicle is likely artifactual due to shrinkage. uterine resistance to cross—sectional expansion (590). Morphogenesis of embryonic disc. There apparently have been no systematic stud- ies in equids of the development of the inner cell mass into the embryonic disc or embryo proper over Days 11 to 21. As noted above, Rauber’s layer (trophoblast covering the inner cell mass) is gone by the early yolk—sac stage (by Day 12). Based on histologic examination of a few embryos 18 days after the end of estrus (575), the characteristics of the developing equine embryo proper are similar to those of other species. The horse apparently presents no surprises in this regard and conforms to the principle that an animal in its individual development passes through a series of constructive stages like those in the evolutionary develop- ment of its species (1248). A dorsal View of Embryology and Placentation 367 an embryonic disc with a zone of meso- derm invading the yolk-sac wall is shown (Figure 9.17). Morphogenesis of the embryonic disc and embryo proper (Days 11 to 40) is a good available research area. 9.7. Transition From Yolk Sac to Allantoic Sac (Days 21 to 40) Placental membranes. The transition between a yolk sac and predominantly allantoic sac is shown diagrammatically (Figures 9.9 and 9.10), by ultrasonic images (Figure 9.16), and by specimens (Figures 9.11 and 9.12). The allantois, which will eventually assume the entire role for physiologic exchange, emerges from the hind gut at approximately Day 21. The allantois moves into a fluid- filled cavity known as the exocoelom (Figure 9.9). The exocoelom is located between outer somatopleure (body wall) and inner splanchnopleure (Visceral wall). The walls consist of mesoderm and a layer of trophoblast (somatopleure) or mesoderm and a layer of endoderm (splanchnopleure). By Days 24 or 25, the allantoic sac is already vascularized and quite large compared to the embryo prop- er (Figure 9.11,B,D). The pulsating embryonic heart and the passage of blood in the circulatory system were readily observed in a newly obtained Day 24 specimen kept submerged at body tem- perature (Figure 9.11,C). Heart pulsa- tions continued for several hours after removal of the embryo proper. The dia- grams (Figure 9.9) and photographs (Figure 9.11,B,D) show that the allantois becomes prominent and forms a cup under the amnion and embryo proper. The union of the allantois and chorion (somatopleure) results in an allantochori- onic placenta. At this stage, the chorionic girdle, an important band of cells approx- imately 1 mm wide, forms around the conceptus. The area of chorion between allantoic sac and yolk sac is the location of the chorionic girdle; the mesoderm in 368 Chapter 9 this area remains avascular as shown in the Day 30 specimen (Figure 9.11,E). The figures depict the manner in which the allantois continues to grow, and the yolk sac gradually encompasses a smaller and smaller proportion of the embryonic vesicle (Figures 9.9, 9.10, 9.11, and 9.16). The embryo proper is carried by the growing allantois toward the opposite pole. By Day 40 (end of embryo stage), the embryo prop- er and its amnion have moved to the oppo- site pole, and apparent functional replace- ment of the yolk-sac placenta is nearly complete (Figures 9.10 and 9.13,A). Because of growth of the allantoic sac, the membranes separating yolk sac and allan- toic sac meet at the dorsal pole of the vesi- cle, resulting in formation of the umbilical cord; for this reason, the cord attaches at the dorsal aspect of the uterus. The cells of the chorionic girdle invade the endometri— um to form the endometrial cups (pg. 370); only a single layer of trophoblast and a few small tags of trophoblastic cells remain (Figure 9.12,A; 1668). Gross anatomy of the embryo proper. A detailed study of the morphogenesis of the equine embryo proper is needed. Apparently the only available detailed report was made by French workers (157) more than 25 years ago. A single 12.5 mm embryo proper (approximate age: one month) was used. Other reports provide only a few notes or lines of description of the embryo for different ages (187, 430, 1015, 1668). The value of some of these reports is diminished by the unavailabili- ty of critical information on the day of ovulation. Dating has involved day of ovulation based on palpation (1668, 1015), day of breeding (187), and last day of estrus (430). The gross changes in the embryo proper that are visible externally have been sum- marized in Table 9.2, and photographs of a few stages are shown in Figures 9.11 and 9.12. The pontine flexure is a translu- cent area that forms an equilateral trian- gle in the cephalic region (Figures 9.11,F and 9.12,D). The flexure is reportedly a TABLE 9.2. Characteristics of the Equine Embryo Proper Day of pregnancy Characteristics 9 Distinct inner cell mass 14 Pear-shaped embryonic disc with a distinct primitive streak (Figure 9.17) 16 Cylindrical with well-developed neural groove; 14 pairs of somites 18 Beginning of C-shape and closing of neural folds; 16 pairs of somites 20 Sharp C-shape 23 Beginning of limb buds; recognizable internal organs; heartbeat (Figure 9.11,C) 26 Pontine flexure appears (equilateral triangle when viewed laterally) and is 3 mm at widest point with 45° angle; eye visible as a 0.5 mm macule; tail 6 mm long 28 Pontine flexure is 2 mm with 30° angle; first pharyngeal pouch 30 Pigmented eye cups; 4 paddle—shaped limb buds recognizable as legs; pontine flexure is 1.5 mm with 200 angle; first evidence of rudimentary ears; heart, liver, and cartilaginous centers visible against bright light (Figure 9.11,F) 34 Rudiments of 2nd and 4th digits; prominent genital tubercle; pontine flexure closed to a slit 36 Pontine flexure nearly closed (Figure 9.12,C) 38 Well developed tail and hind limbs; tapering feet indicating a soliped 40 (Refer to Table 9.4 on page 394 for fetal characteristics) useful aging criterion because of consis- tent appearance at Day 26, near disap- pearance at Day 36 (Figure 9.12,C), and the intervening, methodic, and easily quantitated closure (187). As indicated in the table, the angle of closure and width at the base of the triangle can be used for aging. The remainder of the table should be self-explanatory, especially if used in conjunction with the indicated figures. The equine embryo proper, studied at approximately one month (12.5 mm in length), had a gentle spiral shape with the head twisted to the left and the tail to the right (Figure 9.20). The region caudal to the umbilicus was short and strongly curved in a ventral direction. The limbs were distinct and paddle-shaped. The thoracic limb buds were about 2.8 mm Frontlimb Eye Pancreas Duodenum Vitelline canal ,_'..-\,»_:: 4’-' . \ r ' , Allantoic canal h}, Hindlimb ,- 1.;.;:' Tail . - — / Genital turbercule Cloaca Post. intestine FIGURE 9.20. Drawing by Barone and LaPlace ( I 5 7) of the reproductive organs in an equine embryo at 30 days. Mesonephros Embryology and Placentation 369 long and the pelvic limb buds about 3.2 mm long. Rudimentary ears, eyes, nose, and mouth were discernible. The origins of the Vitelline and allantoic ducts from the midgut and hindgut (Cloaca), respectively, are shown in Figure 9.20. The cloacal area is of special importance because of its eventual differentiation into urogenital and anal structures. Some of the features of the developing reproductive system (e.g., genital tuber- cle, Wolffian duct, gonads) are also shown. A drawing of the embryonic car- diovascular system, including the origin of the vessels in the umbilical cord, also appears in the original report (157) and in the first edition (575). Embryonic reproductive organs. Exten— sive descriptions of the embryologic devel- opment of female and male reproductive organs in mammals can be found in general reproductive physiology books. Specif- ic information for the equine species is, as expected, deficient. The earliest descriptions of the development of the equine reproductive organs are for 30 days after breeding (187) or approximately one month (157). As noted in the next section, gender is not determinable on the basis of gross external examination until the fetal stage (>40 days). The only external struc- ture in the genital region at 30 days is a 1 mm gen- ital tubercle located between the hind limbs. It is very prominent by Day 36 (Figure 9.12,E). It has been stated (187), however, that morpho- Esophagus Stomach 370 Chapter 9 logic sex may be determined, histological- ly, at 30 days on the basis of a second set of cortical cords on the periphery of the female, but not the male, gonads. Others (157) could not find any trace of Mullerian ducts (paramesonephric ducts or forerun- ners of female tubular genitalia) at this time. The Wolffian ducts (mesonephric ducts or forerunners of male tubular geni- talia), however, were evident on the ven- tral surface of the mesonephros (embry- onic kidney) just beneath the coelomic epithelium (Figure 9.20). They extended ventrally and emptied into the central area of the cloaca. Each mesonephros was 5.5 x 1.4 mm and consisted of tortuous epithelial tubes emptying into the Wolffian ducts. The gonads at this time were in the form of germinal ridges on the surface of the mesonephroi covered by coelomic epithelium. The ridges were dense cords about 2.8 mm long and con- tained many small dark-staining cells and large scattered cells with a clear cyto- plasm (presumably the primary gonocytes or their descendents). A description of the gonads at Day 36 is given elsewhere (pg. 401). 9.8. Endometrial Cups Of the many interesting ramifications and unique aspects of mare reproductive biology, one that has attracted much attention is the development, function, maintenance, and ultimate sloughing of the endometrial cups. The cups are dis— crete raised areas a few millimeters to several centimeters in diameter or length and are arranged in circular or horseshoe fashion at the caudal portion of the gravid uterine horn. They produce the well-known hormone eCG. According to Steven (1544), the endometrial cups were first described in 1912. A series of studies in the 1930s and 1940s by Cole and co— workers at the University of California elucidated the presence, source, and nature of eCG (pg. 48) and the nature and function of the endometrial cups (review: 1544). Renewed interest in the cups was generated by the finding by Allen and associates (57, 62, 1113) at the University of Cambridge that the trophoblastic cells of the chorionic girdle invade the uterine epithelium to form the prominent, eCG- producing, decidua-like cells of the cups. Hereafter, the decidua-like cells will be termed cup cells according to precedent (57, 81). One should guard against the fre— quently repeated blanket statement that the endometrial cups are of fetal origin. Only the decidua-like cup cells stem from fetal trophoblastic cells. Other essential components of the cups are of maternal origin, including blood vessels, connective tissue, and uterine glands. 9.8A. Origin of the Endometrial Cups The morphogenesis of the endometrial cups is depicted in Figure 9.21. Based on the English studies (57), the early histoge- nesis of the cup cells can be divided into five phases: attachment, invasion, phago- cytosis, migration, and differentiation. Attachment. Before attachment, areas of the endometrial epithelium become gly— cosylated (1771, 1773). This local reactivity, involving saccharides, may have a role in permitting attachment of the chorionic girdle and also may have an effect on local immuno-responses in the uterus. The girdle initially (e.g., Day 25) consists of shallow folds. The folding process increases, and elongation into villous structures occurs by Day 33. The intervil- lous spaces are filled with an extracellu- lar material that may serve as an adhe- sive between the fetal and uterine surfaces. The apices of the girdle cells are microvillous and many form pseudopodia. As observed at 37 days, attachment con- sists of an interdigitation of the surfaces of the elongated chorionic cells with corre— sponding indentations in the endometrial epithelium. Firm attachment between the embryonic vesicle and the endometrium at this early stage involves only the chori- onic girdle portion of the vesicle. Attachment and invasion of chorionic girdle cells Days 50-60 Mature cup l >Days 70-80 Sloughed cup free in uterine lumen Invasion. The attachment of the girdle to the endometrium is of short duration and soon progresses to the invasive phase by approximately Day 38. Pseudopodia form on the apical surface of the tro- phoblastic cells and broadly appose the plasma membrane of the epithelial cells. Immediately thereafter, the pseudopodia invade the endometrium by entering the cytoplasm of the epithelial cells rather than entering through the intercellular spaces. Late entry. It has been concluded that only a minority of the several million girdle cells are transformed into cup cells (1861); girdle cells that do not invade undergo necrosis. Phagocflosis. As the girdle cells contin- ue to invade, they sequester and engulf the disrupted epithelial cells. Migration. The migratory phase involves penetration of the basement membrane of the uterine epithelium and passage of the trophoblastic cells and their engulfed epithelial contents into the Embryology and Placentation 371 Days 40-50 FIGURE 9.21. Diagrammatic pre- sentation of the morphogenesis of the endometrial cups and their eventual sloughing and conver— sion, in some cases, into allanto- chorionic pouches. -:l IIIJIl] _ Trophoblast Chorionic girdle Endometrial epithelium Endometrium Endometrial cup Uterine milk Sloughed cup enclosed in allantoic pouch endometrial stroma between the glands. The pseudopodia move through, followed by the remainder of the cell. The tro- phoblastic cells migrate down the length of the uterine glands but do not damage or phagocytize the glandular epithelium. Differentiation. The invasion ceases after infiltration of the endometrial stro— ma, and the trophoblastic cells hypertro- phy and differentiate into mature cup cells. The invasive, migratory, and phagocytic properties of the trophoblastic girdle cells are in close accord With girdle cell mor- phology (1113). They contain a large nucle- us and nucleoli. Their migratory ability is compatible with the amoeboid appearance and a layer of fine filaments in the cyto- plasm. The extracellular matrix may act not only as an adhesive during the attachment phase, but it may play a role in the digestion of epithelial cell debris. After differentiation, most of the unique features of the girdle cells are lost. 372 Chapter 9 9.83. Morphogenesis The endometrial cups become grossly visible as pale slightly swollen areas of uterine folds, beginning at about Day 40. They reach maturity at Days 50 to 60 (Figures 9.13,C,D,F and 9.21). The cups slough (separation of degenerated tissue from living tissue) from the uterine wall between Days 70 to 100. After sloughing, the remnants of the cups may be enclosed by invaginations of the allantochorion, termed allantochorionic pouches (Figures 9.14,D,E and 9.21). A gross description of developing, mature, and sloughing cups and their enclosure by allantochorionic pouches has been published, and the development of knowledge in this area has been reviewed (311). Shape and arrangement. The mature cups are raised above the surface of the endometrium and possess, unlike the immature forms, a depressed surface. The round shape, moderately pedunculated base, and depressed surface account for the name cups. The majority of the struc- tures, however, are irregular in shape and many of them are oblong or band—like (Figure 9.13,F). The cups are arranged in a ringlike or horseshoe fashion in the cau- dal portion of the gravid uterine horn. A portion of the ring may extend a short dis— tance into the uterine body. Because the ring of cups originated from the chorionic girdle of the conceptus, the orientation of the ring provides an indication of the ori— entation that the conceptus had within the uterine lumen at approximately Day 36. In all of eight specimens (approxi- mately Days 50 to 300), the umbilical cord reached the uterine wall at the dorsal sur- face of the uterine horn near the mesome- trial attachment; that is, the bilaminar omphalopleure was at the dorsal aspect of the horn within the ring of cups (Figures 9.13,D and 9.14,C; 575). The cups formed a longitudinal ring (as opposed to a trans- verse ring) along the lateral and medial walls of the horn. The origin of the cups from the chorionic girdle explains their irregular shape, their circular arrange- ment, and their location at the caudal por- tion of the gravid horn. The irregular shape is probably due to a lack of girdle contact between endometrial folds. In pony mares (430) and in horse mares (311), the total weight of all cups was approxi- mately 10 g. Sloughing and formation of pouches. Sloughing of the endometrial cups occurs between Days 70 and 100 and is complet- ed at about Day 130 (311). Prior to slough- ing, a material of honeylike consistency and color accumulates between the cup and overlying allantochorion (Figure 9.14,C). The material consists of necrotic debris and the secretion of the uterine glands and endometrial cups. At the time of sloughing, the cups are pale, opaque, and less turgid. The entire cup is slowly detached, and the degenerating cup either lies free between the endometri- um and allantochorion or becomes enclosed in a fold of allantochorionic tis- sue (allantochorionic pouch; Figures 9.14,D,E and 9.21). Allantochorionic pouches were formed in about 60% of the series described in Table 9.3. The pouches are filled with endometrial cup secretion and the sloughed cup. The pouches range from 0.5 to 10 cm. They dangle in the allantoic cav— ity and are first seen at about Day 75. The neck is apparently constricted in some pouches so that the contents likely cannot escape into the uterine lumen but instead are probably retained or absorbed into the fetal circulation. In later stages of preg- nancy, the pouches are muddy-gray and cheesy, in contrast to the amber-colored, sticky material of newly-formed pouches. According to some workers (311), most of the pouches are gone by approximately Day 130, but pouches have been observed as late as Day 200, after Day 300 (575), and in the discharged placenta at term (1770). Pouches in the discharged placenta were up to 2 cm long, and some were patent—that is, the pouches had commu- nication with the endometrial surface. Embryology and Placentation 373 TABLE 9.3. Development and Fate of the Endometrial Cups and Allantochorionic Pouches Endometrial cups (No.) Allantochorionic pouches (No.) Crown rump Approx. Devel— Fully Slough— length (cm) days Total oping developed ing Sloughed Present Absent 2.0 to 2.9 36 to 43 4 4 0 0 0 0 4 3.0 to 5.9 44 to 57 11 3 8 0 0 0 11 6.0 to 10.9 58 to 74 23 0 19 2 2 0 23 11.0 to 14.9 75 to 86 12 0 5 4 3 2 10 15.0 to 20.9 87 to 105 14 0 6 2 6 5 9 21.0 to 29.9 106 to 133 32 0 1 4 27 25 7 30.0 to 39.9 134 to 156 9 0 0 1 8 3 6 40.0 to 81.3 >157 4 0 0 0 4 2 2 Adapted from ( 311 ). Days of pregnancy estimated from crown-rump length (187). 9.8C. Histology of Cups and Pouches Several descriptions have been pub- lished of the histology (80, 81, 311) and fine structure (674, 724, 1113, 1830) of mature endometrial cups. The histologic aspects of the sequence of events from formation through sloughing of cups, formation of the allantochorionic pouch- es, and restoration of the uterine epithe- lium were reported and illustrated in the first edition (575); the photomicro— graphs are repeated here only for mature cups. Day 40. The lendometrial cups are immature and do not have depressed surfaces. Depth of penetration of cup cells is markedby the basal portion of the moderately dilated endometrial glands and a band of lymphocytes. The band of lymphocytes is peculiar to the base of the cups and is not found in other areas of‘the endometrium. Some lymphocytes follow the course of the uterine glands through the decidual mass. Uterine milk from the glands is discharged into the uterine lumen. The uterine epithelium is largely intact and principally lOW columnar to cuboidal. The cup cells are large, pale-staining, vacuolated, and rounded or irregular in outline. Some of them are binucleate. The nuclei are ovoid and vesicular and contain large, prominent, and dense nucleoli. The characteristic euchromatic nucleus and large nucleoli suggest that the cells actively synthesize proteins (674). The cup cells, therefore, closely resemble decidual cells as seen during pregnancy in some other species. They differ in that they apparently do not contain large amounts of glycogen (80). They do, however, contain much lipid material (1830). The lymphocytes are pri- marily small. Polymorphonuclear leuco— cytes (PMNs), plasma cells, and eosinophils usually are not observed at this time. Day 60. A histologic overview and close views of a mature cup are shown (Figure 9.22), and a word description is given in the legend. 374 Chapter 9 Embryology and Placentation 375 Figure 9.22. Day 60. Photomicrographs of mature endometrial cup. A. Overview. The allantochorion encloses a pocket of uterine milk (um) at the depressed center of the cup. The trophoblast is attached just beyond the periphery of the cup. The uterine glands have further dilated and filled, and the cells contain secretory granules simi- lar to protein secretory granules of endocrine tissues (724). The secretion from the uterine glands accumulates in the crater in the center of the surface of the cup between the cup and the overlying, nonattached trophoblastic layer or epithelium of the allantochorion. B. Allantochorion overlying an endometrial cup. The overlying trophoblastic membrane is avillous. The uterine milk (bottom) is acellu- lar. The trophoblastic cells have pronounced absorptive properties and are bathed by the uterine milk. Circular area at the top of sec- tion is a blood vessel. The mesodermal layer is highly vascularized, presumably to carry away phagocytized and absorbed material. C. Lymphatic area at the base of cup. The lymphatic vessels (IV) are highly developed (80), and therefore some of the internal secre- tion of the cups (eCG) may reach the general circulation through lymphatics. D. Cup cells in the area of the lymphocytic band showing intracellular lymphocytes (arrows). The clear area around the lympho- cytes suggests beginning cytolysis and is the first evidence of maternal cell-mediated destruction of cup cells. At this stage, cell destruction is apparently cytolytic rather than phagocytic. E. Degeneration (dark cells) of scattered cells is already occurring even though the cup is just reaching maximum development. The cytoplasm is eosinophilic and vacuolated and the nucleus is pycnotic (dark and condensed). The following observations indicate that the life of the cups is terminated by a mater- nal immunologic defense mechanism (44, 62): 1) The pronounced accumulation of lympho- cytes likely represents a maternal immuno- logic response to the foreign fetal antigens of the cup cells; 2) The maternal lymphocytic response is especially marked when the mare is carrying a hybrid fetus; and 3) Cultured girdle cells function longer than in situ cells. 376 Chapter 9 Day 80. The uterine glands are active and uterine milk fills the deep depression between the cup and overlying allanto- chorion. The interdigitation between allantochorion and the sides of the cup is more developed by this time. The cup cell area is vacuolated with a prominent lym— phocytic band at the base of the cup. Degeneration and vacuolation are pro- nounced by this time. Large vacuoles appear within the cup cells. Large lipid droplets have been demonstrated (80) with special staining techniques, and it was concluded that the droplets may become confluent and displace the cyto- plasm and nucleus. It is not clear whether the appearance of the large vac- uoles is attributable to a physiologic or degenerative process. It is noteworthy that certain viable-appearing cells con- tain large droplets. In addition to the vacuolated cells, many obviously degen- erating cells (eosinophilic or dark stain— ing) are present. Day 100. The cup may still be attached to the uterus, but sloughing may be imminent. A sloughed cup may be enclosed by allantochorion, except at its base. The overlying trophoblastic layer is hypertrophied, due apparently to phago— cytotic action, and the allantochorion is invaginated due to accumulating cup debris; pronounced invagination signals the formation of an allantochorionic pouch. The glands in the underlying area appear functional, whereas the cup area is degenerate. Small and medium sized lymphocytes dominate the underlying lymphocytic zone and plasma cells increase in number. The jellylike coagu- lum of the sloughing cups may consist of vacuolated and anucleate cup cells and PMNs and lymphocyte nuclear remnants, or cellular breakdown may be extensive approaching an amorphous mass. Endometrium at Days 140 to 160. The cup cells are gone, but the uterine glands remain hyperactive and distended. The uterine epithelium overlying the Viable endometrium is restored and separates the uterus from the sloughed cup. Restoration of epithelium appears to involve migration of epithelium into the area from the periphery. In addition, the epithelial lining of the uterine glands may contribute to the restoration process. However, epithelial metaplasia of connec— tive tissue cells cannot be ruled out. The area formerly occupied by the cup cells contains lymphocytes, pigmented macrophages, and connective tissue stro- ma. Plasma cells are more numerous than before and are confined to the endometrial stroma. Macrophages con- taining lipofuchsin are also present and are more apparent than when they first appeared at Day 100. The macrophages are like those seen in degenerating corpo- ra lutea, suggesting that the cup cells had steroid secreting properties. Endometrium at Days 180 to 200. The endometrium at the cup site has been fully restored. The surface epithelium is columnar. The uterine glands are still dilated and active and secreting uterine milk, but resemble uterine glands else- where. The number of lymphocytes is con- siderably decreased to resemble non—cup areas. The number of eosinophils is strik- ing, especially toward the center of the area formerly occupied by the cup. The presence of eosinophils is compatible with the fetal, foreign-tissue origin of the cup cells. The stroma also contains engorged macrophages, leucocytes, and plasma cells. Plasma cells are present in large numbers only after the cup cells are gone. Day 180 allantochorionic pouches. The uterine lumen separates the pouch and the underlying restored endometrium. The lower portion or neck of pouches may be patent or nonpatent (311). If patent, uterine milk may continue to pass into the pouch between the necrotic cup mass and the surrounding allantochorion. The allantochorion is attached to the endometrium by microcotyledons around the periphery of the neck-region of the pouch. The neck of the pouch is nonab- sorptive and contains much connective tissue. Presumably, the connective tissue in this area may eventually close off the pouch. The epithelial lining of the neck of the pouch is stratified squamous. The tro- phoblast of the allantochorion, which is of ectodermal origin, apparently retains the ability to transform from absorptive to nonabsorptive, skin-like, epithelium. The side walls contain phagocytic absorptive cells, and the connective tissue in this area is well developed and vascularized. The allantochorion, in some pouches, is thinnest at the apex, and the absorptive properties of the trophoblast may be well developed. The pouches appear to be one way the fetal membranes can wall off or destroy (phagocytose) and resorb the cells of fetal origin (cup cells). Little material of fetal origin, therefore, is left behind, which may be important in terminating the maternal immune rejection of fetal cells. 9.9. Amnion 9.9A. Embryonic Amnion The origin and formation of the amnion, the membrane in most intimate contact with the embryo proper, is depicted diagrammatically (Figure 9.10). The amniotic fluids serve to suspend and protect the embryo in an aqueous envi- ronment. The functional role of the amnion is emphasized by its absence in species that develop in water (1123). The amniotic membrane forms by folding from the somatopleure as depicted in Figure 9.10. As the allantois develops, it passes around and over the amnion and fuses with it to form an allantoamniotic membrane. The membrane separates the allantoic and amniotic cavities and con- sequently protects the embryo from the urine-like wastes in the allantoic fluid. Embryology and Placentation 377 9.93. Fetal Amnion It has been demonstrated in other species that the developing fetus swal— lows, breathes, and urinates (1474). Swallowing probably occurs in the equine fetus as suggested by ultrasonic and videoendoscopic observations of opening and closing of the mouth (649, 601), espe- cially during the last two-thirds of preg- nancy (648). The newborn foal and aborted fetus have substances in the pulmonary tissues that are derived from amniotic fluid (842), and this observation is consis— tent with breathing-like activity. Amniocentesis (sampling amniotic fluid for diagnostic purposes) has been described (1788, 176). The amnion is attached to the ventral aspect of the fetal body around the yolk stalk. As develop- ment progresses, the ventral opening becomes smaller and finally is reduced to an umbilical ring. In most species, the amnion begins at the juncture of the umbilical ring with the skin. It follows the umbilical cord for considerable dis- tance in the horse, however, resulting in prominent amniotic as well as allantoic portions of the cord (Figure 9.14,A,D). Placental fluid volumes. As pregnancy progresses, the fetal membranes increase in weight in parallel with the increasing weight of the uterus (Figure 9.23). The changes in volume of the amniotic and allantoic fluids have not been adequately detailed. In a limited study in ponies (575), amniotic fluid volume increased rapidly between Days 100 and 160. This agrees with the statement (95) that amni- otic fluid volume in horses is low for the first three months and then increases more rapidly to equal the volume of allantoic fluid at midpregnancy. The allantoic fluid volume in the pony study increased rapidly between Days 40 and 80 and then decreased (Figure 9.23). The need for better information on the vol- ume of the allantoic fluid in relation to the size of the fetus is emphasized by the 378 Chapter 9 Placenta & fluids 1600 Allantochorionic / fluid (ml) .- 1200 Amnionic ,." fluid (ml) ’1' \x E ,0... .I' E 800 /' "a; I5 P ./' ' m . é h / <5 f. K /' Allanto— 400 , -...' j chorion (g) I" .-/ o - - - O -'/ ’r’ ----- -. 57/ \ ’0’- Allantoamnion (g) o Mare-M (2)(3)(3) (2) (3) (2) (1) 4o 80 120 160 200‘ Days of pregnancy FIGURE 9.23. Changes in weight of uterus, weight of placental membranes, and volume of placental fluids in pony mares. Numbers of mares are in parentheses. From (575). recent awareness that the fetal-amniotic unit is highly mobile within the allantoic sac at 2 to 4 months of gestation but not toward the end of gestation (pg. 408). The volume of amniotic fluid and allantoic fluid at term in horses was 3 to 5 and 8 to 15 liters, respectively (95). Histology. The inner ectodermal lining of the equine allantoamnion consists of squamous epithelium at Days 50 to 60 (Figure 9.24). By Day 80, however, the inner ectoderm and outer endoderm of the allantoamnion have a more cuboidal form (Figure 9.25), perhaps reflecting increased ectodermal activity. The base- ment membrane beneath the endoderm is very thick and may serve to decrease the permeability and therefore minimize the passage of materials between the allantoic and amniotic cavities. Striking histologic changes occur in the allantoam- nion during the last third of pregnancy, FIGURE 9.24. Day 60. Blood vessels with red blood cells in the allantoamnion. including hyperplasia and thickening of the ectoderm, endoderm, and intervening connective tissue. FIGURE 9.25. Day 80. Allantoamnion showing cuboidal endoderm with a prominent basement membrane beneath. The inner surface (toward the fetus) of the equine amnion becomes covered with plaques (Figure 9.26; 80). The plaques begin to appear at about Days 70 to 80 and are grossly distinct at Day 100. They apparently contain large amounts of glycogen (80). Histologically, at Day 100, the mature plaques consist of onion-like layers of concentric ectodermal cells, with or without a connective tissue core. The ectoderm covering the plaques is taller (cuboidal) than in other areas of the allantoamnion (squamous). Another striking feature of the equine allanto- amnion is the development of a promi- nent vascular system. The blood vessels are distinct as early as Day 36 (Figure 9.12,C). As late as Day 60, the vessels are straight (Figure 9.14,A) but become tortu- ous by Day 100 (Figure 9.14,D). Some vessels become very convoluted in small discrete areas and form nodules that FIGURE 9.26. Day 300. Allantoamnion showing plaques on the inner surface. Embryology and Placentation 379 project into the amniotic cavity. These nodules appear at about the same time as the plaques and consist of convoluted blood vessels embedded in a jellylike material of very loose connective tissue (Figure 9.27). The material resembles Wharton’s jelly of the umbilical cord. Function of vascular system. The func- tions of the plaques and the vascular system of the equine allantoamnion are not understood. Perhaps the plaques and vessels are manifestations of a mecha- nism for nutrient storage or for salvaging usable materials excreted by the fetus into the amniotic fluid. These possibil- ities are suggested by the simultaneous development of vessels and plaques, the glycogen and/or glycoprotein content of the plaques, and the increased height of the ectodermal cells that separate the plaques from the amniotic fluid. This is clearly a neglected area of study. FIGURE 9.27. Nodule in allantoamnion showing loose mesenchyme with vessel in the center. 380 Chapter 9 9.10. Fetal-Maternal Attachment The placenta of the mare, like that of other eutherian species, consists of fetal and maternal tissues that are in apposi- tion for purposes of physiologic exchange (1123). The functions are diverse and include fetal nutrition, respiration, waste removal, and assurance that the fetus is protected from maternal immune response that is directed against fetal or placental antigens (1123, 88). The placenta of the mare is of the diffuse nondeciduate type with superficial villous attachment. The villi are organized into complex tufts known as microcotyledons which cover the surface of the allantochorion and fit into corresponding complex crypts in the endometrium. This type of placentation is noninvasive and generates minimal maternal cellular response (88). However, the chorionic girdle does form invasive attachment in association with the devel— opment of endometrial cups (pg. 370). The equine placenta is classified as epithelio- chorial since the uterine epithelium is in contact with the outer layer of the chorion (trophoblast). The expansion of the pla- centa into the uterine horns and body during the early fetal stage (Days 40 to 80) is discussed in Chapter 8 (pg. 320). Definition of the mature units of exchange. The units of placental exchange in equids have been called microcotyledons. This term has been used to encompass both fetal and maternal tis- sue. In cattle and other ruminants, the terms cotyledon (fetal), caruncle (mater- nal), and placentome (entire structure) are often used (1333). On this basis, micro— cotyledon, microcaruncle, and micropla- centome will be used in this text when ref- erence is made to fetal, maternal, and fetal plus maternal components. The microplacentomes have been extensively studied in the past 30 years, especially by workers in Europe. Early history. A fascinating account of the early history of knowledge of the equine placenta has been published by Steven (1544). Several remarkable reports appeared in the Italian literature three and four centuries ago. Some of the early descriptions were as follows: 1) 1598—first printed illustration of the diffuse placenta of the mare; 2) 1604—microcolytedons described and illustrated; 3) 187 6—associ— ation made between microcotyledons of the _ horse and the more obvious cotyledons of ruminants, with a vivid illustration of the vascular arrangements of the microcotyle— dons; and 4) 1897, 1915—decription and clarification of the interrelationships of the amnion, allantois, yolk sac, and chorionic girdle. 9.10A. Early Attachment The physical attachment of the surface of the trophoblast to the uterus is a grad- ual process. The early equine placenta consists of simple apposition of fetal and maternal epithelia (1387). Close associa- tion of the vascularized trophoblast and the uterine epithelium and dilated subep- ithelial capillaries has been detected as early as Day 25 (472). Early, localized, and temporary attachment occurs between the chorionic girdle and the endome- trium, but such attachment terminates on Days 36 to 38 after the invasion of the girdle cells into the endometrium (pg. 370). According to one worker (923), the fetal placenta and endometrium are connected at Day 88 or 39 by interdigitating microvilli of the trophoblast and apposing uterine epithelium. The early fetal microvilli often occur in groups and con- siderably outnumber their maternal counterparts (1387). They are usually straight, long, and thin, but short or branched microvilli may occasionally be found. By Day 40, attachment has pro- gressed to the point that the conceptus pours hesitantly from the incised uterus (Figure 14,A; 575). The clinging may be due to the interdigitations of the microvilli or to possible adhesive quali- ties between the apposing fetal and maternal surfaces. 9.1 OB. M icrocotyledons and Absorptive Arcades Fetal macrovilli—not to be confused with microvilli—are the forerunners of the fully developed fetal placental exchange units, the microcotyledons. The macrovilli are rudimentary undula- tions at Day 45. It has been stated (923) that the macrovilli begin to appear on the allantochorion adjacent to the embryo proper. Others state (1668) that the macrovilli begin to develop at Day 45 over the entire surface of the allanto- chorion and give it a hazy appearance. Maximum placental attachment does not occur until completion of the complex folding and penetration of the micro- cotyledonary tufts by approximately Day 150 (1387). Attachment of the fetal pla- centa, therefore, is a gradual process involving transformation of the very sim- ple rudimentary macrovilli to the highly complex microcotyledons and micro- caruncles (microplacentomes) over about 110 days (Days 40 to 150). For this rea- son, attempting to define a day of implantation is not an appropriate approach in this species. The arcade space is a highly absorptive area around the peripheries of the microcotyledons. The arcade space and the microplacen- tomes, therefore, develop in concert. The arcades form a continuous network and are not discrete delineated areas like the areolar regions of swine. Systematic studies of microcotyle- donary development have been made by workers in the Soviet Union (923) and England (1384, 1386, 1387). Samuel and co- workers (‘1387, 1388, 1546) have given Vivid descriptions of the dynamic changes in the ultrastructure of the equine placen- tal barrier. The following description of cotyledonary development is based on these references and on the examination of pony specimens (575). The description of the mature microplacentomes also draws on other published reports (80, 210, 211, 1640). The atlas on fine structure of Embryology and Placentation 381 the placenta by Bjorkman (211) is recom- mended, especially for those interested in comparative ultrastructural details. The fine structure of the placenta of the zebra also has been described (857). Days 50 to 60. The macrovilli, or fore- runners of the microcotyledons, develop a core of connective tissue that contains blood vessels (Figure 9.28). A marked change occurs in the allantochorionic macrovilli between Days 50 and 60; shal- low rounded macrovilli become much thinner and longer (575). The tropho- blastic cells at the apex of the villi are stratified and appear to be mitotically more active than elsewhere on the devel- oping macrovilli, signifying growth and penetration of the macrovilli into the developing maternal crypts (indenta- tions) of the endometrium. The surface cells of the remainder of the macrovilli are primarily tall columnar with large, basally located nuclei, but occasionally pseudostratified epithelium is apparent. The maternal epithelium is cuboidal. Electron microscopy has demonstrated pinocytotic vesicles near the surface of the trophoblastic cells, indicating exchange activity of the macrovilli (1384). Groups of macrovilli on the allantochori- on are separated by arcade areas con- sisting of phagocytic cells. The arrange- ment of rudimentary macrovilli with capillaries just beneath the epithelium and the coating of microvilli favor the passage of nutrients, and some mater- nal—fetal gaseous exchange begins to occur. At this stage, however, the fetal blood vessels are separated from the basal lamina of the trophoblastic epithe- lium by connective tissue (1387). 382 Chapter 9 Day 80. The simple macrovilli have become more complex with secondary folding (Figure 9.29; 575). Interdigitation has progressed so that the maternal and FIGURE 9.28. Days 50 to 60. Arcade areas (aa) between several macrovilli (mv) on the fetal-placen- tal membrane. The endometrium contains maternal crypts (Inc) which interdigitate with the macrovilli. The fetal and maternal tissues were not tightly attached and pulled apart during preparation of the specimen. The close-up shows the nature of the epithelial lining of the apposed fetal and maternal tissues. Note the erythrocytes in the vessel of the allantochorion. Arrow indicates a mitotic figure. fetal tissues do not separate during histo- logic preparation as they did on Days 50 to 60. The epithelium of the maternal crypts has become reduced (squamous to cuboidal with small nuclei). The tips of the chorionic villi appear to be pseudos- tratified suggesting continued growth. Vascularity has increased on both sides. This is particularly noticeable on the fetal side where vascular development has pro- gressed markedly over that on Day 50, especially at the tips of the macrovilli. These increases in vascularity as preg- nancy advances reflect the increasing ability of the placenta to function as an organ of exchange. Clustering of macrovilli has proceeded so that the intervening arcade spaces are more defin- able (Figure 9.29). Uterine milk can be seen in these absorptive spaces. Embryology and Placentation 383 FIGURE 9.29. Day 80. A) Increasing complexity (folding and branching) of the fetal macrovilli (above) and the maternal crypts (below). The clear space between macrovilli and the septum of the maternal crypts is artifactual due to shrinkage during processing. B) Close-up of the association between the projecting fetal macrovilli and the accommodating maternal crypts. C) Absorptive arcade. Note the prominent phagocytic cells of the trophoblast (above). The opening of a uterine gland into the absorptive area is shown (arrow). 384 Chapter 9 Day 100. The microplacentomes have become clearly defined (Figure 9.30). The trophoblastic cells are cuboidal to columnar, and the uterine epithelium is cuboidal to squamous in the areas of interdigitation. A noticeable increase in vascularity also has occurred. Collagen fibers separate the maternal capillaries from the epithelium (1386, 1387). On the fetal side, however, some capillaries con- tact the epithelium so that the basement membranes of the trophoblast and fetal endothelium (internal lining of blood vessels) become fused. The pinocytotic vesicles in the trophoblastic cells have greatly increased in numbers and con- tain electron-dense material, similar to that of the uterine lumen. Based on both light and electron microscopy, there is considerable anatomical indication of continuing refinement of mechanisms for the movement of substances across the placental junction. Lymphocytes occa- sionally appear to accumulate on the maternal side of the arcades (Figure 9.30). The lymphocytic infiltration seems greater in this area than in any other area of the endometrium, except at the base of the endometrial cups (pg. 375). Perhaps the lymphocytes appear in response to fetal antigens that may reach the maternal tissue from the high- ly phagocytic overlying trophoblast. Day 150. Discrete tufts of complex macrovilli form on the fetal side of the placenta (microcotyledons; Figure 9.31). Samuel and co-workers (1384, 1386) sug- gested that the fetal component of the microplacentomes forms by the coales— cence of a number of adjacent macrovilli together with their secondary folds into a complex multifolded structure with a common main stem. They note that Amoroso (80) had previously suggested that each microcotyledon developed from a single macrovillus. The specimens depicted herein support the View of Samuel and co—workers, as demonstrated by comparison of Figures 9.28 and 9.31. The coalescing and branching of adja— cent macrovilli to form a microcotyledon can be appreciated from these figures by using the indicated arcade areas as points of reference. The multibranched villous tuft fits into a correspondingly complex maternal structure. On the maternal side, the epithelium is reduced in height. The phagocytic cells of the arcades are tall columnar and may be of unusual length (Figure 9.31). Pigmented macrophages are more numerous in the maternal stroma at this time than at earlier stages. Day 200. The reduction in height of the maternal and fetal epithelium has progressed to the point where only 12 um of tissue separate the maternal and fetal blood streams. Furthermore, intercellular channels form between adjacent epithelial cells on both the maternal and fetal side, possibly to pro- vide a route for fluid transport. A pro- gressive indentation of the epithelium of the allantochorion by fetal capillaries occurs (Figure 9.32) and by Day 270, reduces the effective thickness of the fetal placenta (1386, 1387). This feature has some similarities to the air blood barrier in the lung and emphasizes the function of the placenta as an organ of gaseous exchange (1546). It should be noted that some localized degenerative changes also occur in the microplacen- tomes (Figure 9.32; 1387). Embryology and Placentation 385 FIGURE 9.30. Day 100. Further complexity of a developing microplacentome. Note the arcade area (arrows) on either side of the microplacentome. The close-up shows the highly absorptive characteristics of the arcade area. Note the accumulation of lymphocytes on the maternal side of the arcade space. 386 Chapter 9 FIGURE 9.31. Day 150. A highly complex microplacentome with the arcade area on either side (arrows). The close-up shows the neck of a uterine gland opening into the arcade area. 9.1 OC. The Mature Microplacentomes Gross appearance. There are thousands of microcotyledons on the outer surface of the allantochorion, and each is 1 to 2 mm in diameter when mature. They are grossly visible and cause the red velvety appearance of the outer surface of the dis- charged placenta. If the outer surface of the allantochorion is viewed under mag- nification, the intricate, branching, or arborescent structure of the microcotyle- dons is readily appreciated (Figures 9.33 and 9.34). A microcotyledon fits exactly into a corresponding capsule (microcarun- cle) in the endometrium, with appropriate crypts to accommodate each branch. Vascularity. Vascularity of micropla- centomes has been described (1543, 1640, 1544). Long straight arteries from the subendometrial vascular plexus pass between the uterine glands to the surface of the globular microcaruncle. Branches pass over the rim of the microcaruncle and form a dense capillary network in the walls of the maternal crypts. The network is drained by a single vein for each micro- caruncle. Vascular supply on both mater- nal (microcaruncle) and fetal (micro- cotyledon) sides provides for effective transfer of gases. It has been suggested that direction of flow in maternal and fetal blood vessels accommodates a coun- tercurrent exchange system (1387, 1544). Embryology and Placentation 387 FIGURE 9.32. Peripheral portion of a mature microplacentome showing three finger-like macrovilli separat- ed by maternal septa. Note the maternal capillaries (Inc) and fetal capillaries (fc). The dark border at the apposed fetal and maternal surface is from the interdigitating microvilli (mv). The close-up of the apex of a macrovillus shows the uterine epithelium (ue), microvilli (mv), fetal epithelium or trophoblast (tr), degenerat- ing trophoblastic cell (dtr), fetal connective tissue or mesenchyme (mcs), maternal capillaries (me), and the indentation of the allantochorionic epithelium by fetal capillaries (fc). Courtesy of N. Bjorkman. 388 Chapter 9 Functional development of micropla; centomes. The development and mature form of the placental microplacentomes present an excellent example of exquisite biologic compatibility between form and function (Figures 9.33 and 9.34). During the period of attachment (Days 40 to 150), the fetus is growing at a tremen- dous rate, and the anatomical changes are dictated by the ever-increasing physi- ologic demands of the fetus. Prior to Day 40 (embryo stage), expansion of the yolk sac and later the allantoic sac and their gradual vascularization are adequate to accommodate the increasing needs. These approaches do not suffice during the fetal stage. The placenta is an organ of physiologic exchange, and the degree of Chorioallanlols Openings of ulenne glands Uterine epilhellum Maternal side of micro- cotyledon Ulerine anery exchange is dependent not only on con— centration gradients but also on the extent of the functional surface area and the nature of the intervening tissues and fluids. The contact surface area between fetal and maternal portions of the placen- ta is therefore increased as the need for exchange grows. This is accomplished by the following: 1) gradually increasing complexity of macrovilli from simple undulations to the intricate multibranch- ing cotyledonary tufts, and 2) intimate interdigitation of microvilli on the two apposing surfaces with increases in num- bers and complexity (increases in height with some branching). These changes are paralleled by corresponding changes on the maternal side. The accommodating Umbilical vessels Uterine vein FIGURE 9.33. Drawing by Steven and Samuel of mature microplacentomes showing the interdigitation of the fetal and maternal aspects and the openings of uterine glands into the surrounding arcade area. From (1546). structural form is one of microvilli over the surface of macrovilli. This results in a surface area many times greater than that possible from simple expansion. In addition to surface area changes, the dis- tance between fetal and maternal capil- laries is reduced by alterations in the thickness of epithelial and connective tis— sue components. As the development of FIGURE 9.34. Microcotyledons and corresponding microcaruncles (below) as viewed through a dissect— ing microscope. The microcotyledons are seen on surface view, but the lower edge has been reflected to show a side View. The endometrium is seen on surface View. Each microcotyledon fits into a micro- caruncle. The pale network surrounding the micro— caruncles represents the arcade area. Embryology and Placentation 389 the microcotyledons progresses, the tro- phoblastic epithelial cells are reduced in height and the basal lamina loses defini- tion (1387). The fetal capillaries become closely applied to the base of the tro- phoblastic cells and may form indenta- tions in the cells (Figure 9.35). The basal lamina of the capillary in the indented area is fused to the basal lamina of the trophoblast, effectively eliminating the fetal connective tissue barrier in this area. The indentation of the trophoblast by the capillaries also shortens the dis- tance between fetal and maternal blood streams and reduces the amount of respiring placental tissue along the diffu- sion pathway (1387). By Day 300, the maternal epithelium is reduced by about a third of its original height. The most prominent changes for reducing the dis- tance and barriers between fetal and maternal blood streams occur on the fetal side. These changes are directed toward maximal effectiveness of the hemotropic route of exchange (blood to blood). Placental exchange. A review (1474) of the nature of equine placental exchange is the source of the following comments. The oxygen gradient between maternal and fetal sides is far narrower (e.g., dif- ference of 4 mm Hg) than in ruminants (e.g., 20 mm Hg). The level of oxygen in the fetal arteries is much greater than for the fetus of other farm species. The high oxygen level in the equine fetus and the relatively small maternal-to-fetal gradient indicate that the oxygen exchange system is much more efficient than in the other farm species. These physiologic differences are reflected by anatomical differences in the placental exchange system. In the mare, a counter— current vascular flow system in the microplacentomes would presumably increase the efficiency of exchange. In addition to the blood gases, some small molecules (e.g., water, ammonia) proba- bly are transferred by diffusion. How- ever, a facilitated transfer probably occurs for the majority of nutrients (e.g., 390 Chapter 9 FIGURE 9.35. Macrovillus (top) showing the mesenchymal core (mes) and fine structure of the allantochori- onic epithelium (tr: trophoblast). The trophoblast and the uterine epithelium (ue) are joined by interdigitat— ing microvilli (mv). Note the layers of tissue separating the fetal capillaries (fc) and maternal capillaries (mc). Fine structural details include nucleus (n), mitochondria (m), basal laminae (bl), and endometrial stroma (es). The close-up shows the details of the interdigitating microvilli on the apposed surfaces of a cell of the tro- phoblast (upper left) and the uterine endothelium (lower right). Courtesy of N. Bjo‘rkman. glucose, amino acids). Direct evidence for this aspect of placental exchange in mares is lacking and is presumed on the basis of studies in other species and indi- rect indicators in mares. Rate of uterine and umbilical flow seem similar to those of other species when weight of uterus and fetus are considered. Avillous areas. Development of the microplacentomes, and therefore attach- ment, does not occur in the following areas (1770): 1) overlying the endometrial cups, 2) at the cervical os (Figure 9.36), 3) opposite the opening of each oviduct, 4) overlying the attachment of the umbil- ical cord, and 5) along invaginated folds of allantochorion. The nonvillous area at the cervix is quite prominent in the full— term placenta. The area consists of a star-shaped arrangement of raised linear avillous ridges and therefore has been called the cervical star. The ridges begin at a small avillous area opposite the internal cervical 0s and radiate out 3 to 12 cm (1770). The avillous spots at the opening of the ‘oviducts are minute. The avillous area at the umbilical cord attachment is a remnant of the bilaminar omphalopleure, demarcated by the sinus terminalis. The curved bands of avillous folds, Which infrequently are seen in the full-term placenta, probably result from folding of the allantochorion such that the area does not contact the endometri- um. These avillous areas indicate that contact of the trophoblastic membrane with the uterine epithelium is necessary for stimulation of formation of micro- cotyledons and microcaruncles. The mor- phology of the equine allantochorion at the tip of the gravid horn has been exam— ined by Japanese workers (1171); hypoplastic villi were described, and it was suggested that they were caused by disorders in formation of microplacen- tomes in this area. Embryology and Placentation 391 FIGURE 9.36. Cervical star or avillous area of allantochorion (right) and excised cranial portion of cervix (left). 9.10D. The Absorptive Arcade Adjacent microplacentomes are sepa- rated by an arcade area which is a contin- uous arched network surrounding the microplacentomes (Figure 9.33). The continuity of the arcade space may not be appreciated from the study of two- dimensional photomicrographs since the cross sections of the space may give the false impression of discrete areas. Groups of endometrial glands open into the arcade area, and secretion is accommo- dated by a raised ridge of smooth (avil- lous) trophoblast. The trophoblastic cells in this area are columnar with all the appearances of phagocytic cells, and the underlying mesoderm is highly vascular- ized. The allantochorion overlying the endometrial cups is similarly equipped. The mare retains the histotropic route (absorption of uterine milk), apparently as a supplementary avenue of fetal nour- ishment through the phagocytic tro— phoblastic cells of the netlike arcade area. Amoroso (80) has drawn attention to the possible importance of the uterine glands and the nature of uterine secretions dur- ing pregnancy. Not until the study by Samuel and associates (1388), however, has detailed consideration been given to the uterine glands. Ultrastructural stud- ies were made at intervals during preg— nancy. The glands secreted actively throughout pregnancy, and the overlying trophoblast contained inclusions resem- bling the secretory product. The nature of the secretion initially appeared to be pro- teinaceous. Cellular debris appeared later in pregnancy, indicating holocrine secre- tion. The presence of a well differentiated network (arcade area) that is capable of absorbing and breaking down the secre- tions of the glands suggests that the uter- ine glands are important in some unde- fined way to the well—being of the fetus. 392 Chapter 9 9.11. Fetus 9.11A. External Reproductive Organs The genital tubercle is the embryonic process that differentiates into the penis in the male and the clitoris in the female (Figure 9.37). It is an important structure to the anatomist and the clinician because it provides the earliest and the most convenient external indicator of gen- der. The genital tubercle is about 1 mm long in the 30—day embryo (Figure 9.12,E). According to one report (187), gen- der cannot be determined on the basis of external features until 40 to 45 days. The location of the tubercle, however, is not a reliable gross indicator of gender until Day 55 (600). Before differentiation, the tubercle maintains a position between the hindlimbs and midway between anus and umbilical cord . When the body lengthens, the tubercle remains near the anus in the female and near the umbilicus in males (Figures 9.38 and 9.39). The distance of the genital tubercle to the anus and to the umbilical cord is shown for removed specimens (Figure 9.38) and for fetuses Figure 9.37. Location and appearance of the genital tubercle (arrow) in male (upper) and female fetuses. examined by ultrasonic imaging (Figure 9.39). The optimal time for diagnosis of fetal gender by transrectal ultrasonography is approximately Days 60 to 70 (360, 359, 361). Diagnoses were also made approximately between Days 70 and 100, but in 13% of Fetal gender (individual observations) 200 a D Crown-rump . Female a Male . ° _L 01 0 Length (mm) 3 o 50 40 Genital tubercle D to anus an 30 20 D D Distance (mm) 10 D Genital tubercle to umbilicus . Distance (mm) 51 57 63 69 75 81 87 93 99 Number of days from ovulation FIGURE 9.38. Individual observations on the effect of fetal gender on distance of genital tubercle to anus or umbilicus. From tabulated data (360). Location of genital tubercle 5 Female Key for location score 1: Near umbilicus 2 3 _ 2: Intermediate 8 ' l 3: Between rear limbs m i 4: Intermediate ‘ 5: Near tail 44 50 56 62 68 74 80 Number of days from ovulation FIGURE 9.39. Changes in location of the genital tubercle in female and male fetuses as determined by ultrasound. Adapted from ( 360). the attempts an adequate View of the tubercle was not obtained. As seen in the removed fetus, by Day 55 a pair of prominent vulvar lips extends dorsally from the clitoris in the female. In the male, a perineal raphe extends caudally on the median line from the base of the penis to the point where the scrotum will develop. At Day 80, the scrotum is a pale empty eminence (7.5 x 1 x 1 mm) 20 mm caudal to the umbilicus. At Day 150, a gubernaculum is palpable in the scrotum on each side. The male prepuce may be noted as early as Day 77, but it does not become pendulous like that of the postnatal foal until Days 115 to 120. In the female, the clitoris has receded to its terminal position inside the ventral commissure of the labia by Days 100 to 120. The mammary papillae are visible in both sexes as pale dots (0.25 mm) in the inguinal region at Day 55. By Day 120, they become incorporated into the prepuce of the male. In the female, the fetal mammary glands become promi- nent and glandlike by Day 300. In the postnatal male, the mammae are repre— sented by teat-like projections on the cranioventral border of the penile sheath. Embryology and Placentation 393 The testes have not completely descend- ed at Day 300 and are Within the inguinal canal, with the cephalic poles at the level of the inguinal ring (187). The gubernacula are quite large after Day 150 and can be mistaken for testes on digital palpation. Complete descent of the testes usually occurs between Day 315 of pregnancy and the first two weeks of post-natal life. 9.113. Other External Features External developmental changes in the fetus are reasonably predictable and are therefore useful in estimating the stage of pregnancy. The more salient external fea— tures have been summarized in Table 9.4, and photographs of pony fetuses are pre- sented in Figures 9.13, 9.14, and 9.40. Estimations of stage can be quite valuable to the clinician, pathologist, and research scientist. Radiographic fetometry can be used for aging of equine fetuses by the time and order of appearance of ossifica- tion centers and diaphyseal growth (656). Bergin and associates (187) have described the external developmental horizons and measurements useful for age determination of equine embryos and fetuses. The mares weighed 409 to 500 kg (900 to 1,100 lbs), and the reference point was the day of breeding. The authors concluded that contour, crown-rump, trunk, forefoot, hindfoot, and head length and Width had similar reliability for aging up to 100 days. Thereafter, breed and individual variations seemed to reduce the accuracy of head and extremi- ty measurements but not contour, crown- rump, and trunk measurements. Tail and ear measurements were more vari- able and therefore less useful criteria. Body weight was one of the most useful criteria throughout the days sampled (>Day 20). The Bergin data for weights of 110 embryos and fetuses from racing and rid- ing horses are compared to data (575) for 19 pony embryos and fetuses (Figure 9.41). Note that body weight changes of 394 Chapter 9 TABLE 9.4. External Characteristics Useful for Determining Age of the Equine Fetus Day of preg- nancy Head Legs Reproductive organs Hair Conformation 40 Ears rudimentary Elbows and Migration of genital None Head between Eyelids stifles tubercle begins forelegs External nares 45 Sex determinable None 55 Ears: triangle Hocks and Prominent vulva None Prominent fold covers fetlocks and penis brain case opening Mammary papillae Eyelids closed (0.25 mm dots) except for 1 mm slit 60 Eyelids closed or Soles and frogs None Unmistakably almost closed equine Nares: 1 X 0.5 mm slits 80 Points of Mammary papillae None Head and neck shoulders are raised buds in normal and hips Scrotum: pale bulge position 2 cm behind umbilicus 100 Ears: 1 cm long Hooves pale Clitoris recessed On lips Muscle groups and curled forward yellow to post-natal easily and down Raised position recognized Eyes bulging coronary band 120 Vulval lips meet at On chin, ventral commissure muzzle, Prepuce pendulous eyelids 150 Ergots: 5 mm Glandular shaped Eyelashes mammae in female Palpable gubernaculum in scrotum 180 Early mane and tail hairs 210 Suspension of mam— Mane hair: mary gland in 2.5 mm female 240 On ears, poll, back of tail Vibrissae on chin, throat, muzzle 270 Body covered with fine hair _ _ Tail switch 300 Prominent pad Full -term coat covering soles Embryology and Placentation 395 DAY 100 1 cm DAY 160 1cm FIGURE 9.40. Fetuses from ponies. Day 40. Attached regressed yolk sac. Day 60. Eyelids closed. Day 80. Head and neck raised to normal position. Day 100. Ears curved forward, eyes bulging. Day 160. Well-developed ears. Day 220. Hair on mane and tail. Day 300. Full hair coat. 396 Chapter 9 Fetal weights 100 50 Kilograms U1 100 —— Horses (n=110) 50 ----- Ponies (n=19) Grams 20 100 180 260 New foal Days of pregnancy FIGURE 9.41. Weight changes in embryo and fetus of horses and ponies (adapted from 187 and 575). horses and ponies seem (not critically determined) quite comparable up to Day 100. Beginning at Day 100, the weight changes of the fetus apparently begin to reflect the extreme differences between ponies and horses in birth and adult weight. Changes in contour, crown-rump, and trunk measurements are shown in the first edition (575). Crown-rump length is commonly used to age fetuses from slaughterhouse stud- ies; data from the Bergin series are depicted in Figure 9.42 and can be used for this purpose. Erupted central upper incisors were found in all newborn foals examined after nursing (187). They were not found, how- ever, in near term fetuses taken by cae- sarean section. Changes in crown-rump 100 ...................... ............. Length (cm) ~l 20 60 100 140 180 220 260 300 340 Days of pregnancy FIGURE 9.42. Changes in crown—rump length for estimating fetal age in horses. Adapted from (187). 9.11 C. Fetal Gonads A review has been published on differ— entiation of the mammalian embryonic gonads and development of steroid pro- duction in various species (271). The gonads of the equine fetus have attracted much interest, primarily because of the unusual size resulting from the massive number of interstitial cells. It has been known for more than a century that there is a marked histologic resemblance among luteal cells, hepatic cells, and the interstitial cells of the equine fetal gonads. It has also been known that the gonads of a 7 — to 8-month horse fetus are larger than the ovaries of the mare at that stage of pregnancy. Early gross and histologic descriptions of the equine fetal gonads have been reviewed, and the salient histologic features have been described (323). These workers associated the development of the fetal gonads with the condition of the maternal ovaries and hormonal concentrations in the mare. Study has more recently been concen- trated on fine structure (635, 636, 695, 1081), biochemical aspects (521, 695, 1001, 1197), and germ cell development (389, 390, 1727, 575). The following account draws on the work of the above-cited authors. Gross changes in ovaries and testes. The gonads of one Day 40 to 45 concep- tus were embedded in the midventral portion of the mesonephros and measured Embryology and Placentation 397 1.4 x 0.5 x 0.5 mm (1727). At Day 40 to 45, the gonads are surrounded by a cortical layer of surface germinal epithelium. At Day 60, when the maternal ovaries have reached maximum development, the fetal gonads are quite small (<1 g; 5 x 5 x 4 mm; 1727). The forerunner of the infundibulum is attached to the perime- ter of the mesonephros. Slow growth of fetal gonads occurs until approximately Day 100 (1.9 g in ponies; Table 9.5). Between Days 100 and 200, however, the gonads grow very rapidly (Figure 9.43) to reach a maximum size of approximately Weight of fetal gonads 100 El 0 - Fetal ovaries 0 Fetal testes . Crown-rump length (cm) 112 165 215 260 Estimated day of pregnancy FIGURE 9.43. Weights of pairs of fetal gonads. Adapted from (323). Day of pregnancy estimated from Figure 9.42. TABLE 9.5. Mean Weight of Fetal Gonads in Comparison to Other Fetal Organs in Ponies Fetal organs Fetus or Day of No. of Gonads Adrenals Thyroids Pituitary Kidneys Liver Foal gestation fetuses (g) (mg) (mg) (mg) (g) (g) (g) 80 3 1 6 5 10 . . . 6 71 100 3 15 32 26 . . . 10 83 140 to 160 8 19 173 252 47 9 42 988 180 to 200 5 48 352 1054 93 26 98 3083 300+ 3 31 841 3046 186 60 238 10235 Newborn 2 6 18614 Adapted from (430, 575) 398 Chapter 9 50 g in ponies and 70 g in horses (weights of both gonads combined). A comparison of the weights of fetus and fetal organs, maternal ovaries, and fetal gonads is shown in Table 9.5. The fetal gonads increase in size at a time when the mater- nal ovaries decrease. Measurements of 14 x 8 x 10 mm have been reported for Days 90 to 120 (1727). At approximately Day 150, the weight of the fetal gonads begins to exceed that of the maternal ovaries. The average dimensions of the gonads for Days 120 to 180 have been reported as 35 x 20 x 19 mm (1727). The great enlarge- ment is caused by hyperplasia of the medullary interstitial cells and, as a result, only about half of the surface remains covered with cortical tissue. That is, the medulla (interstitial area) grows so rapidly that the slower-growing cortex becomes confined to one side of the gonads. By Days 180 to 270, only about a third is covered by the cortex. Between Day 270 and birth, the borders of the ovarian cortex are delineated by the uteroovarian ligament and fimbria on the caudal and cranial poles, respectively. The weight of the fetal gonads decreases after Day 250 to approximately 10 to 20 g in the newborn horse foal and to approxi- mately 5 g in the pony foal. The gonads of the newborn weigh approximately a tenth of those at maximum development in the fetus (Table 9.5). Female and male fetal gonads Show no significant difference in weight (Figure 9.43). Most of the increas— ing and subsequent decreasing weight changes, as discussed below, result from hyperplasia, hypertrophy, and the even— tual degeneration of interstitial cells. The germinal elements are limited to the nar- row cortical zone in fetal ovaries and to scattered cords in fetal testes. The gonads at Days 30 to 60 are yellow- ish-white, whereas they are brown in older fetuses. The cut surface of female and male gonads is a homogeneous brown. When viewed on the cut surface, the ovaries, but not the testes, have a thin rim of white tissue (cortex) contain- ing the germ cells (636). The white germi- nal rim is only on the ventral aspect of the ovary (Figure 9.44). That is, unlike the testes, the germ cells in the develop- ing ovaries are confined to the thin germi- nal crescent or cortex on the ventral sur- face of the ovary. By Day 150, the fetal equine testis has a dense fibrous capsule (tunica albugin’ea) which is continuous with the subcapsular connective tissue. The capsule contains no germ cells. The ovary at Day 150 has a similar though less prominent capsule over the large non-germinal area of the ovary (Figure 9.45). Interstitial cells. Histologically, the interstitial cells are similar in fetal ovaries and testes (635, 695). The intersti- tial cells appear prior to Day 50, either singly or in small clumps distributed throughout the central or medullary por— tion (crown—rump length: 4 cm; 323). In the youngest gonads (Day 60 testes) examined by one group of workers (695), the interstitial cells constituted about two-thirds of the tissue. The remainder consisted of small stromal cells and semi- niferous tubules undergoing early differ- entiation. The interstitial area made up the bulk (approximately 90%) of the gonads in Day 80 specimens (575). The FIGURE 9.44. Maternal ovaries, fetal ovaries, and fetal uterus (top to bottom) at approximately 220 days. Grey portion of fetal ovaries is the thin layer of cortex. FIGURE 9.45. Day 150. Capsule and interstitial cells in the nongerminal area of a fetal ovary. centrally located interstitial mass con- sisted of approximately 90% interstitial cells and 10% connective tissue elements. The growth of the gonads up to 250 days can be attributed largely to the continued development of the interstitial mass which is due to an increase in number and size of cells (323, 695). Mitotic figures were not found. Photomicrographs of interstitial cells are shown for Day 80 (Figure 9.46) and Day 150 (Figure 9.47). The cells contain granular eosinophilic cytoplasm and well— delineated nuclei with prominent nucle- oli. The interstitial cells are morphologi- cally quite similar to luteal cells and contain all the organelles normally asso- ciated with steroid biosynthesis (abun- dant smooth endoplasmic reticulum, mitochondria with tubular cristae, and a well-developed Golgi apparatus). Little change occurs in the ultrastructure between Days 60 and 250 (695). The decrease in size of the gonads after Day 250 can be attributed to degenera- tive changes in the interstitial cells. The Embryology and Placentation 399 m FIGURE 9.46. Day 80. Interstitial cells of fetal gonad. FIGURE 9.47. Day 150. Interstitial cells of fetal gonad. deposition of lipochrome pigments in the cytoplasm is presumptive early evidence of a breakdown of the interstitial cells 400 Chapter 9 (Figure 9.48; 323). This is followed by shrinkage of the cells and pycnosis of the nuclei. Some interstitial cells reportedly persist, even postnatally, and in the peripheral area of the interstitial cell mass may be incorporated into the theca interna of small follicles (323). Seminiferous tubules. The cord-like forerunners of the seminiferous tubules can be found in the interstitial mass in both sexes. Seminiferous tubules under— going early differentiation were described in testes at Day 60 (695). In Day 80 ovari- an specimens, the cells of the seminifer- ous tubules were large with pale nuclei (575; Figure 9.49). The tubules in fetal ovaries at Day 100 were fewer in number and difficult to find. In fetal testes, the seminiferous tubules continue to develop. By Day 150, the ger- minal tissue is associated with a tortuous network of tubules distributed within the interstitial mass (575). At Days 150 and 180, the tubules are surrounded by a cell- rich, compact investment of fibroblasts. A similar but less compact cuff surrounds FIGURE 9.48. Day 180. Fetal testis with forming seminiferous tubule. Arrows point to deposits of lipochrome pigments. FIGURE 9.49. Day 80. Medullary cord extending into the mass of fetal ovary. the many blood vessels in the area. The tubules may have a lumen containing a mucus—like secretion. The walls consist of thin columnar cells with apparently inac- tive nuclei and occasional large cells with prochromosomal-like nuclei. The contin— ued development of connective tissue tra- beculae results in a distinct lobulation of the fetal testes at Days 180 to 200 (Figure 9.50). 9.11D. Fetal Ovary Both mitosis (division of cells with maintenance of a full complement of chro- mosomes) and the initial phase of meiosis occur in the germ cells of the fetal ovary. Mitosis involves multiplication of the pri— mordial germ cells (primitive germ cells appearing in the gonads prior to differen- tiation into ovaries or testes) into many oogonia. Meiosis is apparently limited to the initial phase of the first division (prophase I), wherein members of the 64 pairs of chromosomes approach each other. Many of the various stages of FIGURE 9.50. Day 180. fetal testis. Embryology and Placentation 401 prophase I can be found. Until studies by Deanesly (389, 390), oogenesis in the fetal ovary was described for many species but not for the horse. The following account represents a composite of the Deanesly reports, the results of histologic examina- tion of fetal ovaries from ponies for Days 80, 100, 150 to 160, and 180 to 210 (575), and a report on the development of the cortical area (1727). Days 36 to 45. An overview of a Day 36 gonad attached to the mesonephros is shown in Figure 9.51 (575). Primordial germ cells were found, and some of these cells were undergoing mitosis. Sexual dif- ferentiation appears to occur between Days 39 and 45 (1727). The establishment of sexual differentiation, based on an ultrastructural study, has been discussed (1081). It was concluded that the early dif- ference in distribution of steroidogenic FIGURE 9.51. Day 36. Gonad attached to mesonephros. Close-up shows epithelium (e) and primordial germ cells (pgc). A higher power of a primordial germ cell is shown in the inset. 402 Chapter 9 cells suggests that interactions between somatic and germ cells vary according to genetic sex. Day 80. Meiotic figures, mostly prochro- mosomal, were found in the youngest ovaries studied (Day 73 and Day 80). The ovaries at this time have a thin but dense vascular connective tissue capsule. The germ cells are arranged in layers and cords within a thicker layer of loose sub- capsular connective tissue (Figure 9.52). The cords of germ cells move into the stroma and appear to blend with the medullary tubules which are present in the ovary at this early stage. Mitotic fig— ures can be seen in the outer third of the cortical area, and meiosis of germ cells is evident, especially in the inner two thirds. Most meiotic cells are in the prochromosomal stage, but leptotene and zygotene figures can be found. Some of the germ cells are in a degenerating pyc- notic state; atresia has already begun. FIGURE 9.52. Day 80. Fetal ovary showing the connective tissue capsule (A), a cord of germ cells in the outer portion of the cortical area (B), and mitotic figures in outer cortical area (C). Arrow indicates a cell in anaphase. Day 100. The vascularity and thickness of the cortex has increased by Day 100, and all stages of meiotic prophase can be found (Figure 9.53). Meiotic figures are plentiful in the inner portion of the cor- tex, and many of the figures are undergo- ing degeneration. Primary oocytes and a few primary follicles are present, espe- cially in the inner portions. That is, the germinal area displays both outer and inner layers of oocytes that represent a progression in oocyte development. The activity in the outer layer primarily involves multiplication of oogonia by mitosis. The inner layer is more advanced and consists of enlarged oocytes that have passed through the zygotene, pachytene, and prediplotene stages or are becoming atretic. Massive degeneration of germ cells is reported to occur between Days 90 and 120 (1727). Days 150 to 160. The germinal area has been reduced to an outer layer that shows all stages of meiotic prophase I (Figure 9.54). The number of primordial follicles Embryology and Placentation 403 FIGURE 9.54. Day 150. Germinal area of ovary with meiotic figures and primordial follicles (arrow). FIGURE 9.53. Day 100. Fetal ovary showing cortex and underlying interstitial area. Close-up shows germ cells with meiotic figures. 404 Chapter 9 has increased and the follicles are better defined. Some degenerative germ cells can be found, but degeneration, or atre- sia, is not as marked as it was at Day 100. Days 180 to 200. Cells showing active meiosis are rarely seen. The oocytes are confined to primordial follicles dispersed in the connective tissue of the capsule. Conclusion. Meiotic prophase activity in the fetal equine ovary extends from approximately Days 70 to 160. Atresia of oocytes is massive by Day 100 and can occur at any stage of meiosis. Few pri- mordial follicles emerge from the oogonia that first entered meiosis. Apparently, almost all the oocytes entering the post- natal period as primordial follicles are derived from those that formed near the surface of the fetal ovary and entered meiosis during late gestation. It is not clear when tertiary follicles begin to appear, but a 0.23 mm follicle has been found at Day 300 (389). 9.11E. Tubular Genitalia On a comparative basis, the embry- ologic development of the oviducts, uterus, cervix, vagina, and vulva in the horse can be assumed to be similar to that described for other animals. The principal species difference is amount of fusion between the two female ducts. Considerably more fusion occurs in the horse than in other farm species, as indi- cated by the proportionately longer uter— ine body. Specific sequential changes of the embryology of the reproductive tract of the horse have received only limited study (575). The meager information available indicates that the Mullerian ducts have not yet appeared at one month (187). The following account of the development of the tubular genitalia is based on histologic examination at Days 100, 150 to 160, and 180 to 200 (575). Day 100. The uterine horns were approximately 1.0 x 0.5 cm in cross sec- tion, and the lumen was slit-like with ventral-dorsal orientation (Figure 9.55). The cells of the uterine horns were not completely differentiated, and stromal and smooth muscle cells were quite simi- lar in appearance. The two muscle layers were distinguishable, however. The epithelium was tall columnar, contained many mitotic figures, and had a mucoid coating. Uterine glands and leucocytic or lymphoid cells were not found. The epithelium of the vagina was stratified cuboidal in the cranial portion, and the epithelium of the vulva was stratified squamous. In contrast to the uterine horns, leucocytes were present in the stroma of the vagina. Days 150 to 160. The uterine horns were 2 x 3 mm in cross-section and con- siderably more mature. The lumen had prominent folds (Figure 9.56). Epithelial mitotic figures were still evident, but uterine glands were not found despite the epithelial hyperplasia. The epithelium and stroma were still free of leucocytes, and no luminal secretions were evident, although some leucocytes were found in the stroma of the uterine body. Muscle had differentiated, and the vasculature was more prominent. Days 180 to 200. By this time the horns had assumed many of the features of the adult uterus. The most striking feature was the pronounced folding of the mucosa and the projection of some of the folds deep into the stroma (Figure 9.57). The deeper folds contained some secretory material, and the epithelium appeared glandular and was of the low cuboidal type. The folds of the uterine body were subdivided, and the epithelium of the deeper folds was columnar and glandlike. The smooth muscle seemed more hyper- trophied and appeared more like contrac- tile tissue. Numerous leucocytes were present in the stroma of the uterine body. There appeared to be a time progression in the appearance of leucocytes; leuco— cytes appeared first in the vagina, then the uterine body, and finally the uterine horns. Embryology and Placentation 405 FIGURE 9.55. Day 100. Cross section of fetal uterine horn and a close-up of the uterine epithelium showing mitotic figures (arrow). , q” “wax ,‘w I»: It: ‘4 (‘5 v«‘ , 5; l g N : l_~‘“?f§§:‘“fi\“¢f.a{‘fixx ‘ ’\ igw‘v N’ c. x t‘» FIGURE 9.56. Day 150. Cross section of fetal uterine horn and a close-up of epithelium and layers of uterine wall. 406 Chapter 9 FIGURE 9.57. Days 180 to 200. Cross section of fetal uterine horn. Note the deep folds in the mucosa. 9.11F. Pituitary Gland Detailed descriptions of the embryologic origin of the mammalian pituitary gland may be found in embryology books. The posterior lobe (neurohypophysis) develops from neural tissue, whereas the anterior lobe (adenohypophysis) develops from an evagination (Rathke’s pouch) of the ecto- derm of the roof of the mouth (sto- modeum). The fetal pituitary may be of more importance than generally appreci— ated and perhaps is involved in maternal as well as fetal endocrinologic mecha- nisms. The fetal pituitary and associated hypothalamus and adrenal glands have been implicated in the initiation of partu- rition in sheep. There apparently have been no direct studies in the horse on the physiologic role of the fetal pituitary. Anatomical studies on the development of the equine fetal pituitary have been reported (1385, 1744, 575). It may be helpful to review pituitary cell types described for adult animals (1385) before continuing, particularly with regard to the anatomi— cal appearance of cells with differing secretory functions. Day 41 to 55. Ultrastructural study of the anterior pituitary of the early fetus has been done (1744). Development of Rathke’s pouch is well advanced by Day 41, and differentiation of the infundibular stalk has begun. The lumen of the infundibular stalk is continuous with the third ventricle. By Day 43, the pouch is separated from the oral region by a cartilaginous plate. By Day 55, the growth of the pars distalis is extensive and consists of a mass of cell cords sur- rounded by blood vessels and connective tissue. Day 60. The adenohypophysis consists mainly of undifferentiated cells in a col— lagenous matrix. Day 75. The pars distalis is well devel— oped. The epithelial cells are tightly arranged in clusters and appear to be secretory (1744). Day 80. The gland is still embryonic, consisting of mesenchymal tissue of uni- form cell type. The cells are large with numerous mitotic figures indicating marked hyperplasia (Figure 9.58). The cells are apparently active with granular cytoplasm. Chromophobic or chromophilic cells (type A or B) are not yet definable, and the sinusoids are also poorly defined. Day 100. The hypothalamic-pituitary portal system is established between Days 50 and 95 (cited in 1744), creating a possible functional link between the hypothalamus and pars distalis. Between Days 80 and 100, a marked change occurs in the parenchymal cells wherein the embryonic tissue takes on the appearance of a functional gland. The parenchymal fetal activity. Fetal mobility refers to whole body movement involving position or location changes, sometimes profound, within the allantoic sac. Fetal activity refers to in-place movements (e.g., head, mouth, and limb movements). Fetal activ- ity contributes to fetal mobility, and often these activities will occur together. Either can occur alone, however (e.g., limb move- ment without a change in whole body location or a location change without active participation of the fetus). Days 10 to 16. Mobility of the entire conceptus occurs within the uterine lumen in the early embryo stage (pg. 305). Several studies have indicated that the side of ovulation does not influence the side of fixation at the end of the mobility phase; that is, side of ovulation and side of fixation are independent events. The difference in size of uterine horns, howev— er, does affect the side of fixation and accounts for the more frequent fixation in the previously nongravid horn during the postpartum period. Days 16 to 40. Movement of the entire conceptus between the day of fixation and the end of the embryo stage has not been detected in several ultrasound studies, except in association with embryo loss (pg. 527). It follows, therefore, that side of ovulation (left or right ovary) would be unrelated to side of the conceptus at Day 40 (left or right uterine horn). After fixa- tion and orientation, the conceptus under- goes conversion from yolk sac to allantoic sac over Days 21 to 40 (pg. 367). As a result, the umbilical cord of the develop- ing fetus is attached to the dorsal aspect of the vesicle. Thus, the side of cord attachment is the same as the side of fix- ation, and the endometrial cups are also necessarily located in this area. Days 40 to 60. The umbilical cord lengthens and the fetal-amniotic unit is free to move within the allantoic sac, except that it is tethered to the inner dor- sal surface of the allantochorion by the attachment of the umbilical cord. During this stage, the fetus begins to become Embryology and Placentation 409 active and occasionally, toward the end of this stage, the fetal-amniotic unit moves among the two uterine horns and uterine body (601). In contrast to equids, the amnion in other farm species is fused to the allanto- chorion in certain areas, preventing intra- allantoic location changes (95). Days 60 to 100. The fetal-amniotic unit is highly mobile within the allantoic sac and can be found at any time in either of the uterine horns or in the uterine body without regard to the side of attachment of the umbilical cord (601, 649). Days 100 to 180. The extent of transu- terine mobility gradually diminishes by Day 150 and ceases by approximately Day 180 (648). Days 180 to term. The cessation of fetal mobility is associated with an unknown mechanism that usually assures that the fetus will be in the horn containing the attachment of the umbilical cord when mobility ceases, and furthermore, assures that the fetus will be in cranial presenta- tion. The fetus usually does not change horns thereafter; consequently at birth the fetus is usually in the horn of the umbilical cord—that is, the horn of con- ceptus fixation on Day 16. 9.123. Earlier Studies Transuterine migration. Transuterine migration of the fetus within the allanto- chorionic sac has been suspected because of inconsistencies in agreement of fetal location at the beginning of the fetal stage versus end of pregnancy. In an ini- tial study (40), the fetus at term was in the uterine horn opposite the location of the conceptus at Day 42 in 18% of pony and Thoroughbred mares. In the exten- sive study of Pascoe (1242), the location of the conceptus at Day 42 was compared to the location of the fetus at term, based on postpartum palpation of the involuting uterus and, in some cases, the shape of the discharged placenta; the fetus was assumed to have occupied the largest horn. No transuterine migration was 408 Chapter 9 2% FIGURE 9.60. Day 150. Fetal pituitary showing cord-like appearance with vascular channels. Day 200. The gland is richly cellular‘ (Figure 9.61). The degree of vascularity is similar to that of the adult gland and typ- ical of an active endocrine organ with fen- estrated and thin capillary endothelium (1385). The adenohypophysis of the fetal foal appears to contain all the cell types found in the adult gland, except pro- lactin-producing cells, and there is evi- dence of secretory activity for all cell types (1385). Apparent ACTH-producing cells and thyrotrophs seem fewer in num- ber by Day 200 (1385). In the most recent report (1744), the fol- lowing conclusions were made about the time of appearance of various cells types that differ from the above described find- ings: 1) Prolactin-secreting cells (mammo- trophs) were abundant as early as Day 75; 2) ACTH-secreting cells (corti- cotrophs) were not numerous but were present by Day 75; 3) GH-secreting cells (somatotrophs) and LH/FSH-secreting cells (gonadotrophs) were not differenti- ated from one another before Day 250; and 4) Thyrotrophs were not identified at FIGURE 9.61. Day 200. Richly cellular and vascu- larized fetal pituitary. any stage. The divergent conclusions between this study and the earlier stud- ies are a source of confusion. However, it can be concluded that the cells of the anterior pituitary are active during much of pregnancy, and the morphologic changes indicate a need for studies on the functional role and importance of the equine fetal pituitary. 9.12. Fetal Mobility and Activity 9.12A. Overview of Conceptus and Fetal Kinetics The availability of ultrasound scanners and Videoendoscopes has rekindled inter- est in the fascinating kinetics of the conceptus. Much of the work in this area has been done only in the past two years. Therefore, many readers will not have a current familiarity with this subject, and an introductory overview or summa- ry will be given. A distinction is made between the terms fetal mobility and Embryology and Placentation 407 cells are much smaller and numerous and are arranged in trabeculated fashion with prominent reticular and septal con- nective tissue (Figure 9.59). Chromo- phobes and chromophils of types A and B are discernible. The A cells are few and are at the edge of sinusoids. They con- tain prominent eosinophilic granules. Gonadotrophs and thyrotrophs are pre- sent at Day 100, with gonadotrophs making up about 20% of the cell popula- tion (1385). Somatotrophs (GH producing) are the most numerous as identified by droplet size. Cells that secrete ACTH also appear to be present. Based on mor- phology, the equine fetal adenohypoph- ysis may begin to actively synthesize and secrete trophic hormones between Days 75 and 100. Days 150 to 160. All three cell types are apparent. Overall, the glandular tissue has assumed a cord—like appearance, with increased density of connective tissue and trabecula and invasion of the parenchy- ma by vascular channels (Figure 9.60). FIGURE 9.58. Day 80. Fetal pituitary. ‘ £~ ‘ .ai .s\\\‘.-‘ "§ “a FIGURE 9.59. Day 100. Fetal pituitary showing parenchymal cells are arranged in trabeculated fashion. 410 Chapter 9 detected in 139 maiden mares. In con- trast, migration, as indicated by disagree- ment between conceptus position at Day 42 and fetal location at term, occurred in 18% of 760 lactating mares. In a recent ultrasound study (648), the fetus at term was in the horn in which fixation occurred in 10 of 10 pony mares. These studies indicate that the side containing the fetus at term differs from the side of fixation of conceptus but only in a minori— ty of mares (e.g., 18%), especially those that have had previous pregnancies. Perhaps the larger uterus in older mares accounts for an increased incidence of dis- agreement between side of fixation and side of fetus at term; alternatively, palpa- tion errors to establish side of the concep- tus at Day 42 may be more likely to occur in older mares. Umbilical cord twists and fetal presen- tation. Twisting of the umbilical cord has been described and also indicates fetal mobility, although not necessarily between horns. In a study of 157 concep- tuses (1684), 78% had twisted cords, and the average number of twists was 4.4 per cord. Twists were noted as early as 68 days, but the number of twists did not differ over the various months. Between 4 and 7 months, 50% of the fetuses were in cranial presentation, compared to 97% at 7 months. Fetal changes in direction resulting in umbilical cord twists, there- fore, seldom occurred after seven months. Similarly, according to tabulated data (95), the equine fetus has an equal chance of cranial or caudal presentation during approximately the first half of pregnancy. By 8.5 to 10 months, 11/11 fetuses were in the cranial presentation. In a recent ultrasonic monitoring study (648), the first day that the fetuses were in their final presentation and location (left versus right horn) ranged from Day 151 to 192 (mean: Day 171), excluding two fetuses that temporarily changed presentation on Days 240 and 250, respectively. It has been concluded (1683) that caudal and transverse presentations at term are important causes of fetal malformation and dystocia. Twisting apparently occurred in the amniotic and allantoic portions of the cord independently (1684), suggesting movement of the fetal-amniotic unit with- in the allantochorionic sac and change in relative position of the fetus within the amniotic sac. In recent studies (601, 649, 648,), rotation of the fetus within the amnion was not observed; the existence and incidence of this phenomenon awaits clarification. Fetal activity. Doppler ultrasound has been used to assess apparent fetal activi— ty (534). The fetus was reported to display simple movements (e.g., apparent flexion or extension of limbs or back) over 3 to 8 months and more complex movements during the last three months. 9120. Recent Studies Extensive fetal mobility during two to five months. During transcervical video- endoscopic examination on Days 69 to 78 in seven mares, it was discovered that the fetus underwent astounding feats of activity and location changes within the allantochorionic sac (Figures 9.15 and 9.62; 601). In addition, the openings into the portion of the sac corresponding to the corpus-cornual junctions were some- times widely dilated and at other times tightly constricted, indicating extensive uterine dynamics. A Day 44 fetus was inactive and immobile during six minutes of continuous viewing, a Day 60 fetus was quite active and slightly mobile, and five fetuses on Days 69 to 78 were extremely active and extremely mobile. Subsequent study using both transcervical videoen- doscopy and transrectal ultrasonography confirmed the phenomenon on Days 69 to 81 and indicated that it was not an arti- fact of the examining technique (649). On the average, major location changes (between uterine horns and between a horn and the body) occurred five times per hour. Several instances were Embryology and Placentation 411 observed in which the fetal-amniotic unit was hidden in a uterine horn and then appeared to be forced through the con- stricted horn entrance into a dilated uter- ine body. The side of umbilical attach- ment did not affect the location of the fetus (cord horn versus noncord horn). Yet, as noted above, the fetus is usually in the horn of the umbilical cord at term. The extensive fetal mobility apparently began to decline after Day 100 and ceased (as indicated by cessation of movements between horns) by approxi- mately Day 180 (648). Considerable study will be needed to determine the cause and role of the extensive activity and to char- acterize the manner in which the fetus finally assumes a cranial presentation in the cord horn. Clearly, the fetal-amniotic FIGURE 9.62. Illustration of mobility of the fetal-amniotic unit and shifts in the allanto- ic fluid on Days 60 to 100. The series of depicted changes can occur rapidly (e.g., 30 minutes). Prepared by E. M. Carnevale. unit, unattached except by the long umbilical cord, is free to move within the allantochorionic cavity during the early fetal stage and does so with vigor. There- fore, transuterine migration, described above, is not the result of a one-way trip. Perhaps the extensive activity and mobil- ity in this species at this early stage play a role in fetal development of muscle and nerve coordination; this, in turn, may be related to the advanced limb development in this species. Allantoic fluid shifts. It also was discov- ered (649) that the dimensions of various portions of the allantochorionic fluid com— partment changed dramatically over Days 69 to 78. The resulting allantoic fluid shifts corresponded to changes in the diameter of various portions of the 412 Chapter 9 uterus (Figures 9.62 and 9.63). During continuous ultrasonic Viewing, alternate maximum dilation and constriction (no Visible lumen) of the entrances into the horns occurred a mean of 1.3 times per hour per horn entrance. Sometimes the Fetal Horn uterine wall was constricted around the fetal-amniotic unit with no detectable intervening fluid, whereas at other times the uterus in the same location was wide- ly dilated without regard to the location (left or right horn; cord or noncord horn). FIGURE 9.63. Ultrasonograms of a segment of the nonfetal and fetal horns from a mare on Day 77 of preg— nancy. Ultrasonograms A, B, and C are cross sections of the midportion of the middle cornual segment. A steel ball (large arrows) was attached to the uterine serosa and served to indicate that the three images were from the same area. Ultrasonograms A and B were taken within 1 minute of each other, and C was taken 22 minutes later. Small arrows indicate the periphery of the uterine horn. Nonechogenic areas (black) within the lumen of the horn represent amounts of allantoic fluid. Ultrasonograms D, E, and F are cross sections of the fetus and uterine horn at the junction of the middle and caudal segments. The three images were obtained from same location within three minutes of one another. Arrows delineate the fetus within the uterine horn. Compare the three ultrasonograms for changes in the amount of fluid surrounding the fetus. From (649). Fetal activity. Based on 30-second activity trials, the fetuses were active a mean of 27% and were quiet a mean of 73% of the time on Days 69 to 81 (649). Activity sometimes involved only move- ments of the extremities, head, or mouth and at other times led to whole body movements (fetal mobility) in sudden bursts (startle-type movements). The whole-body bursts of activity buoyed the fetus into the allantochorionic fluid, and movements of the limbs or head caused the fetus to push off of the allantochori- onic wall. These movements often result- ed in dramatic changes in location, recumbency, and presentation (direction faced by fetus). Thus, extensive activity contributed to fetal mobility. Recum- bency changes occurred an average of 10 times per hour, and presentation changes occurred an average of five times per hour. Embryology and Placentation 413 9.13. Other Aspects of Conceptus Development 9.13A. Factors Affecting Growth Rate of Embryonic Vesicle Type of mare. Length of the embryo proper beginning at Day 19 and diameter of the embryonic vesicle beginning at Day 9 are shown for horses and ponies (Figure 9.64; 590). Ultrasound studies have not detected a difference between the two mare types in dimensions of the conceptus through the embryo stage. Limited data indicate that the embryonic vesicle is about 2 mm smaller in jennies than in ponies on Days 11 and 12 (195). The interrelationships between vesicle diameter and uterine diameter have been postulated to account for fixation of the vesicle a day later in horses than in ponies (larger uterus in horses) and a day later in jennies than in ponies (smaller vesicle in jennies; pg. 309). Dimensions of conceptus Embryonic vesicle Days 311-165 3 3 Days 16-28 Days 28-45 Mean height & width (mm) 91317 2125 29 33 Number of days from ovulation Horses (n=9 to 20) ‘ Regression - Actual means . Ponies (n=9 to 80) DE 5 i Regression 1 z ' D Actual means 37 41 30 Embryo proper Length (mm) 45 17 21 25 29 33 37 41 45 FIGURE 9.64. Dimensions of the in situ embryonic vesicle and embryo proper as determined by transrectal ultrasonic imaging. No significant differences were found between horses and ponies for either end point. From (590). 414 Chapter 9 Gender of conceptus. In several species, male embryos cleave and grow faster than female embryos (cited in 1341). In a recent study, no difference in diameter was found between removed male and female equine embryos on Days 6 to 15; sex was established cytogenetically (1341). Similarly, no difference in growth rate over Days 11 to 40 was found ultrasoni- cally in embryonic vesicles that were later determined to be male (n=21) versus female (n=17; 600). Effect of steroids. The ovarian steroids (estrogen and progesterone) have differ— ent effects on uterine secretion of pro- teins. Progesterone is required for the production of uteroferrin, whereas both progesterone and estrogen are required for production of another low molecular weight protein (1751). Therefore, the effect of exogenous steroids on growth rate of the embryonic vesicle was studied (1751). Results of treatment with progesterone versus progesterone and estrogen sug- gested the presence of a differential effect on ultrasonically determined growth rate as indicated by a day-by-treatment inter- action. In another study (194), growth rate of the vesicle did not differ between estra- diol—treated and control mares. 9.133. Other Aspects of Fetal Development Growth rate of the fetus in ponies and horses is discussed elsewhere (pg. 393). Recent information is available on the development of the fetal adrenal (1385, 1831), fetal cardiac growth (1003), fetal heart rate (1028, 1027), growth of fetus (1274), and fetal pulmonary maturity (1788). A review of equine fetal physiol— ogy is also available (1474). An interesting aspect noted in this review is that a ductus venosus is absent in the equine fetal circulation in contrast to most other mammalian species. Thus, small microspheres (15 um) injected into the equine umbilical vein were trapped in the fetal liver. Catheterization problems. Apparently because the placental attachments involve almost the entire uterus, surgical invention, such as for vascular catheteri- zation, is more likely to cause placental damage in equids than in species with large placentomes. Vascular catheteriza- tions have been done after midpregnancy, and these procedures have been reviewed (1473). Entry is made at the bifurcation of the uterine horns where the umbilical vessels are most accessible. Placement of catheters in the amniotic and allantoic sacs and removal of fetal gonads are also discussed. The author notes that it is a mystery why some fetuses are retained for 40 to 50 days after catheterization and then suddenly abort and why abortions are more likely after catheterizations than after gonadectomy. Insertion of catheters within 2 or 3 weeks of term is hazardous because of the large active fetus and the dangers of compromising the uterine circulation. Uterine capacity. In the 1938 study of Walton and Hammond (cited in 1621), reciprocal matings were made between draft horses and ponies. The resulting crossbred foals born to draft mares were threefold larger than those born to ponies. The disparity diminished follow— ing birth but was still considerable after four years. Embryos from ponies trans- ferred to draft mares developed into foals with 48% greater mean birth weight and 37% greater weight gain by 6 to 7 months (1621, 1618, 1619). Even at 53 months of age, the size differences were distinct. These studies have shown that the size of the newborn foal reflects not only its genetic makeup but also the maternal environ- ment. A profound aspect of the maternal environment is the size of the dam or uterus. The effects of uterine environ- ment on fetal size are fortuitous and help assure that delivery can occur. The long— term effects after birth may reflect lacta— tional productivity, as well as skeletal development in utero. In an embryo transfer program, no differential effect of recipient on birth weight of foal was detected (1451). Presumably, the failure to find an effect on foal size was due to less pronounced size difference between donors and recipients than was present in the draft horse-pony study. 9. 130. H ippomanes A hippomanes or allantoic calculus is a large flat body composed of concentric rings of amorphous material. It is consis- tently present in the allantoic fluid in equids. Interest in the hippomanes is more historic (Aristotle refers to them) than physiologic (882). King and associ- ates (405, 882) have done extensive studies of the objects, and the following account is based on their reports. A hippomanes appears at approxi- mately Day 90 as a small whitish object with a length of a centimeter or more. After approximately Day 125, it becomes beige or brown, reflecting the color changes of allantoic fluid. The structure increases in size as pregnancy advances and reaches a length of 10 cm or more (Figure 9.65). It has a putty-like consis- tency. The calculus is not attached to, or pedunculated from, the allantoic wall Imnniumnunmmmnqnmuu m 1 2 3 4 5 FIGURE 9.65. Day 300. Hippomanes. Embryology and Placentation 415 but has a higher specific gravity than the allantoic fluid and therefore settles to the floor of the sac. During videoendo- scopic examination, a hippomanes was observed floating in the allantoic fluid (600); whether or not the hippomanes is floating or resting on the floor of the allantois apparently depends on the degree of disturbance to the allantoic fluid by the fetus, uterus, or mare. Whitwell and Jeffcott (1770) found a large hippomanes in every case in their extensive series at term. Several small pieces of debris of hippomanes also may be found in the allantoic fluid but never in the amniotic fluid. These sometimes adhere to the floor of the allantoic cavity and form a small focal area of necrosis. Hippomanes debris also may adhere to the allantochorionic pouches. The hippo- manes should not be confused with pouches, even though earlier workers (cited in 882) made the faulty assumption that the pouches and the hippomanes were attached and unattached struc- tures of identical origin. A hippomanes apparently originates from the deposition of material from the allantoic fluid onto a nucleus of epithe— lial debris (882). It increases in size by the concentric deposition of material from the allantoic fluid. On cross section of the hippomanes, concentric rings forming stratifications around a central core are evident. Histologically, the con- centric layers consist of cell debris embedded in an amorphous mass. Chemically, it consists of mucoprotein in which minerals, especially calcium phos- phate, have been deposited. It resembles some urinary calculi in other species; in this regard the allantoic fluid may be considered an accumulation of fetal urine because of the connection of the allantoic cavity to the fetal bladder by the allantoic duct (urachus). 416 Chapter 9 9.13D. Reproductive Immunology A term being used increasingly in reproductive biology is major histocom- patibility complex (MHC; review that includes horses: 1540). The MHC is a set of genes that encodes information for the type of proteins found on the surface of cells of each individual (1731). The result— ing proteins (antigens) incite immune responses. Immunofluorescence assays have been used to detect the H-Y antigen on equine blastocysts (1767). The conceptus should be susceptible to immunologic rejection by the mother since paternal as well as maternal MHC genes are expressed by the conceptus. However, a complex system of protective mechanisms has evolved that assures successful survival of the conceptus. It has been postulated that unique proteins and steroid hormones are produced by the trophoblast that impart a privileged status to the conceptus even though cyto- toxic lymphocytes are produced as a response to the foreign antigens (169). Thus, the conceptus is said to generate immunosuppressive activity. The early equine conceptus produced immunosup- pressants on all days studied (Days 9 to 26; 1369, 1368, 1367). Late entry. Further elucidation of a immunosuppressive fac- tor in the horse has been reported (1870). During the development of endometrial cups, the foreign antigens of the fetus stimulate a cytologic immunoreaction against invading trophoblastic cells Leucocytes (lymphocytes, eosinophils, plasma cells, and macrophages) appear at the border of the endometrial cups, invade the cups after Day 80, and destroy the fetal cup cells. Other tro— phoblast cells that did not invade the uterus are spared. Paternal MHC anti— gens have been demonstrated on the chorionic girdle cells but not in other regions of the allantochorion (356). This concept is compatible with the findings that in-vitro cultured girdle cells (62) and the endometrial cups of a mare carrying a fetus sired by her brother (235) have a much longer productive life. In addition, MHC genes attributable to a particular stallion may elicit an especially strong reaction that is further enhanced during subsequent pregnancies to the same stal- lion. For detailed information on the immunologic aspects of endometrial cups, consult the reports of Allen and Antczak and associates (44, 52, 58, 88, 46, 1183, 926, 53, 48, 924, 420). An immunologic barrier appears to ter- minate pregnancies that involve a dam and fetus of different species. Pregnan- cies are called intraspecific when a mated female and male are the same species, interspecific when the pair are of differ- ent species, and extraspecific when a recipient female is a different species than a transferred embryo. For example, embryo transfer of donkey embryos to mares has been used to study immunore- jection (53, 58, 48, 61). About 80% of don- key—in-horse pregnancies abort between Days 80 and 95. The success rate is increased by infusion of serum from nor- mally pregnant mares or immunization of the recipients with lymphocytes from the donkey parents. These approaches apparently alter the recipient’s rejection reaction, and similar approaches are used in organ grafting. Studies on the immunology of interspecies and extraspecies pregnancies are well reviewed (58, 48). A monoclonal antibody specific for the equine trophoblast was recently pro- duced in mice (1183, 87). These studies indicated that the developing equine tro- phoblast gradually expresses new molecules according to its need for devel— oping functions. In addition, the estab- lishment of a monolayer culture of cells from the donkey chorionic girdle has been reported (1772). It is anticipated that these technologies will be an aid in fur- ther elucidating the immune responses and molecular structure of the equine placenta. Embryology and Placentation 417 HIGHLIGHTS: Embryology and Placentation In mares mated before ovulation, sperm penetration of the oocyte occurs 10 to 12 hours after ovulation and the first cleavage at 24 hours; about three blastomeres develop per day for the next few days. Most embryos at Day 5 are still in the oviduct and are morulae, Whereas at Day 6 most are in the uterus and are beginning to form a blastocoele. 3. After formation of a blastocyst, the zona pellucida is shed from a new inner layer called the capsule which remains for a few weeks as the outer coating. Encirclement of the blastocoele with endodermal cells probably occurs by Day 11. The resulting yolk-sac vesicle remains spherical and is highly mobile within the uterus until fixation at Day 16. A portion of the yolk sac becomes encircled with mesoderm to form a three-lay- ered wall. By Day 22, the allantoic sac begins to emerge from the embryo proper and gradually becomes dominant over the yolk sac. Placental attachment occurs gradually over Days 40 to 150. Placentation is dif- fuse, involving complex microcotyledons that fit into corresponding microcarun- cles. The microcotyledons are surrounded by a net-like absorptive area. Secretions from the uterine glands are discharged into the area and are directly available to the overlying trophoblastic cells. Fetal cells from the chorionic girdle, a belt of tissue surrounding the conceptus, invade the endometrium at about Day 38 and form the eCG-producing cells of the endometrial cups. The amnion and contained fetus move within the allantoic fluid, constrained only by the umbilical cord. Fetal activity and mobility of the fetal-amniotic unit are extreme over approximately Days 60 to 120, with active location changes between both uterine horns and uterine body several times per hour. The genital tubercle is the forerunner of the clitoris (female) and penis (male). It can be used to ultrasonically identify fetal gender, optimally between Days 60 and 70, by its relative location near the tail (female) versus umbilical cord (male). The fetal gonads are unusually large in equids, exceeding the size of the maternal gonads at 7 to 8 months. The large size is due to the development of interstitial cells that histologically have the appearance of luteal or hepatic cells. 12. The fetus develops a hypothalamic—pituitary portal system between Days 50 and 95, and the pituitary takes on the appearance of a functional gland between Days 80 and 100. 418 Chapter 9 MILESTONES: Embryology and Placentation Study of development of the fetal gonads (323). Descriptions of equine placentation with emphasis on the role of uterine glands (80). Extensive morphologic and biochemical studies of the hippomanes (405, 882). Report on developmental highlights of the embryo and fetus (187). Atlas on the fine structure of the placenta (211). Initiation of a series of detailed studies on the organization of the micro- cotyledons and microcaruncles (1386, 1387, 1546). Beginning of a series of reports by Canadian workers on the conceptus cap- sule (1015, 516), culminating in an extensive review in 1989 (196). Beginning of a series of reports on the immunosuppressing aspects of equine placentation (44). 1975 First of two detailed reports on fetal oogenesis (389, 390). 1975 Report on development of the fetal pituitary (1385). 1977 Detailed study of the uterine glands during pregnancy (1388). 1980-84 Development of intravascular fetal catheterization and study of maternal— placental exchange (1473, 1474). 1982 Structural study of the embryo up to Day 22 (198). 1982 Accounting of the early history (beginning in 16th century) of placental stud- ies (1544). 1983 Characterization of in vivo growth and shape changes of the embryonic vesi- cle and transition from yolk sac to allantoic sac over Days 10 to 40 (581, 580). Discovery of the extensive mobility of the fetal-amniotic unit Within the allantoic fluid (601). Characterization of in vivo and in vitro cleavage rates, with ovulation deter- mined at hourly intervals (206). —Cfiapter 10— ENDOCRINOLOGY OF PREGNANCY The endocrinology of pregnancy in mares is a good example of the complexi- ty and beauty of biologic mecha- nisms. Several striking endocrinologic phenomena have evolved in mares including the following: 1) the production of chorionic gonadotropin (CG), 2) the production of unusual estrogens, and 3) the appearance, maintenance, and loss of several endocrinologic structures (endometrial cups, supplementary corpo- ra lutea, and fetal gonads with massive quantities of interstitial cells). Sections are devoted to circulating and urinary concentrations of hormones, the roles of the ovaries, conceptus, and endometrial cups, and the regulation of the life and function of follicles and corpora lutea. 10.1. Hormone Concentrations 10.1A. Chorionic Gonadotropin Circulating levels. Investigations on circulating levels of CG have been reviewed (43, 384, 1374). There is general agreement that CG is first detectable on approximately Days 35 to 42 in indi- vidual mares, rises rapidly to a peak at Days 55 to 65, and then declines slowly to low or nondetectable levels by Days 120 to 150 (Figure 10.1). The day of reference is not uniform among reports and can account for several days discrepancy. Reference points include day of ovulation, last day of estrus, day of service, and day of gestation as estimated from crown- rump length. Regardless of the day of reference, most authors agree on a 5 or 6 day difference among mares for the first detectable appearance of CG. There is strong agreement among reports that variation among mares in the maximal concentration is great; differences of ten- fold or more can be expected. There is also strong agreement among reports that the rise of CG concentrations is much more rapid than the decline. An impression of the wide divergence among mares in CG profiles can be gained from Figure 10.1. It would be interesting to quantitate CG in uterine venous blood or uterine flushings to determine if some CG may be released into the uterine lumen 0r mater- nal circulation prior to the invasion of the CG—producing trophoblastic cells into the endometrium (pg. 370). Increasingly sensi- tive assay systems are detecting chorionic gonadotropin in primates at increasingly early stages of pregnancy. Monkey chori- onic gonadotropin is detectable several days earlier in the uterine venous efflu- ent than in the general circulation; it has been detected apparently as early as Day 9 after ovulation (1087). The ques- tion of day of first appearance has impor— tant implications for the functional role of eCG. Temporal association with endome- trial cups. There is a temporal associa- tion between the appearance of CG in the blood and the development of the endometrial cups (311). Growth of the cups and the rapidly increasing con- centrations of circulating CG occur concurrently between Days 40 and 70 420 Chapter 10 Equine Chorionic Gonadotropin (n=58) 160 0.... .:..° 120 no '0... :. ° 80 40 ' a ': .o : o 5" E .g Individual profiles a: E 3 160 I: O o 120 80 40 40 60 80 100 120 140 Number of days from ovulation FIGURE 10.1. Circulating concentrations of eCG. Upper panel: Dots represent individual values taken at weekly intervals, and solid line is regres- sion line best representing the pattern of individual values. Lower panel: Selected profiles for individual mares. Note the extreme variation among mares in maximum values and duration of profiles. From (600). (Figure 10.2). Both sloughing of the cups and decreasing levels of circulating CG occur shortly thereafter, with a temporal association between the two within mares. The concentration of CG in the allantochorionic pouches enclosing the sloughed cups (pg. 372) is very low after approximately Day 100. The concentra— tions in nonvascular body fluids (milk, urine) are noted in Chapter 2 (pg. 51). Effect of genotypes on CG levels. Genotype of the fetus profoundly influ- ences CG productivity of the endometrial cups since the cup cells are of fetal origin (pg. 370). Allen and co-workers have stud- ied extensively the effect of fetal genotype by the use of horse and donkey and other crosses and by cross—species embryo transfers. Compared to mares with a horse fetus (stallion sire), production of CG is depressed in mares carrying a mule fetus (donkey sire; 43, 312) and elevated in jennies carrying a hinny fetus (stallion sire; 43). The ratio of FSH-like to LH—like activities of CG is also affected by geno- type (1556, 1558). The ovaries ofjennies with a hinny fetus had massive follicular growth in addition to luteinization, and the CG had a greater FSHzLH ratio than for jennies with a donkey fetus. It was suggested (1558) that the ovaries of equids are protected from the potential extreme effects of massive amounts of CG when carrying a fetus of their own species and that this protection is mediated by refrac— toriness of the receptors. In donkeys car- rying a horse conceptus (embryo trans- fer), FSH concentrations fell and progesterone concentrations rose sharply, coincidental with abnormally high levels of CG (1655). The donkey receptors responded to the higher FSH—like activity of CG from a horse conceptus, and there- fore the ovaries were apparently hyper- stimulated during this unnatural preg- nancy. In horses carrying a donkey conceptus, there was a complete absence of CG in 7 of 8 mares and no increase in progesterone; these unnatural pregnan- cies were not carried to term even when exogenous progesterone was given. The effect of sire on CG levels also has been studied in horse >< horse matings; some stallions were associated with low levels and some with high levels (cited in 1012). Furthermore, the FSH-like activity was positively correlated with serum CG concentrations and was related to sire of the fetus. Recent studies (1024, 1012) have demonstrated that the mare’s contribu— Relationship between eCG and endometrial cups eCG 150 “4/ 50 Endometrial cups ‘ Concentration (ng/ml) \l 01 N 01 O 40 60 80 100 120 140 160 Number of days from ovulation tion, as well as the sire’s, to the fetal genome affects the levels of CG in the maternal circulation. It is concluded, therefore, that productivity of CG is a her— itable characteristic, and a line of animals could be developed by genetic selection techniques for high CG productivity (1012). Effect of ratio of mare body size to cup mass on CG levels. In addition to fetal genotype, the size of the endometrial cups in relation to size of the mare also would affect circulating concentrations of CG. A large cup mass and a small mare body size would be expected to be positively related to circulating CG concentrations. Cup mass seems similar between ponies and horses; a mean weight of 10 g has been reported for both mare types (pg. 372). In this regard, the conceptus is similar in size between the two mare types at the time of invasion of the chorionic girdle cells (590). Therefore, it follows that the endometrial cups also would be similar in size between ponies and horses, assuming that the size of the conceptus is a reflec- tion of the size of the chorionic girdle. Higher concentrations of CG can, there- fore, be expected in ponies because of the small body size and less dilution of the CG output. This assumes that cup-mass is a direct reflection of number of cup cells and that the cup cells secrete at a 180 Endocrinology of Pregnancy 421 (6) iufiiaM FIGURE 10.2. Temporal association between weight of the endometrial cups (adapted from 430) and circulat- ing concentrations of eCG (adapted from 1373). constant rate without in utero regulatory control. In this regard, CG production in vitro is a function of cell numbers. Perhaps in vivo production is also a reflection of the number of healthy cup cells, with cells secreting a constant rate of CG (1604). An early report concluded that CG concentrations were fourfold higher in ponies (317). Significantly higher levels of eCG were obtained from small ponies than from large ponies (Figure 10.3; 600). Twins and other factors influencing CG levels. Mares with twin fetuses and two sets of endometrial cups produce large quantities of CG (1373). In a recent study (600), eCG levels were approxi— mately twice as high in mares with bilat- eral twins than in mares with unilateral twins or singletons (Figure 10.3); the singletons were from mares that earlier had unilateral twins but underwent embryo reduction. The low levels in uni- lateral twins probably reflect the large area of ineffective apposition between the two conceptuses. Mares mated at the first postpartum estrus had higher CG levels than those mated at a later estrus (171), and mares in their third or subse- quent pregnancies had higher CG levels than primiparous mares (1127). Perhaps, size of the uterus at the time of invasion 422 Chapter 10 Equine Chorionic Gonadotropin Fetal twins Bilateral twins 100 imii } / + (n=10) 80 Singletons ‘ ‘ ,1 (n=13) 34 60 .. ____| 4 f ." .......... _ 0 i Unilateral twins 20 (n=3) Group, P<0.05 0 Fetal gender 100 Male fetus (n=25) 80 /T~\ ' F""r~~.- 60 Female fetus (n=33) E 40 Group, P<0.05 E 20 r c 0 I .g Mare weight (U E- 170 to 288 kg a) 100 0 S o 80 60 """ 40 x 290 to 460 kg T \‘I =2 ,’ (n 9) Group, P<0.05 20 r' 0 Control vs fetus removed 80 Control 60 40 Fetus removed L (n=28) 20 GI'OUp, NS 45-51 Number of days after ovulation 59-68 73-79 87-93 FIGURE 10.3. Effect of various factors on circulat- ing levels of eCG. Data on influence of fetal twins are from horse mares, data on influence of fetal gen— der and mare weight are from a single survey in pony mares, and data on the effect of removal of fetus are from another group of horses and ponies combined. Fetuses were aborted (lower panel) by 6 daily injections of PGF, beginning on Days 38 to 44. From (600). of the trophoblastic girdle cells is also a factor in the resulting cup mass. Nutrition also has been reported to affect CG levels (312), but the effects have not been well documented. Because many fac- tors affect CG levels, studies in this area are difficult; confounding due to interrela— tionships among factors is a challenge. Effect of fetal gender on CG levels. A recent report from Germany presented the interesting observation that CG levels were higher in mares carrying female fetuses than in mares carrying male fetuses (1110). Higher CG productivity for fetal cells of female origin recalls the find- ing that mares with female conceptuses are more likely to show estrous behavior during pregnancy, especially on Days 12 to 20 (pg. 321). It is not known whether these two observations are related, but the similarities are intriguing. The hypothesis of a positive effect of female fetus on CG levels, however, was not supported in a recent study (Figure 10.3, 600); instead, the concentrations were higher (P<0.05) in mares carrying male fetuses. Clarifying study is needed on the role of fetal gender on eCG levels. Pulsatility. The production of CG is apparently constant (tonic secretion) without diurnal variation or pulsatility. This conclusion is based on failure to find fluctuations greater than what was inher- ent in the assay in five mares sampled every 30 minutes for 25 hours (1604). The authors commented that the absence of short-term regulation is consistent with failure to find secretory granules in cup cells and implies that secretion of CG involves rapid turnover with mini- mal storage. Administration of GnRH did not alter CG production (1604, 1148). 10.13. Luteinizing Hormone Circulating levels. Limited study has been done on the circulating concentra— tions of eLH and eFSH during pregnancy. In pony mares, mean LH remained at the low mid—diestrus levels following the ovulatory surge until Day 32, which was the last day studied (1092; Figure 10.4). The continuation of low mean baseline levels of LH up to Days 35 to 40 has been confirmed in both mares and jennies (1654). Available assays do not adequately differentiate eLH from eCG, and conse- quently there is no direct information available on LH levels during the time that CG is present (Days 40 to 120). After the disappearance of CG, LH concentra- tions are comparable to mid—diestrus or baseline levels for the remainder of preg- nancy (1143, 1164, 1757). After the ovulatory surge, it is likely that mean LH concen- trations remain low throughout pregnan- cy. In one study, LH levels after the ovu- latory surge were apparently not related to the secretion of estrogens (1143). Treatment with GnRH failed to increase LH levels during pregnancy, indicating low pituitary content (1148). Prolactin lev— els were found to be extremely variable Gonadotropins (n=11) Concentration (ng/ml) 1 13 24 40 56 72 Number of days from ovulation FIGURE 10.4. Changes in gonadotropin concentra— tions in early pregnant and hysterectomized mares. No significant differences were found between preg- nant (n=6) and hysterectomized (n=5) mares, and data were combined to study the effect of day. However, concentrations of LH for Days 36-72 are for hysterectomized mares only. Vertical bars indi- cate the magnitude of the least significant differ- ences. Adapted from (1092). Endocrinology of Pregnancy 423 during pregnancy, without a clear pattern (1143). Pulsatility (pg. 45) and isoforms (pg. 43) of LH and FSH are discussed in Chapter 2. Fetal blood levels. Higher levels of LH immunoactivity were found in the fetal blood on Days 100 to 160 (4.2 :05 ng/ml) than on Days 160 to 240 (1.1 i0.2 ng/ml; 1757). It was not known whether the LH activity before Day 160 was due to fetal LH or maternal CG. It is noteworthy, however, that there is considerable growth of the fetal gonads following the appearance of high LH/CG activity in the fetal circulation. An abstract (1131) noted that bioactive LH in fetal blood was high when maternal LH activity was high (likely due to CG), but fetal bioactive LH remained high when maternal bioactive LH was undetectable. On a temporal basis, these two studies (1757, 1131) sug— gested that initial development of the fetal gonads could be due to either mater- nal CG or fetal LH but that subsequent development and steroid production is attributable to fetal LH. 10.10. Follicle Stimulating Hormone Circulating levels. Irvine and Evans measured FSH to Day 24 (490) and Day 78 (806) and concluded that surges occurred at 10— to 11-day intervals dur- ing early pregnancy, as well as during the estrous cycle. It was stated that an FSH surge lasting 2 or 3 days occurred consistently in 12 of 12 Standardbred mares on Days 24 to 27, and another peak occurred in 11 of 12 mares on Day 38; FSH surges then continued at 10—day intervals in all mares. Earlier workers suggested that pituitary gonadotropins may peak at lO-day intervals, based on a study of estradiol synthesis and follicular development (1677). In another study (1092), FSH concentra- tions were examined in six pregnant and five hysterectomized ponies from Day 1 to Day 72. The difference between groups (early pregnancy versus hysterectomy) 424 Chapter 10 and the group by day interaction were not significant. Data therefore were pooled to examine day effects. Mean concentrations of FSH were initially high, comparable to the corresponding days of diestrus in agreement with earlier work (490); a decrease occurred between Days 10 and 13 (Figure 10.4). Mean concentrations remained low until Day 24, tended (P<0.l) to increase between Days 24 and 28, and then decreased. Thereafter, fluctuations occurred in mean concentrations, but these were not significant. The lowest mean values occurred on Days 60 to 72, and these means were significantly lower than on Days 7, 10, and 28. The results supported the New Zealand observation of a surge on Days 24 to 27 (806) but did not confirm the presence of a surge at Day 38. Inspection of means (Figure 10.4) and data for individual mares did not support the conclusion (490, 806) that FSH surges occur every 10 to 11 days during early pregnancy. The time of apparent surges was not synchronized among mares, and in some mares, obvious surges did not occur at any time, accounting for the large standard errors (Figure 10.4). Others also have failed to find a regu- larity in FSH surges during pregnancy (1655). Part of the difficulty in characteriz- ing FSH may be due to infrequent blood sampling and the lack of deliberate attempts to associate FSH concentrations with follicular waves in individual mares. In another study, two mares showed apparent 10-day rhythms in FSH concen- trations during Days 0 to 100, whereas in two others, the profiles did not seem to fol- low a rhythm (1654). One jenny appeared to have a 20-day FSH rhythm, whereas three others had erratic fluctuations. Relatively low concentrations of FSH were found in pony mares during the last half of pregnancy (2 or 3 ng/ml over Days 150 to 330; n=7), but large elevations occurred at irregular intervals in some mares (1757); FSH was not detectable during this time in the fetal circulation. FSH Summary. The FSH profile dur- ing early pregnancy sometimes consists of periodic surges which have not been adequately characterized. Initially, the profile is comparable to the profile during the corresponding days of diestrus. The mean concentrations decrease compara- ble to what occurs at the end of diestrus, but the minimal values seem higher than during the corresponding days of estrus. As a generality, the mean concentrations during approximately the first 60 days are relatively high, followed by a decrease to minimal baseline concentra- tions which are reached sometime before Day 150. Thereafter, concentrations remain low with occasional surges. There are surges before parturition and a prominent surge in association with par— turition (pg. 481 ). Better characterization of FSH pat- terns during pregnancy, as well as dur- ing other reproductive statuses, likely will require consideration of season of the year, follicular profiles in individual mares, frequent blood sampling (many times per day), and specialized data pro- cessing techniques. Clarification of the timing of follicular waves (ultrasonic monitoring of individual dominant folli- cles) and the association with FSH surges in individual mares should be a productive research area. There is cer- tainly adequate observational informa- tion available to encourage an extensive investigational effort in this interesting and important area. 10.1D. Progestins Circulating concentrations of proges- terone and other progestins during preg- nancy have been studied extensively by competitive protein-binding assay (38, 550, 762, 1496, 1674) and by radioimmunoassay (265, 550, 1527, 1417). The study of Holtan and associates (762) provided a characteri— zation of peripheral progestin levels throughout pregnancy and included con- sideration of progesterone versus other progestins. The results are used here as the basis for an introductory overview (Figure 10.5). In the following account, one day was subtracted arbitrarily from the published days (based on day of insemination) to approximate a reference with Day 0 as the day of ovulation. Progesterone. Progesterone increased between Day -2 and Day 5, similar to what occurs during the estrous cycle. Values tended to decrease thereafter to Day 30 and tended to increase between Days 30 and 42. High values were attained by Day 62; Days 54 through 118 were higher than for all other days. Progesterone then gradually declined and reached low values (1 to 2 ng/ml) at approximately Day 180. The gradual decrease in progesterone concentrations during approximately Days 12 to 30, fol- lowed by an increase between Days 30 and 40 has been well documented (906, 186, 1592, 32, 762, 1527). An increase in proges- terone at the expected time of initial CG Progestins (n=4 to 9) 8 50¢ -pregnanes 4 0 c 20 E \ 2’ E 16 2 fl '1 _ Progesterone E : i ' ---- ‘-/ E 12 H O C o 0 1701 -hydroxy- progesterone \ \ \ ‘ \ \ ‘1 0 60 120 180 240 Number of days from ovulation Endocrinology of Pregnancy 425 production, but before the appearance of secondary corpora lutea, was first report- ed in 1974 (1527); a significant increase occurred between Days 32 and 44. A recent study using daily sampling found that the initial increase began on Day 35 or Day 36 (186). There is general agree- ment that the progesterone increase reaches a plateau of approximately 15 ng/ml at Day 60 (762, 1527), due primar- ily to the addition of progesterone from supplementary corpora lutea to the out- put from the primary corpus luteum. In the initial study by Holtan and associ- ates (762), the low levels (2 ng/ml) of proges- terone reached at approximately Day 180 were reported to continue and then increase during the last 30 days of preg— nancy. Others obtained similar results but with an even higher level (8 ng/ml) after Day 180 (1527). The discrepancy was attributed to the extent of cross-reactivity of the progesterone antiserum with other progestins. Recently, Holtan and co-work— ers (761), using gas chromatography/mass FIGURE 10.5. Circulating con— centrations of progestins in sad- dle-type mares. Progesterone data are discontinued after Day 180 because more recent study has indicated that progesterone Parturition . - IS seldom detectable after this 4 time. In addition, the levels of I the various pregnanes are much -30 0 higher (e.g., 100-fold) than those indicated. Adapted from (7 62). 426 Chapter 10 spectrometry, found that progesterone was not detectable during the last half of pregnancy. Apparently, progesterone lev- els previously measured in many labora- tories, and attributed to the fetoplacental unit, were due to assay cross—reactivity. Using this new assay approach, the progesterone profile in early pregnancy was similar to previous reports and reflects the output of corpora lutea. How- ever, after approximately Day 180 proges- terone was not detectable except for low levels (0.5 to 1 ng/ml) in a few mares near term. Progesterone concentrations near term were higher in the umbilical vein (flow toward fetus) than in the umbilical artery; the levels were 5 to 20 ng/ml which was low compared to other pro- gestins. Apparently, progesterone detect— ed in the umbilical vein is rapidly metab- olized, and measurable quantities do not get into the maternal circulation. This represents a major conceptual modifica— tion. 5a—Pregnanes. The progestins that cross-react with progesterone antisera have been identified as 5a-pregnanes (550, 113, 114, 760, 761, 1417). These progestins first become detectable between Days 30 and 60 and increase gradually to Day 300 (Figure 10.5). A more rapid increase occurs during the 30 days prepartum. According to the recent study (761), the concentrations of these progestins is many-fold higher than the 3 to 8 ng/ml originally reported (Figure 10.5). The pre- dominant steroids (5a—pregnanes) in maternal plasma near term were 20a-hydroxy-5a-pregnane-3-one (400 to 2100 ng/ml) and 5a-pregnane—BB,20a—diol (100 to 340 ng/ml). Four other pregnanes were detected at levels of 30 to 100 ng/ml. The stimulus for the prepartum rise in concentrations of progestins is not known. The pregnanes are further discussed in Chapter 2 (pg. 69). Other progestins. Circulating concen- trations of 17a-hydroxyprogesterone dur- ing pregnancy have been characterized (762, 1417). Less than 0.2 ng/ ml was found in the plasma, except between Days 40 and 120 (2 to 4 ng/ml) and the last 30 days prepartum (0.5 ng/ml; Figure 10.5). The maximum values thus coincided with the high progesterone concentrations between Days 40 and 120. The source and role of this progestin are not known. Follicular fluid contains high levels (pg. 63), but there was no apparent increase near the time of ovulation or early diestrus. Concentrations of 20a- dihydroprogesterone in the plasma of pregnant mares have been characterized more recently (1417). The concentrations were low (approximately 2 ng/ml) during the first three months, higher during 5 to 10 months, and rose to very high levels (80 to 120 ng/ml) during the last month. The source of this progestin is uncertain. Both luteal and placental tissue contain the enzyme system needed for its produc— tion (cited in 1417). Species crosses. The concentrations of progesterone are extremely high (as high as 800 ng/ml) in jennies carrying a hinny fetus (jenny bred to stallion). Collection of blood samples from various organs indi- cated that the ovaries were the main source of the high progesterone levels (1445). It also was reported that at approx- imately Day 60 the progestin responsible for the high levels was progesterone, and the increase was due to increased produc- tion and not decreased clearance rate. The high levels for this cross between species is associated with high levels of CG (pg. 420). However, in mares carrying a mule (mare bred to male donkey), the CG levels were high, but the progestin levels were apparently unaffected (cited in 1445). Reviews are available and can be consult- ed for further details on the effects of var— ious species crosses and embryo transfers on the levels of progestins and CG (49, 58). 10.1E. Testosterone Blood testosterone has been measured in pregnant mares during the past decade (1472). Concentrations, increased tenfold from the first month to the seventh month and then decreased to basal levels by the time of parturition (Figure 10.6). The rise and fall of testosterone seemed similar to the circulatory pattern of unconjugated and conjugated estrogens. The testosterone profile appeared to be biphasic. The late rise and fall was tem— porally related to development and regression of the fetal gonads, but the early rise preceded the reported time of fetal gonad development (pg. 397). It was suggested that the maternal gonads dur— ing the time of CG production were responsible for the initial rise and that the fetoplacental unit was responsible for the subsequent rise. The early rise seemed temporally similar to the above- described progesterone increases during early pregnancy (Days 35 to 40), but sam- pling was too infrequent for adequate characterization. The roles of the high concentrations of an unbound biologically active androgen during pregnancy in the mare are not known (1472, 1323). Testosterone (18) Concentration (pg/m!) 50 ; Parturition 210 270 345 120 180 0 60 Number of days from ovulation FIGURE 10.6. Circulating testosterone concentra- tions during pregnancy. Numbers in parentheses are the numbers of mares for each day. Adapted from Silberzahn et al. (1472). Endocrinology of Pregnancy 427 10.1F. Estrogens Assay considerations. The following three aspects of the dynamics of estrogen production and circulation must be appreciated: 1) Circulatory estrogens may be free (unconjugated) in blood or plasma or bound to sulfates (conjugated); 2) Estrogens are found in large quanti- ties in the urine and blood, and either source may be used for assay purposes; and 3) The estrogens of pregnancy, as well as the progestins, consist of many forms. In regard to Point 2, the concentration of bound estrogens is 100-fold greater than the concentrations of free estrogens (1592). Reports may be based on a specific free estrogen (e.g., estradiol-17B, estrone) or total conjugated estrogens from the blood or urine. Little is known about the specific roles, if any, of specific estrogens, except that estradiol-17B is probably the most active, and no consideration will be given here to that aspect of estrogen dynamics. However, the time of production of differ- ent forms will be noted. At our present state of knowledge, it appears that mean- ingful biologic concepts can be developed using Widely different assay approaches. The concentrations of total plasma and urinary estrogens appear to follow similar patterns. Determining levels of plasma or urinary estrone sulfate has been advocat- ed as a means of assessing fetal viability or distress (846, 224, 1233) or as a means of pregnancy diagnosis after Day 60 (pg. 333). A recent report (1109) describes the moni- toring of ovarian function and pregnancy by evaluating the excretion of urinary estrogen conjugates in Przewalski’s horses. Urinary conjugated estrogens (sulfates) are being used with increasing frequency to study the basic aspects of estrogen production (1530, 366, 847, 365). Concentration of estrogens and other steroids in the feces are being used for convenient pregnancy diagnosis in feral mares. The simulation of levels of plasma estrone sulfate by intravenous infusion has been reported (1307). 428 Chapter 10 Late entry. A twofold increase in mean plasma estrone and estrogen conjugates and a fivefold increase in urinary estro- gen conjugates were found during early pregnancy (1860). In contrast, concentra- tions of plasma estradiol—1713 did not change. The measurement of estrogen conjugates provided a sensitive assay system that accurately reflected secre— tory dynamics of estradiol-17B in non- pregnant mares and estrone in pregnant mares. Early history. Reports of both circula- tory and urinary estrogenic substances first appeared in 1930. Large quantities of an estrogenic material in the urine of pregnant mares were discovered by Zondek in Germany (cited in 346). Savard and associates (1399) subsequently observed that equilin and equilenin (ring-B unsaturated estrogens) increased in the later months of pregnancy relative to total estrogen titers. Further studies in the 1930s began to elucidate the changes in estrogen concentrations in urine during pregnancy (pg. 67; 167, 326). Estrogens were first detected in the urine at approximately Day 100, increased until Days 200 to 275, and then gradually decreased throughout the terminal portion of pregnancy. Estrogens were not detected after parturition. Although total estrogen titers decreased in the latter months of pregnancy, the relative quantities of equilin and equi- lenin increased. These conclusions have been confirmed in principle (1303, 1399). In the same year that estrogenic sub- stances were found in the urine of preg- nant mares, Cole and Hart (321) reported on the effects of injections of blood serum from pregnant mares into immature rats. Serum collected between Days 43 and 80 produced marked increases in ovary size, attributed to high levels of a gonado- tropin (pg. 321). It also was noted that injections of serum collected later in pregnancy (Days 80 to 180) began Estrogens 16 Plasma estradiol 12 Nonpregnant l \,. (n=5) 4 0 Pregnant (n=3 to 5) 6000 Yolk sac fluid estrogens E ,l U? Estradiol 17B ,’ 3- 4000 ’ I: .2 E 8 2000 2 Estrone O o 0 1000 Uterine fluid estrogens Ill In pregnant mares .’ 800 600 400 200 8 12 14 16 18 20 Number of days from ovulation FIGURE 10.7. Concentrations of estrogens in early pregnancy. Circulating estradiol levels increased between Days 12 and 14 in nonpregnant mares but not in pregnant mares. Estrogen levels were high in the yolk sac fluid and the uterine flushings of preg- nant mares. At least part of the increase in estro— gens in uterine fluid on Days 18 and 20 was attributable to ruptured yolk sacs. Adapted from Zavy et al. (1846). to affect the uterus and vagina more than the ovaries. The appearance of an estrogenic substance (originally termed folliculin) later in pregnancy was there- fore suspected. It was subsequently reported (322) that serum collected from mares between Day 222 of pregnancy and parturition had the following effects when injected into rats: estrous-like vaginal smear, increased uterine weight, and ovarian inhibition. These effects of the mare serum were eventually attribut- ed to estrogens (318). Production by early conceptus. The production of estrogens by the early con- ceptus was discussed previously (pg. 66). These locally produced and used estro— gens do not increase circulating concen- trations measurably since no circulatory increase above diestrous levels was found before Day 35 of pregnancy (1592). Estrogens and androgens have been detected in yolk-sac fluid assayed on Days 10 to 22 and in media used for cul- ture of cells (519). In specific studies (1846, 1843, 1428), increases in estrogens in the uterine lumen and yolk—sac fluids occurred after Day 12 of pregnancy; how- ever, the increases that occurred in the Endocrinology of Pregnancy 429 maternal blood over Days 12 to 20 of the estrous cycle did not occur in pregnant mares (Figure 10.7). These workers also demonstrated production of estrogens by the conceptus in tissue-culture; the increased production over Days 12 to 20 was primarily a function of increasing weight of the conceptus. Early pregnancy. Terqui and Palmer (1592) measured total plasma estrogens (conjugated and unconjugated) from Days 0 to 100. On Days 0 to 35, concen— trations were similar to those during diestrus. An increase in total plasma estrogens occurred on Days 35 to 40, fol- lowed by a plateau (3 ng/ml) on Days 40 to 60 that was higher than the preovula- tory surge (Figure 10.8). The increase at Days 35 to 40 has been confirmed by workers using urinary estrogen sulfates (366). However, no significant increases in circulating unconjugated estrogen levels prior to Day 78 or 88 were detect— ed by radioimmunoassay (Figure 10.9; 1141). This is not surprising considering the low levels of free estrogens during the first trimester. Steroids in early pregnancy (“=9) 15 / Progestins (ng/ml) -10 0 10 Progestins Total estrogens 20 00 N (Ilu /5u) suefionsa —L O 30 40 50 Number of days from ovulation FIGURE 10.8. Circulating progestins and estrogen concentrations during early pregnancy. Adapted from Terqui and Palmer (1592). 430 Chapter 10 Estrogen fractions (n=4) 1000 A 800 /‘~.‘ E Estrone,equilin, \ 600 equilenin {/ ‘ E (E1) P”' u,“ 400 S a: 200 E E 8 4o '5 O 20 EstradioI-17B and17a 0 60 120 180 240 Number of days from ovulation Late pregnancy. Radioimmunoassay technique was developed for free estro- gens by Nett and co-workers (1141) using column chromatography to separate the estrogens into fractions. The concentra- tion of the estrone-equilin-equilenin frac- tion remained low (<20 pg/ml) until Day 78. Values increased to a peak at Day 208 and then declined (Figure 10.9). The concentrations of the estradiol frac— tion followed a similar pattern, although absolute levels were much less. The first significant increase occurred at Day 88 and Day 148 for the two fractions, respec- tively. The pattern of estradiol-17B levels during pregnancy also has been described in an abstract (668), and the description seems similar to that for the free estradi- ol fraction shown in Figure 10.9. The lev— els declined sharply after Day 280, and it was noted that an increase did not occur before parturition as it does in other domestic species. In this regard, it was noted in a short communication (1616) that estradiol-17B levels increased over 14 to 4 days before parturition in donkeys. There is a strong temporal relationship between size of the fetal gonads and circulating concentrations of estrogens but not progestins. 140 120 0 g 100 g 3 80 E 5. s 60 m N 40 8 i 20 V FIGURE 10.9. Circulating con— centrations of estrogens in sad- 0 dle-type mares. The estrogens represent levels of free (unbound) hormones and are '30 0 separated into two fractions. Parturition Adapted from (1141). Estrone and equilin were extracted from the plasma of pregnant Welsh ponies and quantitated by gas liquid chromatography (346). Both estrone and equilin were found to be conjugated as sulfates. Neither estrogen was found before four months, presumably because of a less sensitive assay than the BIA sys- tem used by Nett and co-workers. There was a dramatic increase in estrone during the fourth month, high levels were main— tained until the eighth or ninth month, and then the levels declined (Figure 10.10). During the time of high estrone levels, equilin increased and did not decrease until the last month which was subsequent to the estrone decline. As a result, equilin levels approximated or exceeded the estrone levels during the last few months of pregnancy. After the seventh month, concentrations were lower in multiparous than in primiparous mares. Asynchronous patterns of estrone and equilin levels seem similar to what was found in urine and suggested indepen- dent biosynthetic pathways for the two estrogens. In this regard, isotope studies indicated that equilin and equilenin are derived through a steroidogenic pathway, Plasma estrogens (n=6 to 9) 150 mi + Estrone .1 o o 01 0 Concentration (ng/ml) 120 150 180 210 240 270 305 to 10 tO to to tO to 149 179 209 239 269 304 term Days of pregnancy FIGURE 10.10. Mean plasma estrone and equilin concentrations. Adapted from ( 346). different from that for estrone, estradiol, and progesterone (pg. 67). The equilin con— centrations have been substantiated by RIA (1229). Examples of the patterns of total urinary estrogenic excretion and the variability among individuals are shown in Figure 10.11. Seasonal effects. Haluska and Currie (669) found that estradiol-17B plateaued at high levels over approximately Days 200 to 280 but that the position of the plateau was more related to time of year than to days of pregnancy. Increase in nine mares occurred during a seven-day period between April 13 and April 20 but encom- passed Days 57 to 189 over the nine mares. Decreasing levels occurred during a five—day period between August 24 and August 29. This intriguing observation requires confirmation and additional study and needs consideration of other gestational steroids, the fetal gonads, fetal gender, time of year, and breed. Breed Effects. A recent examination of fecal estrogen during the last three months of gestation found lower amounts in Lipizzan mares than in Thoroughbreds and trotters (1410). Thus, breed is a factor in concentrations of fecal steroids. Endocrinology of Pregnancy 431 Estrogen excretion in individuals 800 0') O O 400 200 Urinary estrogen (pg/24 hr) -240 -180 -120 -60 0 Number of days from parturition 90 150 210 270 330 Approximate days of pregnancy FIGURE 10.11. Patterns of total urinary estrogen excretion for individual pony mares. Adapted from (1303). 10. 1 G Relaxin A major advance in mare reproductive biology in the 1980s was the characteri- zation of circulating relaxin activity dur— ing pregnancy by Stewart and Stabenfeldt (1552). Concentrations reached maximum at about Day 150, seemed to decline approximately 50% by Day 225 and then returned to the high levels until parturi- tion (Figure 10.12). The bimodal profile suggests the development of an addition- al source or stimulus after Day 200. A large prepartum surge was not observed in contrast to findings in some other species (cited in 1552); the authors noted that this may have been due to infre- quent sampling. In a recent comparison of breeds (1548), Standardbreds, unlike Thoroughbreds, did not exhibit the midpregnancy nadir, but instead, relaxin levels steadily declined from Day 150 to parturition. There was no observed dif- ference due to gender of fetus. 432 Chapter 10 Relaxin (n=1) N -b 0') O O O eCG concentration (ng/ ml) 0 120 160 200 240 Day of pregnancy The corpora lutea were not believed to be an important source of relaXin since immunoactivity was not detected until after Day 75. Relaxin levels reached detectability at approximately the time when the placenta begins major steroid synthesis (Day 80). Relaxin is likely syn- thesized within the utero-placental-fetal unit, but a more precise site has not been established (1554). The biologic actions of relaXin in mares are not known, but on the basis of studies in rodents, relaXin may synergize with progesterone in main— tenance of pregnancy. The well-known role of relaXin in loosening the pelvic liga- ments in association with parturition has not been established in mares. 10.1H. Prolactin Only limited study has been done on circulating prolactin levels in pregnant mares. During development of a homolo- gous radioimmunoassay for ePRL, con- centrations of 1.2 to 12 ng/ml were detect- ed during the last few weeks of pregnancy (1346). Recently, a mean level of 0.9 ng/ml (range: 4.6 to 0.1 ng/ml) was found during days 14 to 80 (1408). Concentrations dur— ing the periparturient period are further discussed in Chapter 11 (pg. 471). (|w /6u) uonenueouoo ugxe|au Figure 10.12. Plasnia relaXin activity and eCG 0 levels during pregnancy in a mare. Adapted from Stewart and Stabenfeldt (1552). 280 320 10.2. Survival Mechanisms Initiated by the Conceptus Overview. A fascinating aspect of the endocrinology of pregnancy concerns the mechanisms employed by the developing embryo and fetus to assure its own sur— vival. Reviews are available on the con- trol of early pregnancy by steroid hor— mones originating in the preimplantation embryo in nonequine species (407). Consideration is given to whether the early conceptus contains steroids, whether the steroids are produced by the conceptus or sequestered from the uterine lumen, and whether such steroids control certain phenomena. It is postulated in pigs that two waves of steroidogenic activity occur that would temporally cor— respond to hypothetical roles in oviductal ova transport and the prevention of lute- olysis. Suggested mechanisms (407) that could involve the steroids of the blasto— cyst include the following: 1) morula to blastocyst transformation, 2) shedding of zona pellucida, 3) implantation or attach— ment of blastocyst, and 4) metabolically oriented activities. The possibility that the early conceptus produces LH—like substances to stimulate steroidogenesis is also discussed. Such a hypothetical mechanism seems worth exploring in mares because of the well-established, CG-producing capabilities of trophoblas- tic cells after endometrial cup formation. Mechanisms used by the equine con- ceptus to ensure its survival include the following: 1. Substances are produced for selective passage of the fertilized ovum through the oviduct (pg. 305),- 2. Substances are produced for stimula— tion of uterine contractions and embryo mobility (pg. 307),- 3. A factor is produced to prevent regression of the primary corpus luteum (pg. 441); 4. A factor, probably estrogen, is pro- duced to stimulate uterine turgidity, thereby terminating embryo mobility (pg. 315); 5. Trophoblastic cells invade the endometrium for the production of CG (pg. 370); and 6. Steroid hormones are produced by the trophoblast (pg. 66) and later by the fetoplacental unit to assure that the tubu— lar genitalia function and develop in accordance with the needs of the concep- tus (e.g., uterine vascularization and growth, and production of uterine milk). Conceptus removal before CG produc- mm. Removal of the conceptus and induc- tion of abortion have been used as research techniques in mares. Results of early experiments on the effects of removal of the conceptus on reproductive function were reviewed and tabulated in the first edition (575). Removal of the con- ceptus resulted in a prolonged interovula— tory interval when done on Days 15 or 20 but not when done on Day 10 (726). Re— moval of the conceptus after the critical period for luteal maintenance and after the initial development of uterine turgid- ity (Day 16) resulted in maintenance of the corpus luteum and the establishment of pseudopregnancy (pg. 228 and pg. 528. Removal of the conceptus by hysterotomy at Day 24 resulted in maintenance of the corpus luteum and turgid uterine tone for Endocrinology of Pregnancy 433 2 or 3 months postovulation (906). In another study, loss of pregnancy was induced by crushing the fetus, aspirating placental fluid, administering hCG, or infusing intrauterine saline (33); when pregnancy failure was induced before Day 36, the corpus luteum persisted and mares did not recycle (ovulate) until Days 38 to 60. However, if removal of the conceptus or induction of abortion before Day 38 is accompanied by the induction of luteolysis, an immediate return to estrus and ovulation can be expected. In one such study, pseudopregnancy did not occur when embryonic death was induced on Day 24 by an injection of colchicine, a potent anti-mitotic agent, into the concep- tus (906). The colchicine treatment appar- ently not only caused immediate embryo death but resulted in luteolysis. Subse- quent preliminary trials indicated that luteolysis occurred when the drug was given by intrauterine injection during diestrus. A single injection of a luteolytic dose of PGan or one of its analogues before the appearance of CG is followed by an immediate decline in progesterone (906, 1509), a rapid loss of pregnancy, and return to ovulatory estrus (589). Conceptus removal during CG produc- ti_on. When the conceptus is removed after the trophoblastic girdle cells invade the endometrium (by approximately Day 38), the situation is more complex. Indications are that CG production continues appar- ently unaltered (44, 1657, 1317). In a recent study (600), termination of pregnancy by six daily injections of PGFZoc beginning on Days 38 to 44 did not alter the circulating levels of eCG (Figure 10.3); PGonc treat— ments began after circulating eCG was detected in the maternal circulation. Similarly, fetal death in eight yearling mares between Days 50 and 115 resulted in continued positive CG pregnancy tests, comparable to What would be expected in a normal pregnancy (1103). These findings have important implications for CG-based pregnancy tests. It has been concluded (44) that removal of the conceptus during 434 Chapter 10 the time of eCG production results in maintained luteal function in some mares but not in others. Luteal regres- sion occurred in some mares despite the persisting high CG levels (44, 1657). No information was available on the pres- ence of supplementary corpora lutea or on whether the removal process resulted in a luteolytic surge of PGFZoc. These observations demand confirmation and much additional study because, as sug- gested by the authors, they indicate the existence of a luteotropin of conceptus origin other than CG. It has been stated (44, 64) that when luteolysis is induced by treatment with a PGan analogue, the mares will return to estrus and ovulate only after CG dis- appears from the blood. Four mares with the conceptus previously removed or lost between Days 38 and 45 were treated with a PGan analogue. All had high serum CG when treated. Repeated treatment resulted in complete luteoly— sis as indicated by peripheral proges- terone levels, yet the mares did not return to estrus. It was noted that the situation seemed paradoxical in that high levels of CG failed to stimulate the ovaries in the absence of functional luteal tissue. From the above discussion, it must be concluded that the literature is beclouded on the effect of endogenous CG on the ovaries of mares in which luteolysis has been induced. Carefully planned experi— ments involving removal of conceptus, corpus luteum, or ovaries could provide needed practical and basic information on the controlling interrelationships between the endometrial cups and ovaries. 10.3. Roles of CG Temporal indicators. It is clear that much follicular activity occurs long before CG is detectable. Some or all of the follic- ular activity occurring after Day 40, however, may involve CG. Curiously, CG has considerable FSH-like activity in nonequine species but not in mares. Apparently, equids have evolved a method for protecting their ovaries against the high levels of FSH-like activity in CG, rather than evolving a CG with low FSH activity. The folliculotropic role of eCG in mares has not been clarified. However, temporal relationships strongly support a luteo- tropic function in stimulating the resur- gence of the primary corpus luteum and the formation of supplementary corpora lutea. The maintenance and continued function of luteal structures until Day 160 usually exceed the availability of CG; research is needed on the mecha- nisms that maintain the life of the corpo- ra lutea after the disappearance of CG. Studies involving extraspecific pregnan- cies (embryos transferred from horses to donkeys and donkeys to horses) have led to the conclusion that CG does not have a negative feedback effect on the pitu- itary gonadotropins (1655). An exquisite enigma. Evolution of the endometrial cup-CG system appears to be an exquisite system for providing an ovarian source of estrogens and proges- terone until the fetoplacental unit is able to provide amounts adequate for the needs of pregnancy. Thus, with the release of CG, the primary corpus luteum resurges to produce estrogens (pg. 446), increased progesterone (pg. 443), and possi— bly testosterone (pg. 427). Furthermore, CG leads to the formation of supplementary corpora lutea from ovulatory and anovu— latory follicles to produce additional pro- gesterone and possibly estrogens and androgens. The output of these ovarian steroids gradually decreases as produc— tion by the fetoplacental unit increases. The enigma is that detectable levels of CG are not absolutely essential for a suc- cessful pregnancy (58). The resurgence in progesterone productivity of the primary corpus luteum on Day 35 and the subse- quent formation of supplementary corpo- ra lutea (58) do not seem essential. The solution to this enigma may center around the findings that the ovaries may be removed on Days 50 to 70 without detriment to pregnancy in some mares, whereas others abort (pg. 437); this is also the period of transition between ovarian and fetoplacental steroid production (pg 446). Furthermore, circulating levels of progesterone in early pregnancy are high- ly variable among mares. The CG steroid- stimulating system, therefore, may be essential only in those mares with low circulating steroid levels. It seems unlike- ly that the complex CG system is vesti- gial. It is possible, however, that the ovar- ian responses to CG are secondary to a more profound role for CG that has not yet been elucidated. One such possi- ble role is in immuno-protection, a mech- anism that is currently under study (pg. 416; review: 58). Injections of exogenous CG. It will be most helpful to learn whether luteotropic and folliculotropic activity of CG can be demonstrated in vivo. It appears, howev- er, that it will be difficult to study the effect of CG on various ovarian compo- nents in mares by systemic administra- tion of the hormone. Tremendous quanti— ties are required to mimic the natural state since the blood may contain several million units. The ovaries of other farm species are more than a thousand times more sensitive to CG than are the ovaries of mares. No ovarian effects were detect- ed in mares given 4,000 iu of eCG (414). A single injection of 1,500 iu of eCG, for example, stimulates the ovaries of cattle, whereas a single injection of more than one million units of eCG was without apparent effect in limited trials in mares (575). Injections of 20,000 to 30,000 iu to mares carrying donkey conceptuses caused serum elevations of 1 to 12 iu/ml for 5 to 8 days after each injection, and an apparent formation of supplementary cor- pora lutea occurred in 1 of 7 mares (58). These initial trials offer hope that the role of CG can be studied directly by administration of large systemic doses. Endocrinology of Pregnancy 435 Perhaps the sensitivity problem could be overcome for research purposes by the infusion of CG into the ovarian artery or injection into the ovarian stroma. Another approach could involve artificial- ly injecting or grafting portions of the chorionic girdle to the endometrium to induce endometrial cup formation in, for example, diestrous mares, pseudopreg- nant mares, and pregnant mares at a time when they would not ordinarily be exposed to the hormone. In a preliminary trial (575), segments of a 36-day chorionic girdle were sutured to the endometrium of several mid-diestrus mares. Grossly obvious cups formed in one of the mares, and the blood plasma contained very high levels of CG. Refinement of this experi— mental approach could lead to needed information on the effect of CG on the ovaries and the factors controlling the life span of the cups. In a limited study (62), grafts of girdle cells into the endometri— um or testis developed into cuplike cells, but it was not reported whether CG was produced. Secretion into uterine lumen. In the early stages (Days 40 to 42) of cup devel— opment, the cups act mainly as endocrine glands since exocrine secretory material is not present. Later, they also act as exocrine glands since considerable amounts of CG—rich secretion pour out of the uterine glands into the space between the allantochorion and the depression in the surface of the cups. This area of secre- tion is directly continuous with the exten- sive arcade area which forms an absorp- tive network around the microplacen- tomes (pg. 391). More than a million units of CG may be found in the cup secretion, compared to a maximum total blood con- tent of 3 million units (311). It is question- able whether the CG secreted into the uterine lumen re-enters the maternal sys- tem (1372). The possibility that some CG is absorbed into the fetoplacental circula- tion was noted previously (pg. 423). Perhaps the CG that is discharged into the uterine lumen provides a stimulus for 436 Chapter 10 hypertrophy of the fetal gonads and a stimulus for initiation of steroid produc- tion by the fetoplacental unit. The tempo- ral relationships for such a possibility seem reasonable (Figure 10.13). The dis- charge of placental steroids into the uter- ine veins apparently begins approxi- mately when CG is discharged into the uterine lumen. The arcade area could provide a system for channeling the CG over a large surface area of the allanto— chorion. Although there is no direct indi- cation for such a role of CG, it seems rea- sonable to assume that CG is discharged into the arcade area for some purpose, and the development of an appropriate hypothesis for testing is indicated. In this regard, it has been proposed that hCG may exert a regulatory role in steroidogenic processes in the fetoplacen— tal unit of women (408), but definitive support for the hypothesis is lacking. The mare could be a useful model for investi— gating the relationships between gonado- tropins of fetal origin (from endometrial cups and fetal pituitary) and the produc- tion of steroids by the fetoplacental unit. Specific studies are needed on the fate and function of the CG that is secreted into the uterine lumen. Relationships among eCG, fetal gonads & estrogens Fetoplacental estrogens gonad weight (g) I," Fetal gonads eCG concentration (iu/ml) and 0 40 Number of days from ovulation \ \ 7".---. \ \ 80 120 160 200 240 280 320 360 10.4. Sources and Roles of Luteal Progesterone The importance of progesterone to the maintenance of pregnancy has been established in many species. Species dif- fer, however, in the mechanisms they have evolved to continue generating pro— gesterone. The corpus luteum in swine and cattle serves as a progesterone source throughout pregnancy. Corpora lutea in mares no longer serve as a progesterone source after approximately Day 180. Extraovarian sources begin contributing to the progestin pool prior to the regres- sion of corpora lutea and continue to pro- duce progestins throughout pregnancy. Primary versus supplementary corpora lutea. It has been demonstrated that the primary corpus luteum as well as the supplementary corpora lutea (secondary and accessory), if present, secrete proges— terone into the ovarian vein throughout the period when the ovaries are essential to pregnancy (1506). The concentrations of progesterone in the tissue of the primary corpus luteum from single mares on each of Days 41, 55, 68, and 90 of pregnancy were similar to concentrations during diestrus (1677). In vitro studies also have demonstrated the capability for proges— terone production by primary and supple- 800 600 400 200 FIGURE 10.13. Temporal associ— ations among circulating estro- gens (based on Figure 10.9), eCG (adapted from 762), and weights of fetal gonads (based on Figure 9.43). (ml /5d) suonenueouoo uafionsa mentary corpora lutea as late as Day 100 (1521) and Day 198 (1021). In vitro produc- tion decreased markedly after Day 160. Considerable progesterone production by supplementary corpora lutea has been shown by high levels in the ovarian venous effluent from ovaries containing only supplementary corpora lutea (1506). In another study (1527), progestin concen- trations in six pregnant mares between Days 44 and 180 were significantly differ- ent among mares. The high mean pro- gestin concentrations between Days 90 and 150 were due primarily to two mares; 4 and 8 ovulations were detected in these two mares, whereas the mean number of detected ovulations for the remaining four mares was 0.8. Thus, the number of secondary corpora lutea and the level attained by circulating progesterone were positively associated. Roles of progesterone. Progesterone from the primary corpus luteum is needed for physical embryo-uterine interactions (embryo mobility, fixation, and orienta- tion; pg. 305) and for uterine secretions (pg. 317). Recent studies suggest that it is possible to influence the concentrations of nutrients in the Day 18 yolk sac and to alter diameter of the conceptus by admin- istering steroids (1753, 1751); it is unknown whether this approach would affect embryo survival. Eliminating the corpus luteum by ovariectomy or an injection of PGan on Day 12 resulted in a decrease in the rate of mobility and failure of fixa- tion (850). The embryonic vesicle contin- ued to traverse the uterus after fixation failure but at a reduced rate. The mobili- ty continued until the vesicle was lost, presumably through a patent estrous cervix. In mares treated with PGonc on Day 21, the vesicle was lost within 1 to 3 days without prior ultrasonic indications. Loss probably occurred through the cervix since the cervix was patent in all mares; in one mare a collapsed vesicle was recov- ered from the cervix (589). A continued progesterone source is needed for contin- ued turgidity of the nongravid portions Endocrinology of Pregnancy 437 of the uterus and, therefore, continued fixation of the conceptus. In mares with induced luteolysis or ovariectomy on Day 30, the embryonic vesicle became dis- lodged in 1 or 2 days. It became mobile within the uterus and was frequently found in the uterine body. Dislodgement seems to be a sensitive indicator of inade- quate levels of progesterone (pg. 527). In another study (32), exogenous proges- terone maintained pregnancy in some mares but not in others after the induc- tion of luteolysis with a prostaglandin analogue on Day 27. Embryonic loss in association with progesterone deprivation is discussed in Chapter 12 (pg. 525). Ovariectomy on Days 25 to 45 consis- tently resulted in abortion, whereas ovariectomy on Days 140 and 210 consis- tently resulted in maintained pregnancy (Table 10.1; 764). The interval between Days 50 and 70 was the crucial period since 9 mares aborted and 11 did not when ovariectomized during this time. It would be informative to learn whether mares that abort and those that don’t dif— fer in levels of hormones (e.g., CG, 5a—pregnanes). Pregnancy was maintained by exogenous progesterone in three ovariectomized embryo-transfer recipients (1420); discontinuation of progesterone at 100 days did not result in abortion. TABLE 10.1. Effect of Ovariectomy on Pregnancy Maintenance in Ponies Day of No. mares ovariectomy ‘ that aborted 25 4/4 35 5/5 14/14 45 5/5 50 0/1 55 3/5 9/20 70 6/14 140 0/6 210 0/6 0/ 12 Adapted from (763). 438 Chapter 10 Abortion occurred approximately 2 to 6 days after ovariectomy on Days 25, 35, and 45, and 10 to 15 days after ovariecto— my on Days 55 to 70 (764). Furthermore, there was no significant effect on gesta- tion length in mares that did not abort (764, 1420). Progestins were assayed in the peripheral plasma in five of the ovariec- tomized mares (764). A rapid decline occurred in all mares, followed by an apparent slight rise at the approximate time of reduction in size of the fetal bulge; these preliminary observations on pro— gestin levels suggest an interesting area for further research. It can be concluded that an ovarian source of progesterone is necessary for maintenance of pregnancy in some mares as late as Day 70 but is not essential after Day 140. Additional study is needed, especially to encompass Days 70 to 140. 10.5. Regulation of the Corpora Lutea This section involves the first and sec— ond luteal response to pregnancy, the intervening period, and the third response. The first response involves maintenance of the corpus luteum through blockage of the uterine luteolytic mechanism; the second involves resurgence of the primary corpus luteum; and the third involves the devel— opment of supplementary corpora lutea. The latter two responses are attributable to CG. 10.5A. First Luteal Response to Pregnancy Days of divergence. The factors regu- lating the corpus luteum for the first 11 to 14 days of pregnancy are presumably similar to those regulating the develop— ment and maintenance of the corpus luteum during the corresponding days of the estrous cycle (pg. 265). As noted earlier, progesterone concentrations are similar for pregnant and nonpregnant mares until approximately Days 11 to 14 when a rapid decline occurs in nonpregnant mares (Figure 10.14; 38, 1527). The mecha- nisms responsible for the divergence in the progesterone curves between preg- nant and nonpregnant animals have been under intensive investigation in many species. Prevention of regression of the corpus luteum in the presence of an embryo is called maternal recognition of pregnancy. However, the more specific and appropriate terminology for mares, first luteal response to pregnancy, will be used here. The critical period—when the concep- tus must be present to prevent luteo- lysis—must occur before the divergence in luteal progesterone production between nonpregnant and pregnant mares. An initial study on the critical period involved removal of the embryonic vesicle (726). It was concluded that the critical period was between Days 14 and 16 since luteal life span was prolonged only when the vesicle was removed on Day 15 or later. However, it appears that a critical period of Day 14 to 16 is ques- tionable since luteolysis occurs before this Progesterone 10 8 E E 6 . S l E 4 9‘ Pregnant/1 E \| ([1:16) CD | O o S “ 0 2 fl‘ Nonpregnant ‘. ‘yo. .‘C..‘ . v \ \ 0 4 8 12 Number of days from ovulation 16 20 24 28 30 FIGURE 10.14. Comparison of mean progesterone concentrations in pregnant and nonpregnant mares. Adapted from (38). time in many nonpregnant mares (pg. 238). More recently it was reported that the conceptus had a PGan-inhibitory effect as early as Day 11 (202, 633); oxytocin administration to nonpregnant mares on Days 11 to 17 caused a 34—fold increase in PGFZoc production but only a fourfold increase when the mares were pregnant. Days 11 to 15 also comprise the period of maximum mobility of the embryonic vesi- cle (pg. 306). Results of the experiments involving prevention by the conceptus of the luteolytic effect of exogenous oxytocin and the time of occurrence of conceptus mobility are compatible with the hypothe- sis that continual direct exposure of the endometrium to the conceptus is needed over Days 11 to 15. Role of embryo mobility in the first luteal response. It has been postulated that the extensive mobility of the equine conceptus is comparable to expansion of the bovine trophoblast in the uterine horn ipsilateral to the corpus luteum (587, 1055). The two species have evolved different approaches for conceptus—endometrial contact in the prevention of luteolysis (Figure 10.15). In mares, the embryonic vesicle must prevent production of PGan throughout the endometrium because of Species differences in conceptus-endometrial contact Cow Endocrinology of Pregnancy 439 the systemic uteroovarian pathway for delivery of the luteolysin (pg. 268). In cat- tle, on the other hand, only one side of the uterus needs to be blocked at the critical time. Direct indications of the Vital role played by the mobility of the equine con- ceptus in preventing luteolysis have been provided by uterine ligation experiments (1054). Restricting the conceptus to a small proportion of the uterus resulted in lute- olysis, apparently by preventing direct contact between the conceptus and the remaining portion of the endometrium. The ligations, however, presumably pre- vented potential intraluminal movement of uterine fluids, as well as the embryo, and therefore the test for an association between embryo mobility and luteal maintenance was not definitive. Nature of first luteal response to preg- nancy. The postulate that PGFZoc acts as a uterine luteolysin in nonpregnant mares has been supported by studies described elsewhere (pg. 271). The role of prostaglandins and their inhibition in luteal response to pregnancy in mares has been reviewed frequently in the past decade (e.g., 1440, 1428, 1438). The preven- tion of luteolysis by the equine conceptus theoretically can be attributed to any of FIGURE 10.15. Diagrammatic species comparison between mares and cows, showing the postulated manner in which systemic uterine-induced lute— olysis is blocked by a mobile embryon- ic vesicle in mares and unilateral uterine-induced luteolysis is blocked by an expanding vesicle in cows. Adapted from (587). 440 Chapter 10 the following (1440): 1) preventing trans- port of PGan to the corpus luteum, 2) preventing binding of PGFZOL to the corpo- ra lutea, 3) overriding the luteolytic effect of PGFZoc with a luteotropin, and 4) pre- venting synthesis or release of PGFZa. Preventing PGonc from reaching the cor- pus luteum has been proposed for swine (165); uterine venous PGF2a was reduced Prostaglandin an 10 In endometrium 8 6 Nonpregnant f‘xi A 4 g *1 (I) .g 2 t _____________ O) E o k 3: Pregnant 5 C o a: 100 Endometrial in vitro , : production g 80 i O I g 60 " o " )1 Concentration (ng/ml) 4 8 12 16 20 Number of days from ovulation FIGURE 10.16. Endometrial content, in vitro pro- duction, and concentrations of PGF in uterine flush— ings from pregnant and nonpregnant mares. Adapted from Zavy et al. (1845) and Sharp (1428). and concentrations in the uterine lumen were increased at the critical time. In pregnant mares, however, luminal PGonc levels were low (Figure 10.16), failing to provide support for the redirection of PGFZOL output (1843). The second possibili— ty is also unlikely. Results of studies on the PGan binding ability of equine corpo- ra lutea during early pregnancy are com- patible with the hypothesis that luteal maintenance results from the failure to release luteolysin (PGF2a), rather than from decreased binding ability of the corpus luteum for PGFZa (1699). Indications for the third possibility are weak, although in a preliminary trial, pituitary extracts seemed to prolong luteal life (pg. 265). Recently, an antibody response against eCG was reported to occur in the blastocyst fluid and peripher— al maternal blood as early as Days 8 to 15 (1702). This interesting observation requires confirmation. Indications that minimization of endometrial production of PGFZoc by the conceptus (point 4, above) is involved in the first luteal response to pregnancy in mares are as follows: 1. On Day 14, concentrations of prostaglandins of the F-series in the uter- ine vein were lower in pregnant mares (Table 10.2; 433); TABLE 10.2. Concentrations of Prostaglandins of the F-series in Uterine Veins of Pregnant and Nonpregnant Ponies Prostaglandin F in uterine veins (ng/ml) Day after Nonpregnant Pregnant ovulation mares mares 10 8.2be $0.8 4.38 10.9 14 14.9d :05 9.3C :20 18 6.4b i1.3 7.3bc 11.0 Means among the six groups that do not have at least one common superscript letter are signifi- cantly different (3 or 4 mares/group). Adapted from (433). 2. Peripheral concentrations of a prostaglandin metabolite (PGFM) were minimal or undetectable in pregnant mares (881, 1136); 3. PGFZoc in the uterine lumen, endometrial content, and endometrial production in vitro were reduced in preg- nant mares (Figure 10.16; 1845, 1428); 4. Cultured Day 14 endometrial tissue from pregnant mares produced PGonc, but production was reduced when endometrial tissue was co-incubated with conceptus tissue (189, 1739); 5. In vitro measurement of PGFZa on the luminal and myometrial sides of the endometrium indicated that PGonc secre- tion from the endometrium of pregnant mares was reduced at Day 14 (531); and 6. Late entry. Results of an in vitro study (1874) indicated that an endometrial cytosol from pregnant mares suppressed microsomal synthesis of prostaglandin F, but endometrial cytosol from nonpreg— nant mares increased the synthesis of prostaglandin F; bovine cotyledonary microsomes were used as the prosta- glandin-generating system. These studies indicate that the first luteal response to pregnancy in mares involves inhibition of PGan secretion and thereby protection of the corpus luteum from luteolysis. The term luteostasis has been recommended (1436) to describe the process of luteal maintenance in this species. Neither luteal maintenance nor luteostasis are entirely appropriate terms, however, since they imply stability or a plateau in function. The corpus luteum, in reality, gradually diminishes in size and function until resurgence at Day 35 (second luteal response to preg- nancy). The mechanism initiated by the embryo is more literally an antiluteolytic mechanism. The search for the antiluteolysin. A current major research area involves identification of the substance produced by the embryonic vesicle that inhibits production of PGFZa by the endometrium. In ruminants, a low molecular weight Endocrinology of Pregnancy 441 protein has been implicated and is called trophoblastic protein-one (TP-l; 1660). Luteal life is extended when TP-l is infused into the uterus, and TP-l is now regarded as the longtime elusive antilute- olysin in ewes (1331). The amino acid sequence of TP-l is close to that of an interferon (substances that are produced in response to a Viral stimulus). In swine, an interferon-like protein also seems involved in the luteal response to preg- nancy. There are currently no indications that interferon—like or TP-l-like proteins are involved in the luteal response to pregnancy in mares (1437). In this regard, a recent study (134) failed to find indica- tions that the equine conceptus on Day 13 or 15 expresses interferon genes. Initial studies have been done on the molecular weight of the antiluteolysin in mares. Endometrial tissue was co-incu- bated with conceptus membranes that were placed into dialysis tubing with various molecular-exclusion limits (1752, 1436). This approach suggested that the antiluteolysin molecule produced by the equine conceptus is smaller than a steroid or a prostaglandin. The Day 14 equine conceptus produced low levels of PGE2 and PGIZ during in vitro culture (1739). In other domestic species, these prostaglandins apparently antagonize the luteolytic effects of PGan (cited in 1739), but there apparently are no indications of such an effect in equids. Tissue culture studies indicate that many polypeptides are synthesized and released by the' equine conceptus (1052), as well as by in vitro-prepared tro- phoblastic vesicles (77, 143). Day 14 was a transitional day for changes in the types of proteins secreted by the conceptus. It was noted by these authors that Day 14 is approximately the time when meso- derm and blood-forming islets are devel- oping (pg. 365) and that this morphologic change could signal the change in protein production. A protein (termed mare pregnancy pro- tein—one) has been detected in the blood of 442 Chapter 10 pregnant mares in the earliest samples taken (Day 30) but has not been detected in stallions or nonpregnant mares (564). Other workers (938) have detected preg- nancy specific proteins as early as Day 6 and thereafter until the end of the experi- ment at three weeks (pg. 332) It is not known, however, whether any of these polypeptides have a role in the antilute- olytic mechanism. In conclusion, the nature of the antiluteolysin produced by the equine conceptus has not yet been elucidated. Role of estrogens in the prevention of luteolysis. Trophoblastic estrogen seems to be the principal antiluteolysin in swine and acts to redirect PGFZoc into the uter- ine lumen, thereby decreasing its avail- ability to the corpus luteum (164). In mares, estrogen does not appear to be an active antiluteolysin even though it is produced by the early conceptus (pg. 66). Furthermore, there are no indications that PGFZoc is redirected toward the uter- ine lumen in mares during the critical time (1845, 1440). From the comparative viewpoint, estro— gens play a role in luteal maintenance in pregnant pigs and rabbits but apparently not in cattle, sheep, guinea pigs, or ham- sters (765). The hypothesis that a similar mechanism functions in mares seems worthy of test for the following reasons: 1. Small quantities of exogenous estro- gens stimulate the uterine tone charac- teristic of early pregnancy (pg. 315); 2. Enzymes normally associated with steroidogenesis have been demonstrated in the equine trophoblast as early as Day 12 (pg. 66); 3. The equine conceptus is capable of in vitro estrogen production as early as Day 12, with production increasing markedly during the critical period of the first luteal response to pregnancy (Figure 10.7, pg. 428); 4. Uterine flushings at Day 20 are higher in estrone and estradiol concentra- tions in pregnant mares than in nonpreg- nant mares (pg. 428); 5. Transient edema of the endometri- um, perhaps in response to estrogens, occurs in early pregnancy (590); and 6. Preparation of recipients for embryo transfer by administration of proges- terone and estrogen regimens suggested that estradiol may be important for the establishment of pregnancy (1281). Early studies (178, 1153) supported the possibility of a luteotropic role for estro- gens in mares. More recent studies, how- ever, have cast doubt on the concept that estrogens have a luteotropic effect in mares. Daily administration of various doses of estradiol did not prevent luteoly- sis (1808, 885). Very high doses (10 mg/day) did interfere with follicular development and ovulation. This study indicated that in mares, in contrast to other species, exogenous estrogen prolongs the inter- ovulatory interval by suppressing the follicles, rather than by prolonging the life of the corpus luteum. In another study (1144), administration of diethyl- stilbestrol on Days 84 to 142 did not alter progesterone production. These findings and the mounting evidence that estrogens are part of the cascade of events leading to endometrial PGFZoc production and luteal demise in the absence of an embryo (pg. 274) seem contrary to a role of estro— gens in luteal maintenance in the pres- ence of an embryo. 10.5B. Interval between First and Second Luteal Response After the corpus luteum of pregnancy successfully passes the point of diver- gence between maintenance and regres- sion, it continues the gradual decline in progesterone output until the appear- ance of CG. It is not known whether a continuous positive pituitary stimulus is needed between Days 14 and 35 to assure that the decline in function does not proceed too rapidly. This question is open for study, and an approach is sug- gested by experiments in which adminis- tering an antiserum against a pituitary fraction during diestrus resulted in lute- olysis (pg. 265). In this regard, a high posi- tive correlation exists between LH and progesterone concentrations on Days 5 to 28, suggesting that the moderate decline in progesterone during this time may be related to low concentrations of LH (1144). The yolk sac and the chorioallan- tois produce an array of proteins on Days 15 to 28, but their role, if any, in luteal dynamics is not known (1052). Progesterone (n=6/group) 25 c 20 Pregnant E U) \ 5 15 .5 l ‘6 h 10 E i 8 T? . .. g 5 1T i 3%., ifl“‘+~+.¢\q 0 Hysterectomized 0 0 20 40 60 80 100 120140 Number of days from the end of estrus FIGURE 10.17. Progesterone concentrations in pregnant and hysterectomized mares. Lines are mathematical regression lines. Adapted from (1527). Endocrinology of Pregnancy 443 10.50. Second Luteal Response to Pregnancy During approximately Days 35 to 120, CG is present and is a necessary stimulus for maintenance of the primary corpus luteum. In one experiment (1504), the pri- mary corpus luteum, marked with India ink, regressed between Days 7 O and 140 in hysterectomized mares but not until Days 140 to 210 in pregnant mares. Similarly, peripheral concentrations of progestins increased or were at high levels in preg- nant mares during a time when levels con— tinued to decline in hysterectomized ponies (Figure 10.17). These findings indi- cated that a factor associated with preg- nancy, most likely CG, was needed for maintenance of the primary corpus luteum. Addition of CG to a culture medi— um containing slices of Day 100 primary or supplementary equine corpora lutea stimulates progesterone production (1521). The interval between approximately Days 35 and 40 is of special interest since the circulating levels of progesterone increase during this time (pg. 425). In early reports, it was suggested that this increase in progesterone was at least part- ly attributable to the primary corpus luteum (575, 1527) since it occurred before supplementary corpora lutea were detect- ed; progesterone levels increased in 6 of 6 mares between Days 32 and 44, and Day 44 preceded the first detectable ovu- lation in 5 of 6 mares (1527). The studies described in the next paragraph strongly support the hypothesis that functional and structural resurgence of the primary corpus luteum begins on Day 35, on the average, in response to CG. Other recent studies indicate that the resurgence of the corpus luteum results not only in increased progesterone secretion, but in the production of estrogens (pg. 446). A resurgence of the primary corpus luteum also has been reported in primates and has been attributed to chorionic gonado- tropins (900). 444 Chapter 10 In a recent study (186) using daily blood sampling, the increase in progesterone between Days 35 and 40 occurred before ultrasonic detectability of supplementary corpora lutea. Furthermore, the primary corpus luteum increased in size after Day 35, based on ultrasonic measurements (Figure 10.18; 186). Thus, the term resur- gence applies both to form and function of the primary corpus luteum. Daily blood sampling for progesterone and CG detectability has shown a close temporal association between these two end points (186). The first detectable appearance of CG was on Day 35 or 36. Progesterone Luteal resurgence 700 Corpus luteum (n=2 to 6) 400 + ‘ 300 12 Progesterone (n=11) —l 0 co Concentration (ng/ml) 15 20 25 29 Number of days from ovulation 33 37 41 FIGURE 10.18. Regression lines characterizing cross-sectional area of the primary corpus luteum and circulating progesterone concentrations. Luteal areas were significantly higher on Day 39 than on Day 33 and progesterone concentrations were high— er on Day 38 than on Day 32. Adapted from (186). began to increase from the day of first detection of CG and reached a significant elevation two days later. In conclusion, the second luteal response to pregnancy is rep- resented by resurgence of the primary cor- pus luteum due to the appearance of CG. 10.5D. Third Luteal Response to Pregnancy Control of the formation and mainte- nance of supplementary corpora lutea is attributed to CG and is designated as the third luteal response to pregnancy. There is ample evidence that eCG stimulates fol- licular development and superovulation in nonequine species. In the mare, an ovula— tory and luteinizing role of eCG is indicat- ed by the following: 1) the close temporal association between CG and the develop- ment of supplementary corpora lutea, 2) the continuing increase in number of cor- pora lutea in the presence of CG, 3) and the regression of the corpora lutea after CG is no longer detectable. Ovulation with the subsequent forma- tion of secondary corpora lutea did not occur by Day 70 in any of 12 hysterec— tomized pony mares, based on both necrop- sy and transrectal palpation (1504). However, ovulations have been reported for hysterectomized horse mares. In this regard, however, horse mares are more likely to ovulate while under progesterone dominance than are pony mares (pg. 223). The formation of accessory corpora lutea from anovulatory follicles also did not occur in hysterectomized pony mares necropsied at Day '70. In contrast, 12 of 17 pregnant mares ovulated by Day 7 0, based on transrectal palpation, and this was sup- ported at necropsy by the presence of sec- ondary corpora lutea. As noted above, CG stimulated the production of progesterone by secondary corpora lutea in vitro. It seems likely, therefore, that CG contributes both to ovulation and to the formation and progesterone production of the resulting sec— ondary corpora lutea (ovulatory) and acces- sory corpora lutea (anovulatory). Endocrinology of Pregnancy 445 SUMMARY: Luteal Progesterone [Output - - - Circulating progesterone concentrations Supplementary corpora lutea 60 90 120 150 t 180 Primary corpus luteum Output D. Equivalent to diestrus Output 1. Due to first luteal response Output 2. Due to second luteal response Supplementary corpora lutea Output 3. Due to third luteal response 5 oc-Pregnanes (fetoplacental source) 21 0 240 270 300 330 Number of days from ovulation FIGURE 10.19. Postulated sources of progestins in the maternal circulation during pregnancy. The relative proges- terone outputs of the primary corpus luteum and supplementary corpora lutea are indicated by the shaded areas labeled D, 1, 2, 3. ’ Output D. Progesterone production by the primary corpus luteum can be attributed to the same mechanisms (pitu— itary gonadotropins) controlling diestrus levels. ' Output 1. First luteal response to pregnancy. The mobile embryo prevents luteal demise on Days 11 to 15 by inhibit- ing release of PGan. The resulting pro- gesterone output is considered equivalent to the gradual progesterone decline in hysterectomized mares. Luteal mainte- nance results from blockage of the lute— olytic mechanism in pregnant mares and removal of the mechanism in hysterec- tomized mares. ° Output 2. Second luteal response to pregnancy. Resurgence (hypertrophy and productivity) of the primary corpus luteum begins on Day 35 due to CGp ‘ stimulation. ° Output 3. «Third lutealresponse‘to , pregnancy. Under continued CG stimula- ‘ ‘ tion, supplementary corpora lutea form. This response involves oyulatiens‘lsec- ondary corpora lutea) over Days 401201 '70 and increasing numbers of luteinized fol- licles (accessory corpora lutea) over Days) I 40 to 150. \ 5 The fetoplacental uni-t gradually i assumes the progestin productivity role during the time the luteal output gradu- ally declines. However, the progestins released into the maternal circulation are ' primarily 5a—pregnanes (progeSterone metabolites) and not progesterone, except , for low progesterone leVels near term in, some mares. ° The quantities of pregnanes in the the maternal circulation can be massive, especially during the last month of preg- nancy. Circulating fetoplacental preg~ nanes can reach IOU-fold higher levels than for luteal progesterone (e.g., 1500 versus 15 ng/ml). 446 Chapter 10 10.6. Sources and Roles of Estrogens Early conceptus and estrogens. Production of estrogens by the early con- ceptus is discussed elsewhere (pg. 66). The estrogens produced by the early concep- tus (up to Day 20) are probably essential, but their roles remain to be elucidated. Possibilities include enhanced develop- ment of uterine turgidity and thereby embryo fixation (pg. 315), production of uterine secretions (pg. 317), and other local functions. Follicles and estrogens. Mare ovaries are very active during the first half of pregnancy. The role of the follicles before formation of secondary and accessory cor- pora lutea is not known. Removal of the ovaries on Days 12 (589, 850), 25 (764), or 34 (1454) resulted in loss of the conceptus but not when exogenous progestins were given. Furthermore, ovariectomized mares that are maintained on exogenous progesterone are being used as recipients for embryo transfer (747, 741,). These stud- ies have demonstrated that progesterone is the only ovarian hormone that is abso- lutely essential to the early conceptus. On this basis, the essentiality of ovarian folli- cles and the estrogens that they poten- tially produce are open to question. Other indications of the nonessentiality of the Estrogens in early pregnancy —L N _L o Ovarian intact Altrenogest (n=5) No altrenogest Estrogen conjugate (ng x103 mg Cr) 0 o p -l> 07 on P N .0 o 20 25 30 35 40 45 50 55 60 65 70 Number of days from ovulation Ovariectomized, altrenogest (n=6) follicles are that early pregnancy does not appear to be jeopardized when follicular development is minimal (603, 182), and cir- culating estrogens do not rise until after Day 35 (1592). The surge in estrogens at Days 35 to 40 was attributed to the ovaries since it did not occur in ovariectomized mares (1592, 364). A second surge occurring after Day 60 was not suppressed by ovariectomy and was therefore attributed to the feto- placental unit (1592). The rise at Days 35 to 40 from an ovarian source, followed by a second rise from a nonovarian source, has been confirmed (366). The increase beginning on Day 35 can be attributed to CG. Corpora lutea and estrogens. It was initially postulated (1592) that the CG stimulation of ovarian estrogens was analogous to the synthesis of estrogens during the growth spurt of the preovula- tory follicle under the influence of LH. Recently, however, California workers hypothesized (366) and successfully tested the hypothesis (365) that the primary cor- pus luteum is the ovarian source of the estrogen surge that begins at approxi- mately Day 35 in temporal association with secretion of CG. Urine was analyzed for conjugated estrogens in the following three experimental groups: 1) pregnant and untreated, 2) pregnant and treated FIGURE 10.20. Results of an experi- ment that demonstrated the role of the ovaries in production of estrogens after Day 35. Estrogen conjugates were measured in the urine. Adapted from Daels et al. (366). with a progestin (altrenogest), and 3) ovariectomized on Days 18 or 19 of preg- nancy and treated with progestin. Results are shown in Figure 10.20. A surge in estrogens occurred on Days 35 to 40 in the ovarian-intact groups but not in the ovariectomized group, indicating the surge was from the ovaries. A gradual increase occurred in all groups beginning on Day 45, and this increase was attribut— ed to a nonovarian source. Ultrasonic monitoring of follicles did not suggest that the ovarian estrogens were related to increased follicular activity. In a subse- quent experiment (365), the estrogen surge did not occur in progestin-treated mares in which luteolysis of the primary corpus luteum was induced with PGFZOL, impli- cating the primary corpus luteum as the source of the estrogens. In mares with a Endocrinology of Pregnancy 447 regressed primary corpus luteum, estro- gen levels increased when secondary ovu— lations occurred. These results indicated that the corpo- ra lutea (primary and probably the sec- ondary) are the source of the estrogens presumably due to CG secretion. In preg- nant women, a similar increase in estro— gen levels occurs in association with hCG secretion (cited in 366). In another study (1530), exposure of mares to an endotoxin before Day 70, but not after Day 80, resulted in decreased levels of plasma estrogen conjugates. This result was attributed to endotoxin—induced PGan release (pg. 522) and the resulting luteoly- sis, rather than to a direct effect on the fetoplacental unit. The postulated sources of estrogens during early pregnancy are summarized (Figure 10.21). SUMMARY: Estrogen Sources in Early Pregnancy Follicles? Postovulatory Ovulatory surge surge .‘ Conceptus Uocaluse -10 0 10 20 30 40 50 60 70 80 . 90 - - - Circulating estrogen concentrations \ ¢’ I ’ I i Supplementary /' corpora lutea? ¢ ’ i ’ ’ ’ _ _ — ' Fetoplacental unit 100 110 Number of days from ovulation FIGURE 10.21. Postulated sources of estrogens in the maternal circulation dur- ing pregnancy. 0 A secondary postovulatory surge pre- sumably results from stimulation of the corpus luteum by the ovulatory LH surge. ' By Day 12, the conceptus begins to produce estrogens that do not reach the maternal circulation. Their effect is local (e.g., stimulate uterine turgidity). 0 By Day 36, the resurging primary cor» pus luteum produces estrogens under CG stimulation; levels exceed those of estrus. 0 Perhaps estrogens are also produced by the supplementary corpora lutea, pre- sumably as a response to CG. 0 The extent of contribution from large follicles during CG production is unknown. 0 The fetoplacental output inclines as the ovarian output declines. 448 Chapter 10 Role of fetoplacental estrogens. The roles of the massive quantities of estro— gens produced by the fetoplacental unit and excreted in the maternal urine are not clear. The hypertrophy and vascular— ization of the gravid uterus are probably dependent on steroids. Removal of fetal gonads in late pregnancy caused reduced estrogen levels without altering gestation length (1246). However, estrogen-deficient mares had smaller foals which could be attributed to inadequate vascularization of the uterus and placenta. Parturition also seemed weak, and levels of PGFZa were reduced. The roles of progestin and estrogens as parturition approaches are discussed in Chapter 11 (pg. 468). A recent study (1020) found a decrease in corticosteroid-binding globulin (CBG) in the plasma of mares despite the increase in estrogens, in marked contrast to data on most other mammalian species. It is generally accepted that the increase in estrogens in pregnant women is responsible for a rise in corticosteroid- binding globulin (cited in 1020). It is known that horse CBG specifically binds cortisol, but it is unclear whether the lower levels of CBG in pregnant mares indicate increased or decreased glucocor- ticoid activity. Research is needed on the roles of various types of estrogens, as well as CBG, in equine pregnancy. 10.7. Regulation of Follicles 10. 7A. Before CG Production The temporal disparity between the growth of ovarian follicles during early pregnancy and the earliest detectable appearance of CG in the blood is far too great to suggest that the former is caused by the latter. Follicular growth commences before Day 20, whereas CG has not been detected until 15 to 20 days later. For that matter, follicular growth precedes the appearance of the chorionic girdle (Day 25) which provides the CG— producing cells of the endometrial cups. Furthermore, eCG has an extremely low binding affinity for equine ovarian gonadotropin receptors (1557). We there- fore must look elsewhere for the gonadotropic impeller of follicular growth during the embryo stage. This conclusion has been reached by workers using both horses (129) and ponies (27, 1504). Hysterectomy studies. The uterine- removal approach provided initial infor— mation on the regulatory control of fol- liculogenesis during pregnancy. The rationale for this approach is that it results in the morphologic and func- tional maintenance of the corpus luteum (pg. 267). The progestational state is thus maintained in the absence of influences associated with pregnancy. The follicu- lar changes occurring in hysterec- tomized pony mares closely paralleled the changes occurring in early pregnant mares (Figure 10.22; 1504). These results indicate that factors other than CG are responsible for follicular devel— opment in early pregnant mares. Follic- ular activity has also been observed in four hysterectomized Thoroughbreds, Follicles >20 mm (n=6/group) ———— Number 0— Pregnant 0- - - Hysterectomized 0 20 40 60 Number of days from the end of estrus FIGURE 10.22. Similarity in follicular changes between pregnant and hysterectomized ponies. Lines for each group are mathematical regression lines. Adapted from (1504). and a large number of follicles attained ovulatory size (1536). Considerable follic- ular development apparently is associ- ated with both naturally occurring and induced pseudopregnancy in mares. Although not directly tested, the num- ber of large follicles at corresponding stages seems comparable for pseudo- pregnancy, pregnancy, and hysterec- tomy. Plasma LH concentrations are low in hysterectomized mares, comparable to levels for mares in diestrus and early pregnancy. Several authors have sug- gested that surges of follicular activity occur during early pregnancy (pg. 322; 27, 490, 806, 1116) and that such surges are associated with surges in the circulating concentrations of FSH (490, 806). The occurrence of follicular surges has not been adequately documented. It has been stated that a surge of follicular growth on Days 24 to 27 coincided with an FSH surge, and an FSH peak on Day 38 was followed by an increase in num- ber of follicles (806). The time of appear- ance of apparent follicular surges in another study seemed quite variable among mares, and some mares showed no indication of such activity. Consider- able additional study will be needed to document the existence and nature of follicular waves and FSH surges and their interrelationships. Administering proteinaceous follicu; lar fluid. Every 12 hours beginning on Day 14, pregnant mares were given charcoal-extracted follicular fluid (steroid content reduced by >98%; 182). Treatment suppressed the circulating concentrations of FSH and reduced the number of follicles 11 to 15 mm, 16 to 20 mm, and >20 mm and reduced the diam- eter of the two largest follicles (Figure 10.23). The hypothesis that the follicu- lar activity before Day 40 is due to FSH was supported. The depressed FSH and follicular activity did not alter the circu— lating concentrations of progesterone and did not result in pregnancy loss. Endocrinology of Pregnancy 449 Follicular fluid treatment Largest follicle N M Diameter (mm) 3 a; —L O .1 N m 0 Concentration (ng/ml) E; 14 16 18 20 22 24 26 28 30 Number of days from ovulation FIGURE 10.23. Mean diameters of the largest folli- cle and mean concentrations of FSH for mares treated with charcoal-extracted follicular fluid (treated) or saline (control). Adapted from (182). 10. 7B. During CG Production Levels of FSH were high and fluctuat- ing for approximately 40 to 50 days in both pregnant and hysterectomized mares (1092). Change in diameter of largest follicle seemed characterized by a surge (e.g., threefold rise) in diameter encompassing approximately Days 50 to 70 in 46 pregnant mares. There was no apparent tendency for a similar surge in hysterectomized mares. Perhaps the surge in diameter of largest follicle was due to the presence of large quantities of CG at this time. In a histologic study (445), ovaries were examined on Days 33, 43, and 63. On Day 43 (after appearance of CG), there were significant increases in number of small antral follicles, mitotic index of preantral 450 Chapter 10 follicles, and rate of antral formation. This effect diminished by Day 63. On a temporal basis, therefore, the release of CG may stimulate small follicles, includ- ing those in the preantral stage. The insensitivity of large equine follicles to CG is attributable to the low binding of CG to the gonadotropin receptors (1557). Results from transfer of donkey embryos to horses and vice versa have indicated that FSH, and not CG, stimulates growth of follicles during CG production but the LH-like activity of CG induces the ovulations (1655). Further support for an overall relation- ship between number of large follicles and level of FSH is the marked decline in both follicular activity (pg. 322) and FSH levels (pg. 424) by midgestation. Both the number of follicles and level of FSH remain low until the end of pregnancy, except for occasional surges of FSH. 10. 7C. Efi'ect of Progestins on F ollicles Considerable follicular activity occurs in all natural and experimental condi- tions studied to date in which mares are under prolonged progesterone domi- nance. As noted above, these conditions include hysterectomy and pseudopreg- nancy, as well as early pregnancy. This association between progestins and follic- ular activity occurs in ponies as well as horses, yet ovulation seems to occur more readily in horses than in ponies during certain progestational conditions. Both types of mares ovulate during pregnancy; however, 2 of 4 hysterectomized horses ovulated during the prolonged luteal phase even though plasma progestin lev- els were 2 to 6 ng/ml (1536). In contrast, no ovulations occurred in 12 hysterec- tomized pony mares during the progesta- tional state as determined both by trans- rectal palpation and necropsy (1504). Similarly, ovulations occur in horse mares during prolonged luteal phases, but they did not occur in pony mares that have been monitored (pg. 227). As a third point of contrast, ovulation during diestrus occurs in horses despite high progesterone levels but such ovulations have not been detected in hundreds of ponies subjected to frequent palpation and necropsy (pg. 223). In addition, the incidence of ovulations during proges- terone dominance varies considerably among horse breeds. The occurrence of follicular activity and ovulation in vari- ous progestational states in horse mares has been pointed out by Stabenfeldt and associates (1536). These workers also noted that progestins may not increase in the peripheral blood following an ovula- - tion during a progestational state. Limited experimental work involving long-term treatment of horse mares with progesterone (pg. 283) suggests that 50 mg beginning in mid-cycle may not suppress follicular growth and ovulation but it does inhibit estrus. Similarly, endoge- nous progesterone in hysterectomized, pregnant, and pseudopregnant mares has a more pronounced inhibitory effect on estrous behavior than on follicular growth and ovulation (1536). More detailed study is needed on the follicular and FSH patterns accompanying the prolonged administration of proges- terone. 10.8. Elective Induction of Abortion Before CG production. During the embryo stage, before the emergence of CG, pregnancy is entirely dependent on a luteal source of progesterone. Abortion is readily induced with a single injection of PGan or one of its analogues. During CG production. Abortion is more difficult to induce with PGFZoc and its analogues in the presence of CG, fre- quently requiring multiple injections, and a return to ovulation may be greatly delayed. However, adequate information is not available for the time when a luteal source of progesterone is essential (e.g., Days to 35 to 70). Studies are need- ed to determine whether a single injec- tion of PGan will effectively cause luteal demise during this time. A possible com— plication is that newly forming supple- mentary corpora lutea may not be sensi- tive to PGFZoc; this is a neglected research area. A recent study (1715) indi- cated that supplementary corpora lutea do not consistently respond to a luteolytic dose of PGFZOL; results indicated that repeated PGFZoc treatment may be need- ed for luteolysis of supplementary cor- pora lutea. A single injection of a PGFZa analogue on Day 70 failed to cause abortion in 8 of 8 mares (1509); however, treatment every 12 or 24 hours terminated preg- nancy in all mares. The interval from first injection to abortion was 4 to 5 days, regardless of whether injections were given once or twice daily. Apparently the function of the endometrial cups was not altered since CG concentrations remained elevated for at least a week. Although luteolysis occurred, as indicat- ed by progesterone concentrations, estrus and ovulation were delayed for approxi- mately 40 to 50 days. However, 6 of 8 mares displayed anovulatory estrus prior to this time; the ovaries were small and inactive, similar to what occurs during the anovulatory season. Another worker reported that induc- tion of abortion after the appearance of CG was followed by erratic estrous behavior during the continued presence of CG (33). Results were described as fol- lows: 1) rapid return to estrus after luteal regression; 2) massive follicular development during estrus without an ovulation; 3) increasing progesterone concentrations and continued follicular growth during the subsequent inter- estrous interval, with follicles becoming firmer; and 4) recurrences of estrus until CG was no longer detectable. In another study (436), a single injection of PGFZa during the period of expected CG produc- tion caused abortion in some pony mares; Endocrinology of Pregnancy 451 multiple injections in two mares resulted in abortion of 80 and 90 day fetuses (age estimated) and ovulation 6.5 (mean) days after abortion. A preliminary trial involving induction of abortion with daily PGFZa treatment beginning on Day 42 has also been done (1316). In a recent study (1111), daily treat- ment with a PGFZoc analogue at 82 to 102 days resulted in abortion in two or three days. The following suggestions or con- clusions were made: 1) Treatments did not act directly on the fetus or placenta because estrogen values remained unchanged; and 2) Treatment stimulated secretion of endogenous PGFZoc which led to fetal expulsion through cervical relax- ation and uterine contractions. In a recent study (820), fetal death was induced on Day 45 by injection of 20 to 45 ml of a 24% solution of saline into the allantoic sac through an abdominal inci- sion. Plasma estrone sulfate concentra- tions decreased within 3 to 10 days, and progesterone and CG profiles over the 10 days post-treatment were similar between treated and sham-operated groups. After cessation of CG production. In a clinical trial (575), 25 horse mares on Days 100 to 245 of gestation were treated daily during the winter (January) with PGFZoc until abortion occurred. The per- centage of mares aborting during the first, second, third, and fourth weeks of treatment were 76%, 12%, 4%, and 8%, respectively. During the first week, abor- tions were approximately evenly dis- tributed over the third and sixth day of treatment, with one abortion occurring on the second day. Similar results were obtained in a companion trial in 21 pony mares. The percentage aborting for each of the four weeks, respectively, was 62%, 24%, 10%, and 4%. Day of pregnancy did not seem to be related to interval to abortion. A dystocia occurred in one horse mare and in one pony mare due to fetal malposition (deviation of head and neck). In a German study in mares used 452 Chapter 10 for commercial eCG production (932), an average of three daily injections of PGan analogues was necessary but not more than four; successful induction of abor- tion, however, did not occur in 10% of the mares. Improved regimens or methods are needed to reduce the necessity for prolonged treatment in some mares. The cause of abortion from repeated injections with PGonc is not known; luteolysis can- not be directly responsible since luteal progesterone often is not necessary for pregnancy maintenance after Day 75 (pg. 437). Other abortion techniques. In a recent preliminary trial (1006), abortion was induced at approximately Day 150 in two mares by twice daily injections of PGan and in two other mares by a treatment with estradiol and oxytocin. The fetuses were expelled in intact membranes with- in three days in the PGFM-treated mares. Expulsion occurred more rapidly (13 to 2'7 hours) with the estradiol-oxy— tocin regimen; the uterine contractions were strong and the fetal membranes were disrupted. A large increase in PGFM occurred with the estradiol-oxy- tocin regimen. The authors suggested that this may have been an indication of damage to the uterus. There are indications that abortion can be induced in mares by treatment with estrogens or hCG. One reviewer (1332) cites a report that estrogen implants given on Days 26 to 41 were fol- lowed by abortion in 60 to 90 days. It is also noteworthy that abortion can be induced with hCG treatment (31); four Welsh pony mares aborted when treated with 2,000 iu for three alternate days, beginning before Day 39. Six mares in which treatment started on Days 40 to 97 did not abort, perhaps due to the pro- tective influence of eCG. These findings may be related to the report that high doses of hCG apparently cause reduced pregnancy rates, an effect that has been attributed to the excessive production of estradiol (pg. 281). The results of these ini- tial investigations with estrogens and hCG should be confirmed and expanded as possible approaches to the study of the endocrinology of pregnancy. Abortions can be done quickly by digi— tal entry into the allantoic sac and induc— tion of fetal hemorrhage by tearing of the umbilical cord or a portion of the fetus. It is not known whether simple perforation of the sac reliably induces abortion. It was reported recently (1714) that intracer- vically administered prostaglandin—E2 is a potent cervical dilator and allows easy access for manual removal of the fetus at any stage of pregnancy. Antiseptic solutions, such as peracetic acid (267) or hypertonic saline solutions (820), also have been infused to induce abortion. In a report of a comparison of abortion techniques (267), it was stated that better results were obtained with an antiseptic solution containing peracetic than with Lugol infusion or PGFZa injec- tions. Other reported abortifacient tech- niques include intra-allantoic injections of dexamethasone (1774) or providone- iodine (1694). Other reports and reviews can be consulted for further discussion of the use of multiple treatments of prostaglandin analogues for induction of abortion (1509, 225, 1694). 10.9. Seasonal Effects on Ovaries The length of gestation is approxi- mately 10 days longer in mares mated early in the breeding season than in mares mated late (pg. 329). There has been inadequate attention, however, to the possibility that ovarian activity dur- ing pregnancy may be affected by sea- son. In one study, the number of corpora lutea during pregnancy appeared to be greater for spring-mated than for late- mated mares (27). In a planned study of seasonal effects on the ovaries, mares were mated early (June 1 to July 10), middle (July 11 to August 20), or late (August 21 to September 29) in the Follicular fluid treatment Largest follicle (n=3/group) Summer, control \ Diameter (mm) M o 16 12 _______ 14 18 22 26 30 34 38 Number of days from ovulation ovulatory season (1504). On the average, based on regression analyses, the number of large follicles before Day 30 appeared to increase more gradually in mares mated late. This could be attributed to a seasonal effect on FSH output. Another worker (27) grouped mares into those con- ceiving between April 25 and July 2 (11 mares) and those conceiving between July 11 and February 29 (9 mares). It was concluded that there was no obvious seasonal influence on CG production, although ovarian activity was reduced in the late-mated group, and secondary ovulations in both groups seemed restricted to May to October. The conclu- sions are difficult to evaluate, however, because of the absence of statistical han— dling and a lack of information on the distribution of mares within the two intervals. An effect of season on follicular devel- opment during pregnancy was observed during a study on the effects of a pro- teinaceous fraction of follicular fluid on FSH concentration and follicular devel- opment (182). Follicular activity (e.g., number of follicles >20 mm, diameter of largest follicle) was greater over approxi- 42 46 50 Endocrinology of Pregnancy 453 FIGURE 10.24. Mean diameter of largest follicle in mares treat- ed with charcoal-extracted follic— ular fluid (treated) or saline (con- trol) during summer or fall. Note the greater follicular activity during summer than during the fall in control mares. Adapted from (182). mately Days 20 to 40 in mares mated in the summer than in mares mated in the fall (Figure 10.24). In another study (603), mares induced with GnRH to ovulate during the winter had depressed follicular activity during the ensuing pregnancy (pg. 168). Mares induced to ovulate, therefore, apparently revert after ovulation to the effects of season on pituitary gonadotropin output. These results demonstrate an effect of season on follicular development during pregnancy and provide indirect support for an effect of pituitary gonadotropins (FSH) on the follicular development of early pregnancy. Considerable addition- al information could be gained through slaughterhouse material, for example, by characterizing the associations among month, estimated age of fetus, and ovari- an activity. A possible effect of season on circulating estradiol levels during preg- nancy was noted previously (pg. 431). 454 Chapter 10 SUMNIARY AND HIGHLIGHTS: Endocrindlogy of Pregnancy Ovarian M progesterone . _ i l Fetoplacental 5 i estrogens E s x \K Ovarian estrogens Fetoplacental i l..- E Soc—pregnanes 3/" l . . 3. . 'K- ....... --" 3 ““~..-..“.~.. _, ----- ~ Relaxm ”a.-.“ . I“ ’ ”\K "" i . .' ._ .. l- :" \ .a‘ ll i 3 .i' I: I \_ " a || .’ \ . I: I. . \ ,.’ ’ n .' """"""" l: I n u. 'z .’ n . I: cu--o.—.-.-.--o.—.on--—--u--u.-.w.-o—.—o-o—o—u—o—u—.uu-.--—-—.-.-.-.-.—.-.--- iFolIicles g >20 mm ;’ .' R \ ,If': ”'2“ E Fetal gonads -' K .'<— Cups é— Sup. CL («Wk-— Prim. CL : ' 5 ~ 0 40 80 1 20 1 60 200 240 280 320 Number of days from ovulation FIGURE 10.25. Conceptual summation of the temporal interrelationships among concentrations of circulat- ing hormones and number or weight of endocrinologic structures. Hormonal fluctuations and surges, especial- ly for FSH, are not shown. Prim. CL = primary corpus luteum. Sup. CL = supplementary corpora lutea. 1. After the ovulatory LH surge, the mean concentrations of LH remain at baseline levels throughout pregnancy; however, direct assay during CG produc- tion has not been done because of assay cross-reactivity. Periodic surges of FSH occur during early pregnancy, but pat— terns have not been adequately charac- terized. Considerable follicular develop- ment occurs before the production of CG and is attributable to FSH. Both follicu— lar activity and FSH concentrations are minimal during the last half of pregnan- cy, except for pronounced FSH surges at parturition. 2. Estrogens are produced by the con- ceptus by Day 14 but apparently at this stage do not enter the maternal circula- tion. The uterus (turgidity and secre- tions) is a likely target. 3. The mobile embryo prevents luteal demise on Days 11 to 15 by inhibiting release of endometrial PGFZa into the cir- culatory system (first luteal response to pregnancy); the factor produced by the conceptus that blocks release of PGF2oc has not been determined. The second luteal response begins on Day 35 and is characterized by resurgence (growth and productivity) of the primary corpus luteum. The third luteal response involves formation of supplementary cor— pora lutea. 4. Massive quantities of CG from the endometrial cups account for the second and third luteal responses to pregnancy. Progesterone, estrogens, and perhaps androgens are produced by the CG-stim- ulated resurging primary corpus luteum and developing supplementary corpora lutea. 5. The CG—stimulated ovarian steroids are gradually replaced, in cross-over fashion, by fetoplacental steroids. On a temporal basis, the fetoplacental steroid Endocrinology of Pregnancy 455 production could be initiated by CG through activation of the fetal gonads. However, high levels of CG in the fetal circulation have not been documented. The estrogen and androgen circulating levels parallel the weight changes of the fetal gonads. 6. The fetoplacental unit discharges high levels of {Soc-pregnanes, but appar- ently not progesterone, into the maternal circulation. 7. Rel‘axin is produced somewhere Within the uterine—fetal-placental unit. The bimodal profile suggests the develop— ment of a second stimulus or source With- in the unit at about Day 200. Based on the absence of a temporal relationship in mares, the luteal glands are not a source, unlike in other species. 8. The ovaries are essential for preg- nancy up to Day 45. Progesterone is the only ovarian steroid essential to early pregnancy. The period of Days 50 to 70 is transitional in that ovariectomy results in abortion in some mares, but not in oth- ers. 9. Concentrations of CG vary greatly among mares and are affected most by fetal genotype, size of mare, and presence of bilateral twins. 10. Removal of the conceptus between Days 15 and before the appearance of CG results in pseudopregnancy (maintenance of the corpus luteum and uterine turgid- ity). Removal after approximately Day 38 results in continued production of CG and a complex, poorly understood ovarian response. 11. Follicular development is reduced when pregnancy occurs in association with ovulation induced during the anovu— latory season and when conception occurs late in the ovulatory season. 456 phapter 10 MILESTONES: Endocrinology of Pregnancy 1962 Discovery that fetal genotype altered eCG levels (312). 1973-75 Characterization of circulatory estrogen (1141) and progestin (762) concentra- tions throughout pregnancy. 1974 Finding of a rise in circulating progesterone concentrations after Day 32 and before the occurrence of secondary ovulations (1527). Demonstration that pseudopregnancy occurs after surgical removal of the early conceptus (Day 24; 906). First report that presence of the embryo is associated with release of reduced levels of PGan into the circulatory system (433). Initial study of a series on the changing ratio of FSHzLH activity of eCG over days (637). Discovery that the surge in estrogens on Days 35 to 40 is from the ovaries and a second surge after Day 60 is attributable to the fetoplacental unit (1592}. 1979-84 Demonstrations that the early (Days 12 to 20) conceptus raises the intra- luminal concentrations of estrogens without increasing the circulatory con- centrations (1843, 1846). 1981 Characterization of circulating relaxin levels (1552). 1982-85 Reports on a series of in vitro studies on the antiluteolytic role of the concep— tus during the first luteal response to pregnancy (1845, 189). Initial study of a series indicating that the conceptus has a PGFZoc-inhibitory effect as early as Day 11 (202). First report that resurgence of the primary corpus luteum in both size and function (progesterone production) occurs in temporal association with the appearance of eCG (186). 1990 Successful test of the hypothesis that the primary corpus luteum was the ovarian source of the estrogen rise on Days 35 to 40 (365). — Cflapter 11— PARTURITION, PUERPERIUM, AND PUBERTY Discussion of parturition considers its characteristics with emphasis on the three stages of labor. The discharged pla- centa, endocrinology of parturition, and parturition induction are also discussed. The crucial postpartum interval is given special attention because of its tremen— dous applied, as well as basic, ramifica- tions. The prepuberal period and puberty in fillies has been a neglected research area; available information is discussed. 11.1. Characteristics of Parturition Parturition in the mare is more dra- matic and rapid than in other farm species (1245). An account of behavioral patterns during the last two weeks of pregnancy (1443) and detailed descriptions of the act of parturition in the mare are available (95, 1333, 1362). 11.1A. Predicting the Imminence of Parturition External signs. The signs of approach- ing parturition are useful although too inconsistent to permit reliable and close predictions. Relaxation of the sacrosci- atic ligaments and especially the condi- tion of the mammary glands are Widely used indicators. The glands develop noticeably 3 to 6 weeks before foaling (1333) and distend with colostrum in most mares about 2 or 3 days before foaling (818). The colostrum oozes from the teats, forming a bead of waXlike material at each teat orifice (waxing; Figure 11.1). Waxing occurs in about 95% of mares 6 to 48 hours before foal- ing (1333). Waxing sometimes does not occur, or it may precede parturition by many days. The honey-colored waxy sub- stance usually remains for 12 to 24 hours, then softens and falls away in a Viscous stringy form (1059). Colostrum then drips or streams from the teats. The behavioral signs associated with the first stage of labor (restlessness, sweat- ing; pg. 459) are also useful indicators. FIGURE 11.1. Ventral View of beads of colostrum (waxing) at each teat orifice. 458 Chapter 1 1 Testing mammary secretions. Rossdale and associates have stressed the concept of readiness for birth (1364) and have reported a method for predicting the imminence of birth by testing mammary secretions for calcium and magnesium (1191, 939, 289). The equine prepartum milk test was developed on the basis of studies of the electrolyte content of mammary secretions (1251). The test strips were developed by the water industry for assessing water hardness. Evaluations can be performed rapidly and simply. Concentrations of calcium ions in the milk increase, and zones on the test strip sequentially change color as parturi- tion approaches. The test does not predict the exact time of parturition but substan- tially decreases the time needed for prepartum examinations. Information on sources and use of test strips in France (504) and the United States (918, 955) has been published. Temperature and other aids. Predict- ing the time of parturition by a decrease in body temperature on the day before parturition has had limited success. Some researchers have not found a useful tem— perature predictor, whereas others have found significant, but small, decreases (older literature cited in 1443 and 79). In one study, a significant drop occurred on the day before parturition, but the mean decrease was only 01°C (02°F; 1443). In another study (671), a circadian rhythm in body temperature was found, and there— fore time of day had to be taken into account in using body temperature as a predictor of parturition. Prepartum ultra- sonic evaluation is a potential aid to assessing fetal health and development. This approach has been productive for humans but is in its infancy as a research and development area in horses. An ini— tial project has been done (7), and further studies are inevitable; this will be an area that likely will develop in the 1990s. A number of commercial warning devices are being promoted as aids for monitoring mares near the end of gestation. One warn— ing device, for example, uses prolonged elevation of the tail as an index of parturi- tion (259). Closed circuit television is also being used as an observational aid. A tech— nique that uses examinations of vaginal smears also has been reported (121). 11.13 Nocturnal Parturition Mares foal predominantly at night (131, 1363, 919, 276, 818). Hour of parturition is shown for Thoroughbreds in Figure 11.2. In one of the depicted studies, 86% of 501 mares foaled at night. Ponies tended to foal at night even though lights were on in the stable throughout the hours of darkness (818). Monitoring parturition in stalls that were continuously lit, begin- ning just prior to parturition, did not alter time of parturition (919). However, in a recent study (223), the light—dark cycle was artificially reversed for 40 days before parturition in 27 mares. For controls and treated (inverted light) mares, 13% and 58% foaled between 8:30 and 16:30 hours, respectively, and 80% and 17% foaled between 22:30 and 8:30 hours. Thus, inverting the light- dark cycle for 40 days caused a majority of mares to foal during the day while in artificial darkness. Hour of parturition 120 Australia 100 ([1:1185) 80 60 40 Number of mares 20 12:00 Noon 18:00 24:00 6:00 Midnight 12:00 Noon FIGURE 11.2. Hour of parturition in Thorough- breds in England (1363) and in Thoroughbreds in Australia (131}. Studies of diurnal hormone variation immediately before parturition could help elucidate the nature of the control of noc- turnal parturition. It has been reported that progestins and estrogens did not show diurnal variation prior to parturi- tion, whereas corticoid levels were higher in the morning (341). It is noteworthy that 11 of 12 mares in which abortion was induced with PGFZa aborted at night, even though there was considerable vari— ation in the interval from onset of treat- ment to abortion (436). There apparently have been no other studies on the mecha- nisms involved in the mare’s ability to foal at night, and this could be a fruitful research area. 11.10. The Three Stages of Labor For descriptive purposes, labor or par- turition is commonly divided into three stages. Stages 1, 2, and 3 culminate in passage of the allantochorionic fluid, foal, and placenta, respectively. Mares seem more constant than other farm species in duration of the three stages, the position and posture of the fetus during delivery, and the time of day in which parturition occurs. The mare, thus, should make a good research model for comparative study of the nature and mechanisms of parturition. Some characteristics of par- turition are listed in Table 11.1. Rotation of fetus. A radiographic study of the fetus during late pregnancy and parturition indicated that the fetus ini- tially was in dorsal recumbency with fore- limbs and head flexed (822). Before first stage labor, the fetal movements were confined mostly to head, neck, and fore- limbs. During first stage labor, the head and forelimbs extended, and the cranial portion of the fetus rotated into dorso- sacral position (i.e., dorsum of fetus next to sacrum of dam). During second stage labor, the caudal aspect of the fetus also rotated to the dorso-sacral position. An account of the forces believed involved in delivery is available (1362). Parturition, Puerperium, and Puberty 459 TABLE 11.1. Characteristics of Parturition in Ponies Item No. Mean iSE Length of gestation (days) 24 339 11.0 Teat waxing (%) 24 63% Duration (min) lst stage 22 63 115.3 2nd stage 22 12 i1.3 3rd stage 24 60 110.1 Interval from foaling to: Standing (min) Mare 20 8.7 12.1 Foal 24 2 :27 Nursing (min) 9 65 19.3 Passage of meconium (min) 24 145 i115 First urination (hr) 17 8.5 10.8 Adapted from (818). First stage of labor. In general, the behavioral signs of the first stage are those of restlessness and abdominal pain. According to Arthur (95), a good indicator of the onset of the first stage of labor is the appearance of sweat patches in the flanks and behind the elbows, commenc- ing approximately four hours before foal- ing and increasing in intensity as foaling approaches. The first stage of labor includes relaxation and dilation of the cervix and onset of uterine contractions. Uterine contractions during the first stage are painful, causing restlessness and signs of abdominal discomfort. Mounting pressure of the uterus causes the allantochorion to bulge into the cervix (Figure 11.3). The membrane is thinner at this point and is characterized by an avillous area known as the cervical star (pg. 390). The allantochorion normally rup- tures at the cervical star prior to passage of the fetal-amniotic unit through the allantochorion. Occasionally the allanto- chorion may fail to rupture and begins to emerge through the labia; this sometimes occurs in premature and induced placen- tal separations. Failure of rupture of the allantochorion is distinguished by the 460 Chapter 11 . HORNS OF UTEPUS *7 é WALL OF U'TERUS FIGURE 11.3. Presentation of fetus and overview of fetal—placental circulatory system. Allantochorion is pro- truding into cervix just before loss of allantochorionic fluid. From (1007). prominent cervical star (Figure 11.4). The end of the first stage in normal parturi- tion is marked by rupture of the allanto- chorionic membrane and escape of the urine-like allantoic fluid. Escaping allan- toic fluid can be assumed to play a lubri- cating role for the amnion and contained fetus. The mare may show flehmen (upcurled upper lip; pg. 94) in apparent association with sniffing of the allantoic fluid (532). The role of flehmen and odors associated with parturition needs to be researched since it is possible that odors or pheromones may act to coordinate sequential events (e.g., stimulation of abdominal contractions). Second stage of labor. The second stage of labor features delivery of the foal and starts with forcible straining. Delivery is aided by pressure from the abdominal musculature (1362). Abdominal contrac— tions become apparent shortly after the escape of allantoic fluid. Most mares strain only when recumbent, which likely increases intra—abdominal pressure. The mare generally lies On her side with limbs extended during delivery. The straining efforts consist of a series of 3 or 4 power- ful contractions followed by a few minutes rest. The amnion emerges as a bluish- white, fluid—filled structure at the labia (Figure 11.4). One forelimb appears in the amnion, preceding the other forelimb by about 15 cm. Asynchronous emergence of the forelimbs probably facilitates pas- sage of the elbows and shoulders through the pelvic canal. The greatest straining effort is associated with emergence of the head; the chest and hips present less dif— ficulty. The foal is born with the umbilical cord intact, and the foal is usually encased within the amnion. The contin- ued enclosure of the fetus by the amnion may minimize the resistance to passage of the foal through the pelvic canal. The amnion ruptures due to movements of the foal, and the cord ruptures from move- ments of either the foal or mare. Second x?” "I 4 333 ,6» w E ALLANTOCHORION FIGURE 11.4. Upper panel: Emergence of an intact amnion through the labia during a normal parturition; the allantochorion ruptured internally, permitting the escape of allantoic fluid and emer- gence of the fetal-amniotic unit. Lower panel: Emergence of the allantochorion through the labia; the allantochorion failed to rupture internally. The cervical star (avillous area) is prominent. Courtesy ofD.C. Sharp. stage of labor requires about 17 minutes, with a reported range of 10 to 70 minutes (95); a mean of 12 minutes has been found in ponies (818; Table 11.1). Third stage of labor. The third stage of labor features the expulsion of the Parturition, Puerperium, and Puberty 461 placental membranes. The membranes are expelled by myometrial contractions that are believed to originate at the tip of the horns, causing inversion of the allan- tochorion during expulsion (818). Horses, unlike cattle, do not usually attempt to eat the placenta. Expulsion of the placen- ta generally occurs rapidly in the mare (within three hours, with a mean of approximately one hour; 95, 818, 276). It has been recommended that corrective mea- sures be undertaken if the placenta is retained beyond 6 (110) or 10 hours (1362). Retention of fetal membranes is said to have an incidence of 5% to 10% and is con- sidered serious because it is believed that it can lead to endometritis and toxemia (reviews: 110, 212, 1615). However, in a recent study (1294), retained placenta was observed in 11% of 3,456 parturitions; no effect was found on subsequent pregnancy rate, embryo-loss rate, laminitis, or gener- al health of the mares. Further informa- tion on etiology, pathogenesis, therapy, and consequential effects of retained pla- centa is given in the cited reviews. 11.1D. Myometrial Activity Activity of the equine myometrium pre- ceding and associated with parturition has been examined electromyographically in two mares (670). During the week before parturition, contractile activity increased and was greatest at night. Activity further increased as parturition approached. On the day before parturi- tion, the uterus was active 55 to 80% of the time; this activity was considered to represent Stage 1 of labor. There was a subsequent transient decrease in activity occurring 2 to 4 hours before Stage 2 (expulsion of fetus). This transient quies- cent period has not been described for other species (cited in 670). Activity dur- ing delivery consisted of 10 to 13 bursts in rapid succession. Activity diminished after foal delivery and again increased in association with placental discharge. An initial attempt to further characterize 462 Chapter 11 myometrial activity was not successful on a statistical basis; variability was large (451). Use of intravaginal pressure trans- ducers did not detect activity that could be attributed to the advancing pregnancy (629). Recent reports discuss the use of radiotelemetry to measure uterine elec- tromyographic activity in prepartum horses (352) and the effect of an oc-receptor agonist (used as sedative) on uterine elec- trical activity (817). 11.2. The Discharged Placenta The morphology of the discharged fetal placenta should be thoroughly understood by the clinician, pathologist, and physiol- ogist. Close examination of the fresh pla- centa can indicate abnormalities and the extent of possible retention of placental tissue (1293). It is a very useful structure for teaching and learning purposes and provides a stringent test of one’s knowl— edge of the intricacies of placentation. An excellent account of the morphology of the terminal fetal membranes has been made by Whitwell and J effcott (1770), and much of this section is based on their work. The freshly discharged placenta can be studied by spreading it on a flat surface with the villous side out (Figure 11.5). General shape conforms to that of the dis— tended uterus. In one study, the non— gravid horn was as long or longer than the gravid horn in 24% of the placentae (1770). The allantochorion is usually dis- charged inside-out. In the series of Whitwell and J effcott, the nonvillous side was outermost in 79% of 84 placentae. Placentae in mares with long nongravid horns tend to be shed with the villous side out (not inverted). Outer surface. The outer surface of the allantochorion has a red velvety appear— ance due to thousands of tufts, the well- vascularized microcotyledons (pg. 386). The overall appearance of tufts varies in differ- ent areas, depending on density and height and amount of residual blood. The area between the two horns where the cord attaches is especially dark due to dense tall villi. Microcotyledons may be viewed and studied with a magnifying glass to provide an impressive View superi— or to verbal descriptions. Adjacent tufts are separated by smooth allantochorion. It is this area that lays over the openings of the uterine glands into the intercotyle- donary network or absorptive arcade (pg. 391). There are several areas where the surface of the placenta is smooth, that is, avillous (pg. 390). The star-shaped arrange- ment of avillous ridges at the cervical pole is especially prominent. The cervical star marks the point of the amnion’s exit through the allantochorion and is there- fore ruptured during parturition. Inner surface. The inner surface of the allantochorion consists of transparent endoderm. An exquisite vascular bed in the underlying mesoderm is clearly visi- ble (Figure 11.5). Three types of invagina- tions into the allantoic cavity have been described (1770): 1) folds over large ves— sels, 2) allantochorionic pouches, and 3) small pockets of exocoelomic remnants (Figure 11.6). The areas of exocoelom result from incomplete fusion of the meso— dermal layers of chorion and allantois (Figure 9.9, pg. 356). These cavities may be demonstrated for teaching purposes by pumping air at the site of cord attach- ment alongside the larger allantoic ves- sels. The resulting inflated remnants of Inspissated remnants of endomelrial cup vessel Al|anloic pouch Fold over a large vessel Allantochorionic pouch FIGURE 11.6. Diagrams by Whitwell and Jeffcott of three types of invaginations into the allantoic cavity of terminal placenta. Adapted from (1770). Parturition, Puerperium, and Puberty 463 FIGURE 11.5. Placental membranes showing uterine or outer surface (A) and fetal or inner surface (B) of allantochorion. Inset shows close-up of two stumps of uterine arteries at end of the umbilical cord. In part A, the umbilical cord is passing though the rupture in the allantochorion. The edge of the ruptured area in the allantoamnion, through which the foal emerged, has been folded back for clarity; aa, allantoamnion; alc, allantochorion. 464 Chapter 11 exocoelom can be seen along the yolk sac and around the larger vessels. The amnion has a white translucent appearance and contains many tortuous blood vessels (Figure 11.5). Near the base of the amnion, a small area of exocoelom can be found that extends along the cord for a short dis- tance. Pale amniotic plaques can be seen along the umbilical cord on the inner sur- face of the amnion (pg. 379). A triangular-shaped remnant of exo- coelom is present within the umbilical cord, and the vestigial yolk sac can be located within this cavity. This portion of the exo- coelom occasionally communicates with the rectangular exocoelom remnant within the wall of the amnion. The base of the funnel- shaped cavity (exocoelom) represents the remains of the bilaminar omphalopleure at the placental attachment of the umbilical cord. This area is not covered by allantois and is externally demarcated by the circu- lar vascular channel, the sinus terminalis (pg. 365). The relationships among bilaminar omphalopleure, yolk-sac remnant, and cav- ities of the exocoelom will be difficult to relate to the discharged placenta without a thorough review of placentation, as given in Chapter 9. Umbilical cord. The umbilical cord is usually attached to the greater curvature of the fetal placenta, corresponding to the dorsal wall of the uterus near the junction of the two horns. Two portions of the umbilical cord (amniotic and allantoic) can be readily distinguished. The amniotic por- tion contains two arteries, one vein, the urachus, and the stalk of the regressed yolk sac (vitelline stalk). The urachus con— nects the fetal bladder to the allantoic cav- ity. It is very thin walled and sometimes becomes compressed or twisted to cause distention of the bladder from accumula- tion of fetal urine (1770). The remnant of the vitelline stalk may be located as a pale threadlike cord beneath the layers of the amnion. The allantoic portion of the cord is covered by the glistening endodermal lin- ing of the allantoic cavity. It contains the prominent umbilical vessels which devel— oped in the splanchnic mesoderm of the allantois. The two major arteries within the allan- toic portion of the cord diverge so that one primarily supplies the membranes in the gravid horn and cranial portion of the uterine body, while the other supplies the membranes in the nongravid horn and remainder of the body. The veins tend to accompany the arteries. The two veins converge in the allantoic portion of the cord, although the arteries do not. For this reason, the amniotic portion contains two arteries but only one vein (Figure 11.5). Distribution of blood supply to the pla- centa by the two arteries was studied recently by dye injection (343). When the umbilical cord is allowed to break naturally, the vein and urachus break at the naval stump, whereas the two arteries break within the foal near the apex of the bladder. The arteries therefore project wormlike from the placental stump of the cord for a distance of up to 6 cm (Figure 11.5). The length of the cord in relation to the length of uterine body has not been adequately described; however, the foal can lie outside the mare with an intact cord, probably due to an inversion of the allantochorion. Some attendants believe that premature severance of the umbilical cord from the discharged placen- ta may deprive the foal of blood. In a com- parison of natural and immediate (within 10 seconds after birth) mechanical separa— tion of umbilical cords, no significant hematologic or developmental differences in foals were found between groups (417, 416). Blood flow was not detected in the umbilical vessels even though the cord was pulsating. No indications were found to favor the practice of delaying separation of foal and discharged placenta. Yolk sac and hippomanes. The size of the yolk—sac remnant at term is variable, ranging from threadlike to 4 or 5 cm in diameter. The largest in the Whitwell and J effcott series was 20 cm in diameter. It is commonly stated that the yolk sac regresses and is replaced by the allantoic Parturition, Puerperium, and Puberty 465 TABLE 11.2. Size of Various Components of the Discharged Placenta Thoroughbreds Ponies Placental component No. Mean iSEM No. Mean iSEM Total weight (kg) 139 5.7 i008 10 2.38 $0.23 Allantochorion Weight (kg) 144 3.6 i005 10 1.6 i014 Surface area (cm2) 142 16,700 i150 10 10,200 i900 Allantoamnion Weight (kg) 140 1.85 i004 10 065 i011 Surface area (cm2) 9 22,600 i824 Adapted from (1770). sac during Days 20 to 40. The prominent yolk sac in the terminal placenta, howev— er, demonstrates that considerable hyper- trophy often occurs, raising questions on the apparent development and role of the fetal yolk sac; this is an open research area. The wall of the yolk sac may become calcified and can be mistaken for an anomalous twin (1770). The fluid remain- ing in the allantoic cavity of the dis- charged placenta is turbid and yellow- brown. A large hippomanes (up to 200 g) can be found either in the allantoic cavity or in the expelled allantoic fluid (pg. 415). The amniotic fluid is clear and may con- tain “golden slippers” (protective material shed from the foal’s hooves). Linear dimensions of various portions of the allantochorion have been published (1770; review: 575), and weight and surface area of placental components are shown (Table 11.2). 11.3. The Endocrinology of Parturition , Studies in nonequine species have indicated that the fetus controls the time of parturition through maturation of the hypothalamic-pituitary-adrenal axis and the resulting increase in cortisol (review: 963). Implication of the fetal adrenal glands in the initiation of parturition in sheep and other species is based on the following: 1) Increased secretion of cortisol by the fetal adrenals occurs during the last 48 to 72 hours of gesta- tion; 2) Destruction of fetal pituitary or sectioning of the pituitary stalk elimi- nates the rise in cortisol and the prolon— gation of pregnancy; and 3) Early parturi- tion can be induced by administration of cortical hormones to the fetus or dam (332, 963). Increase in fetal cortisol is believed to activate placental enzyme systems involved in conversion of progesterone to estrogen; the estrogen, in turn, is needed for PGan production. The relationship between the fetal hypothalamic—pituitary- adrenal system and parturition has not been well defined in mares; this species presents special challenges to parturition research because of the difficulty in establishing indwelling vascular cath- eters (1473, 1475). Discussion of the endocrinology of parturition in mares (1245) and the timing of birth in domestic animals, including horses (1475), are included in recent reviews. 11.3A. Adrenal Cortical Hormones The contrast between ruminants and horses in fetal circulating levels of corti- sol and ACTH is striking (1475). Levels are much lower in equids during the last few weeks of gestation, and the prepar- tum rise occurs more abruptly. Also, the fetal adrenal does not respond as well to exogenous ACTH. There is some evi- dence, however, for fetal adrenal hyper- trophy (approximate doubling in weight) 466 Chapter 11 immediately before parturition in mares (331). In a study in Thoroughbreds, the maternal peripheral levels of total llfi-hydrocorticosteroids remained con— stant throughout the prepartum period (Figure 11.7; 985). Concentrations were higher in foals than in mares during the first 18 hours after parturition and were highest during the first hour. Another worker found that corticoid concentra- tions in the maternal circulation increased significantly on the day of foal- ing (341), but this was not confirmed in a subsequent trial. English workers (332, 1133) concluded that there appears to be little placental cortisol transfer in either direction, fetal plasma cortisol remains low throughout the last third of pregnancy, and postnatal values are highest immediately after birth. These workers suggested that there was probably some increase in fetal adrenal activity near term although not as dramatic as the increase in the fetal lamb. In the most recent study (1478), plasma cortisol levels in the fetus were high at birth (67 ng/ml) and rose to a maximum during the following two hours (141 ng/ml). A similar cortisol surge did not occur in premature births. The thy— roid hormone, T3, showed positive cor- relations with cortisol for both normal and premature births. In oxytocin- induced parturitions (728), the corticoid concentrations were greater in the umbil- ical artery (flow from fetus to placenta) than in the umbilical vein. These studies implicate the fetus in the production of corticoids. It has been suggested (728) that oxytocin activates the fetal adrenal, and the resulting corticoids are involved in induction of parturition, as suggested for other species. Recently, concentrations of androstenedione, an androgen of adrenal origin, were reported to rise before partu- rition, peak at parturition, and then decline (1284). The hypothesis that adrenal cortical hormones are involved in initiation of parturition in mares has been tested by Periparturient circulating hormones Pregnancy Progestins (ng/ml) Estrogens Corticosteroids -60 -50 -40 -30 -20 -10 Postpartum ‘ 500 50 3 ? 3' ‘< 400 40 e rn O U) X 3’ E 300 ‘8 3o 3. 3 6' 1’1 8 8 a 200 5 2o 9 _ O. V U) 100 10 E i o o 0 10 20 30 Number of days from parturition FIGURE 11.7. Means of circulating concentrations of hormones in Thoroughbreds during the periparturient period. Adapted from (985). administration of the glucocorticoid dex- amethasone. Initial attempts were not supportive, however, since single injec- tions of up to 80 mg or 40 mg/day for four days failed to induce parturition (264, 275). First and associates successful- ly induced parturition with higher doses of 100 mg/day for four days, beginning on Day 321 (73, 74). This regimen result- ed in birth of live foals in approximately seven days after initiation of treatment in horses and in 3 or 4 days in ponies. Results supported the hypothesis that the adrenal cortical hormones play a role in the initiation of parturition. The dose was massive in comparison to that used in other species, however, and the results may have been due to a pharma- cologic action (i.e., may not occur physi- ologically). 11.3B. Progestins and Estrogens As described in Chapter 10, circulating progestin values increase over the last 30 days of gestation, followed by a rapid decline at the time of parturition. In con- trast, circulating estrogens gradually decrease during the last 2 or 3 months of pregnancy. Both progestins and estrogens decrease precipitously at the time of par- turition; the precise temporal relation- ship to parturition is discussed below. Foal versus mare. Pennsylvania work- ers (550) concluded that prior to and during parturition, progesterone com— prised 30% of the total progestins, com- pared to more than 80% after parturition, and that immediately postpartum there was a higher percentage of progesterone in the foal than in the mare. These stud- ies may need to be repeated, however, in View of the recent report that much of what was called progesterone in earlier studies was probably due to nonspecifi- city of the assays (pg. 69). California work- ers (985) concluded that circulating con- centrations of total estrogens and Parturition, Puerperium, and Puberty 467 progestins were higher in foals than in mares during 18 hours postpartum. Workers in England (156) have exam- ined progestins and estrogens in the feto- placental unit by long-term catheteriza- tion of umbilical and uterine vessels in ponies. Most (apparently 10 of 12), how- ever, delivered stillborn fetuses. Both total estrogens and progestins were high— er in the uterine venous plasma than in peripheral plasma. Progestins in the umbilical vein were much higher than in the uterine vein or umbilical artery. The high estrogen concentration in fetal plasma greatly exceeded the concentra- tions in the uterine vein. In another study (341), progestin levels in newborn foals decreased during the 48 hours post- partum, whereas estrogen levels re- mained unchanged. In induced parturi— tions, progesterone and estrogen were higher in the umbilical vein (flow toward fetus) than in the umbilical artery (728). The high concentrations of progestins and estrogens in the uterine veins and their rapid drop in the peripheral blood during and immediately after parturition support the concept of fetoplacental involvement in the production of these steroids. The higher levels in the umbili- cal vein than in the artery indicate a role of the placenta. Furthermore, the higher levels in the foal during the first day are probably residual from the fetoplacental source, as is the precipitous decline in the maternal circulation within one-half hour after parturition. Further discussion of ' the role of the fetoplacental unit in the production of progestins and estrogens is given in Chapter 2 (pg. 66). Temporal relationships between steroid levels and parturition. Recently, Illinois workers examined progesterone and estradiol every half hour in the plas- ma of periparturient mares (1284). A sig— nificant drop in progesterone and estra- diol was not detected until a half hour after parturition, further indicating a 468 Chapter 1 1 role of the fetus/foal in the production of these steroids. However, although not significant, the levels of progesterone appeared to decrease from 1 day before foaling to 4 to 12 hours before foaling. The decline in estradiol was most dra— matic after foaling. In a later study, other workers found a significant prepartum decrease in progesterone (669); during days -3 and —2 preceding parturition, mean values fluctuated between 6 and 8 ng/ml and during the last 24 hours dropped to approximately 3.5 ng/ml by the time of parturition. There was no sig- nificant change in estradiol—17B near par- turition, but slight increases seemed to occur in each mare. These recent studies indicate that the general statement that progestins increase and estrogens decrease preced— ing parturition in mares may need modi- fication. Although concentrations of pro— gesterone increase over many days before parturition and the concentrations of estrogens decrease over many weeks, these changes do not reflect the changes immediately preceding parturition (during the last day of pregnancy). That is, progestins, but not estrogens, decrease immediately before parturition so that a high estrogenzprogestin ratio is present at the time of parturition. Roles of progestins and estrogens. The functions of progestins and estrogens in late pregnancy and in the initiation of parturition are not known. A recent study (761), using a highly specific assay, indi- cated that some of the pregnanes attained very high levels (e.g., 2000 ng/ml) near term and therefore could be involved in readiness for birth. The progestins may play a role in suppressing the activity of the myometrium in late pregnancy, but this has not yet been studied in the mare. Perhaps the reported increase in preg- nanes plays a role in the myometrial qui- escence that is reported to occur just before Stage 2 of labor (pg. 460); enhanced relaxin production could also be involved in the transient quiescence (cited in 1475). The relationship between the act of par- turition and the recently reported drop in progestins during the 24 hours prepar- tum and the drop in estrogens immedi- ately after parturition is consistent with a role of the resulting high estrogenzpro- gestin ratio in parturition. The role of the progestins and estro— gens in parturition also has been studied by the administration of exogenous hormones (74). Gestation length was shortened to a similar extent by dexa- methasone alone, progesterone alone, dexamethasone plus progesterone, and dexamethasone plus estradiol. Estradiol alone, however, did not alter gestation length. It is interesting that progesterone reduced gestation length in the mare, rather than extending it as it does in some other species. Perhaps the effect of progesterone was mediated by conversion of the injected progesterone to corticos— teroids. Failure of estradiol to alter gesta- tion length is inconsistent with the above statement that a high estrogen2progestin ratio favors parturition. Furthermore, administration of epostane, a competitive inhibitor of 3B-hydroxysteroid dehydroge- nase, altered steroidogenesis in mares but had little effect on the length of ges— tation (528). Removal of the fetal gonads in late pregnancy resulted in low estro- gen levels but did not alter gestation length (1246). Although gestation length was not altered, the act of parturition seemed weak and levels of prostaglandin were reduced. This study indicated that estrogens play a role in mechanisms of PGFZoc production, and the authors cited studies in other species that indicated a close association between estrogen con- centrations and PGan production. It also was found that estrogen deficient mares had smaller foals, perhaps due to inade- quate vascularization or blood flow of the uterus and placenta. Parturition, Puerperium, and Puberty The ovaries do not play an essential role in parturition, based on the findings that gestation length was not altered sig- nificantly in ovariectomized mares (764, 1420). Foals were delivered alive, and the mares lactated. The passive status of the equine ovaries in parturition contrasts with an important active role in other farm species (cited in 1419, 1475). In conclusion, manipulation of circulat- ing concentrations of the progestins and estrogens indicates that these steroids probably do not play a crucial role in determining when parturition will occur; that is, they do not determine the length of gestation. The two steroid types, how— ever, are probably crucial to the continu- ing preparation of the uterus and the fetal environment as pregnancy proceeds. Progestins may also favor uterine quies- cence before parturition, and the estro- gens or an increased estrogenzprogestin ratio may play a role in production OfPGFZOL. 11 .3C. Prostaglandin F20: Prostaglandin F2a likely plays a role in parturition through the stimulation of PGFM, oxytocin & parturient events (n=1) I l I,\ \’1‘ '2 '2 Q: I I Oxytocin A 30 PG FM N O PGFM concentration (ng/ml 10 o' s ,_ X’Aliantoic 4 ~. . rupture g EParturition 0 : : 'i—T—I—I—I—LI—Y—I—TH—I—r—r-l -12 -8 -4 0 4 20 60 100 Number of minutes from parturition 469 myometrial contractions. Concentrations of PGFZoc increased in the peripheral blood of 5 mares during foaling (1426). Concentrations were low at 30 minutes, approximately doubled 5 minutes before the fetus was in the cervical canal, and further increased consistently when the foal was in the canal. Fetal and maternal plasma concentrations of PGFM also increased during late pregnancy and par- turition (155). There was a gradual increase in the metabolite during the last 3 to 4 months of pregnancy. The greatest increase occurred during the second stage of labor. In a study involving frequent sampling (669), PGFM values at Day 270 were significantly greater than those ear- lier in pregnancy. During the 15 days prepartum, average concentrations did not fluctuate in a consistent manner, but concentrations increased suddenly just before rupture of the allantochorion. In one mare with a comprehensive set of samples, concentrations surged to a peak 4 minutes before the allantoic fluids were discharged, decreased transiently, and then increased again along with a surge of oxytocin at the time of foal delivery (Figure 11.8). The periparturient profile 320 M J) O 160 (lui md) uonenuaouoo ugooMxo 80 FIGURE 11.8. Maternal oxytocin and PGFM concentrations associ- 0 ated with the time of allantoic rupture and parturition (deliv- ery). Adapted from Haluska and Currie (669). 470 Chapter 1 1 of PGFM from another study is shown in Figure 11.9 (1246). One study of PGFM levels, however, failed to find an increase in concentrations during the final 10 days of pregnancy (1550). Concentrations did abruptly increase before foaling, and a surge occurred within two minutes after oxytocin injection for induction of parturi- tion. Maximal concentrations were sus— tained for up to one hour, and the surge was about 10 times greater than what occurs during luteolysis. Sharp releases of prostaglandins also occurred in the days following foaling, and it was suggested that these surges may be involved in uterine involution (1550). In catheterized animals, there was an increase in the metabolite in both fetal and maternal plasma during the last two hours before delivery. As indicated above, fetal gonadectomy studies indicated that estrogens are needed for proper PGcmx production. These studies in mares and comparative studies in other species Peripheral PGFM (n=8) 60 J) 0 Concentration (ng/ ml) N O Number of minutes from parturition FIGURE 11.9. Peripheral plasma PGFM concentra— tions during spontaneous foaling. The initial rise corresponded with the onset of first stage labor, and the second sharp increase corresponded with second stage labor. Adapted from Pashen and Allen (1246). emphasize the need for detailed investi- gations of the factors involved in the ele- vation of PGFZcx levels in association with parturition. Recent preliminary study using radiotelemetry indicated that PGonc caused an increase in myometrial activity in prepartum mares within 8 minutes of administration (352). Silver and associates (1476, 1477) have shown the occurrence of a 3- to 5-fold rise in PGFM in association with fasting or intrauterine surgery. The concentrations rose when feed was withdrawn for 12 to 30 hours. The rise was virtually abolished by pretreatment with a prostaglandin synthetase inhibitor. The PGFM rise was associated with a 70% decrease in glucose uptake. Basal levels were restored rapid— ly (within 1 to 3 hours) after feeding or infusing glucose. Some mares (5 of 8) foaled prematurely, within one week after a period of food withdrawal. These stud- ies have shown a close relationship between prostaglandin metabolism and plasma nutrient levels and have drawn attention to the possible consequences of an inadequate feeding schedule on pre- mature delivery. A gradual rise in PGEZ, as well as PGonc, has been detected in the allantoic fluid (1476). Increases in both prosta- glandins in the fetal fluids preceded pre- mature delivery, whereas changes in the maternal plasma were minimal. Studies in other species indicated that locally pro- duced PGE2 is important for cervical ripening (cited in 1475). In this regard, a recent report indicated that exogenous PGE2 can be used to soften the equine cervix (1714). 11.3D. Oxytocin The ability of oxytocin to stimulate milk letdown and myometrial contrac- tions has been well documented in many species. Oxytocin is considered the final hormone in the maternal cascade leading to parturition (review: 1475). Studies in other species indicated that it is not secreted in large amounts until the sec- ond stage of labor; the final stimulus to oxytocin release is from physical disten- tion of the cervix and vagina. However, oxytocin receptors do increase near term, and therefore the amount of oxytocin needed for initial myometrial activity is not great. Oxytocin receptor formation is elicited by changes in estrogenzprogestin ratios and increased PGFZoc production (cited in 1475). Results of a limited study (37) in three Welsh ponies suggested that little oxy- tocin was released during the first stage of labor, but there were indications of oxytocin and vasopressin release during the second stage (expulsion of fetus) and third stage (expulsion of fetal placenta). More recently, oxytocin was found to remain basal, with an increase only at the beginning of the expulsive second stage of labor (Figure 11.8; 669). Concen- trations peaked when the fetal head was Visible at the labia and remained elevated above basal levels for 2 to 4 hours post- partum; that is, until the placenta was delivered. It has been suggested that oxy- tocin could be the trigger to parturition (1550) by causing cervical dilation, uterine contractions, and release of prosta- glandins. Oxytocin is a neurohormone under the control of the central nervous system and therefore could be a factor in the mare’s ability to select time of partu- rition according to presence of a favorable environment (669, 1550). 11.3E Prolactin Circulating concentrations of prolactin were measured in five mares during the periparturient period (1828). Concentra- tions rose markedly in the last week of pregnancy and remained high until declining to basal levels at one to two months after parturition. A role of pro- Parturition, Puerperium, and Puberty 471 lactin in mammary development and lac- tation in mares is presumed on the basis of this study and studies in other species. A recent study (799) suggested that pro- lactin concentrations in periparturient mares are higher in the evening than in the morning. The interrelationships between equine prolactin and other hor- mones centering around parturition are not known. Recently, circulating prolactin was monitored in oxytocin-induced and spon- taneous parturitions (1347). Prolactin appeared (P<0.1) to rise before parturi- tion in relation to the change in colostrum composition. Oxytocin treatment induced prolactin release in preterm mares but not in term mares; the authors suggested that this result may have reflected preterm estrogen levels. Prolactin also appeared to exhibit transient elevation in the foals associated with oxytocin-induced parturitions. The authors suggested that this may have resulted from premature disruption of placental exchange so that the maternal prolactin entered the fetal circulation. Stimulation of elevated prolactin levels by administration of a dopamine antago— nist did not alter LH and FSH profiles (168). These authors concluded that short- term elevation of serum prolactin, similar to what occurs during the postpartum period, does not alter ovarian function. Bromocriptine, a dopamine agonist, has been used to reduce prolactin and proges- terone concentrations in late pregnancy and to thereby induce clinical signs simi- lar to those of fescue toxicity (agalactia, birth of weak or stillborn foals, placental abnormalities; 799). Tall fescue may be infected with an endophyte fungus that inhibits the release of prolactin (cited in 799). This experiment with bromocriptine and fescue toxicity (cited in 799) have raised important questions about the role of ePRL in late pregnancy. 472 Chapter 1 1 SUMMARY: Control of Parturition GRAVID UTERUS HYPOTHALAMUS .IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AND CERVIX POSTERIOR FETUS AND PITUITARY LIGAMENTS Neural signals Maturation l Cortisol ® Oxytocin PGF CE) External signals (e.g., light) HYPOTHALAMUS AND ANTERIOR PITUITARY ACTH ADRENALS PLACENTA ® Relaxin Estrogens Progestins .IIIIIIIII @ Relaxation @TOxytocin receptors C5) Contractions @ Enzyme activation TPGFQQ synthesis @ Softening i Blood flow FIGURE 11.10. Summary of the postu- lated events near the end of gestation and leading to parturition in mares. Some of the suggested sequences are based on studies in nonequine species, especially those involving maturation of the fetal hypothalamic-pituitary area. This simple diagram should not be allowed to obscure the complex sympho- ny of hormonal and neural events required for the birth of a newly complet- ed individual. Each of the following com- ments refers to a letter designation (A—F) in the diagram. A. Based primarily on studies in nonequine species, maturation of the fetal hypothalamic-pituitary-adrenal axis likely is the paramount event lead- ing to preparation of the fetal and mater— nal systems for impending parturition. B. Fetal cortisol and possibly fetal LH (not researched) may be involved in acti— vating the fetal—placental enzyme sys— tems for the appropriate synthesis with- in the estrogen-progestin complex. C. Progestins and estrogens presum— ably are the principal hormones crossing from fetal to maternal systems, but the nature of fetal-maternal signals has not been elucidated for this species. Apparently, progestin levels decrease during the 24 hours preceding partu- rition, Whereas biologically active estrogens remain at a high level until just after parturition. As a result, the estrogenzprogestin ratio increases. The high proportion of estrogen leads to the following: 1) softening 'of the cervix aided by local cervical production of PGE2, 2) build-up of myometrial oxytocin recep— tors, 3) synthesis of PGan in the wall of the gravid uterus, and 4) maintenance of uterine vascular bed. D. A complex interplay among neural signals (fetal movements, uterine con- tractions), a slight increase in basal levels of oxytocin, and an increase in PGFZoc leads to the first stage of labor. A surge in PGFZoc and resulting uterine contractions play a major role in culmi~ nation of the first stage with rupture cf the‘allantochorion. The discharging allantoic fluid lubricates the birth canal. After a period of uterine quies— cence, a burst in oxytocin leads to a fur- 4 ther increase in PGFZOL, strong myome- trial contractions, and delivery of the foal (end of second stage). The levels of oxytocin remain adequate for a few hours for expulsion of the placenta (end of third stage). E. Oxytocin release, which plays a key and pivotal role in all stages of labor, can be blocked by external stimuli impinging upon the central nervous sys- tem and the hypothalamic-posterior pituitary area, allowing the mare to temporarily short circuit the fetal sig— nals for birth. F. The role of relaxin has not been determined in this species, but it may be involved in softening the cervix and liga- ments and in preventing inappropriate or premature uterine contractions. All aspects of the control and mecha— nisms of parturition in mares need research attention. Informational voids in this species include the role of the fetal pituitary and adrenal and the role of the nervous system in coordinating the physical aspects of the birth process. Parturition, Puerperium, and Puberty 473 11.4 Induction of Parturition The applied aspects of induction of parturition in mares have been reviewed (729, 954, 175, 821). Discussion of the triad consisting of the mare, farm manager, and veterinarian can be found in these and other reviews. Induction is usually motivated by managerial convenience, although there occasionally may be other indications, such as rupture of the prepu- bic tendon or prolonged gestation. To reduce the risk of premature induction, it has been recommended (821) that ges- tation length should exceed 320 days and that there be substantial mammary development with the presence of colostrum before induction is attempted. Extent of cervical dilation also has been used as a criterion (256, 1296). More recently test strips for ionic concentra- tions (hardness) of mammary secretions have been used to select time of initia- tion of treatment (pg. 458). Good success for routine induction of daytime foaling has been reported when test sampling was done once or twice daily beginning 10 days before the expected foaling date (954). Silver (1475) states, however, that even with the aid of the preterm milk test and monitoring plasma progestin levels, induction of parturition in mares is not yet fully reliable. Oxytocin. Mares and women are extremely sensitive to the inducing action of exogenous oxytocin (1475). Perhaps the high estrogen and progesterone metabo- lite levels during the last month create a hormonal environment for build up of oxytocin receptors well before term. Oxytocin induction of foaling is expected to be completed in an hour if a proper previous evaluation is done (review: 729). Use of oxytocin for parturition induction in mares was first reported by Britton (255), who used it to induce labor in mares that were described as having uterine atony. Results of using posterior pituitary extract and synthetic oxytocin in planned parturitions have been reported (1296). 474 Chapter 11 Estrogen was given 12 hours prior to the oxytocin treatment in mares with a closed cervix. Details of the protocol including obstetrical procedures are given (1296). Such techniques also have been used in a few cases to induce abortion during the last months of pregnancy (256). Hillman and associates (727, 728, 729) evaluated various doses of oxytocin with and without stilbestrol for the induction of foaling. Time of appearance and degree of expression of the signs of parturition, time of delivery of foal, and time of pas— sage of placenta were influenced signifi- cantly by increased doses of oxytocin. Fetal membranes, for example, appeared between the labia in an average of 42 minutes after 20 iu of oxytocin and in 30 minutes after 100 iu. It was concluded that estrogen was useful in softening and relaxing the cervix when it was tight but was not essential when the cervix was already soft and dilated. These workers have recommended an oxytocin dose of 40 to 60 iu and have published detailed information on the protocol (729). Several other authors favor the use of oxytocin for parturition induction (1750, 1339). Clinical uses of oxytocin, as well as estrogens, have been reviewed (1691). Prostaglandins. The abortifacient prop— erty of PGan is discussed in Chapter 10 (pg. 450). The use of PGFZOL for induction of parturition has not been studied as exten- sively as oxytocin. In the initial abortion study (436), mammary engorgement occurred in most of 27 treated mares, even in mares that were in early preg— nancy. Multiple injections (2.5 mg/12 hours until abortion occurred) resulted in fetal expulsion in 7 of 7 pony mares in which the estimated stage of gestation was >300 days. Other workers (74), how- ever, failed to shorten gestation with a single injection of 12 mg to horse mares on Day 321, and some foals died shortly after birth. In one trial, 3 of 5 mares given 5 mg on Day 338 foaled in a mean of 20 hours (893). Studies by Rossdale and associates of a synthetic prostaglandin analogue (fluprostenol) have been encouraging (1357, 940, 1360). A11 mares (9 of 9) treated with this compound produced viable foals within four hours of initial injection. In a subsequent study (1190), sequential injec— tions were given over the last 50 days of pregnancy. The analogue did not induce parturition in mares until after Day 320. In parturition-induced mares that deliv- ered viable foals, the hardness (calcium concentrations) of milk samples was char- acteristic of normal full-term mares, but the milk test did not meet the full—term criteria in those that delivered premature or stillborn foals. Induced parturition was accompanied by elevated circulating con- centrations of cortisol and andrenocorti- cotrophic hormone, suggesting a centrally mediated action. Apparently endogenous production of PGan did not occur since PGFM levels were not altered. The authors concluded that fluprostenol can- not be recommended unequivocally; low doses of oxytocin may be preferable because the interval from injection to par— turition is shorter and less variable. Use of fluprostenol, as well as clo— prostenol, also has been studied by French workers (1583); they reported that all foals from induced parturitions were viable. More recently, two other PGonc analogues, prostalene and fenprostalene, were used for induction of daytime foaling (954). Two 0.5-mg doses were given two hours apart to mares that met the milk test criterion. All mares (17 of 17) treated with fenprostalene delivered viable foals at a mean of 3.9 hours after the first injection; 12 of 16 mares treated with prostalene delivered viable foals in an average of 3.7 hours, and the remaining mares delivered in 30 to 56 hours, appar— ently spontaneously. The authors con- cluded that the daytime foaling manage— ment system was reliable, but they stressed the importance of critical moni- toring to assure that the mare is ready. Corticosteroids. Four high doses (100 mg/day) of dexamethasone beginning on Day 321 significantly reduced the mean and variation in the interval to parturi- tion (73, 74). Mares foaled in several days. The mares produced and weaned appar- ently normal foals, and there was no evi— dence of retained placentae. However, the foals from treated horses weighed less at birth than those from controls (39 versus 44 kg). Growth rate from birth to 13 weeks was similar for the two groups; that is, during this time, the foals from treated mares did not compensate for the lower birth weights. In contrast, dys- tocia occurred in ponies that were treat— ed with dexamethasone after moderate mammary development had occurred but before colostrum could be expressed (1356). It does not Seem likely that administration of corticosteroids will become a favored method of induction of parturition. The repeated treatments and long interval to parturition will be likely deterrents. Clenbuterol. Drugs that prevent uter— ine contractions and therefore delay par- turition are being used for parturition control in cattle (cited in 175). Clen- buterol has been given in the evening to minimize the occurrence of calving at night. This approach has apparently not been investigated in mares. 11.5. Puerperium The puerperium is the period from par- turition to the return to a condition con- ducive to initiating and maintaining another pregnancy. Herein, the term postpartum interval also Will be used for the period delineated by parturition and the first ovulation. The uterus involutes during this time and the ovaries change from relatively quiescent to active. The interval in mares from parturition to ovulation must be relatively short to favor an interparturition interval of approximately 12 months, despite the Parturition, Puerperium, and Puberty 475 11-month gestation length. In cattle, as a comparative example, gestation is short- er (9 months), but the postpartum inter- val is longer (2 to 3 months), likewise resulting in a 12-month interparturition interval. Perhaps because of the long ges— tation and evolutionary pressures for a 12-month foaling interval, the mare is the only common farm animal with a short postpartum interval. Sows frequently show an estrus 1 to 3 days after parturi- tion, but the estrus is usually anovulato- ry; ovulatory estrus usually does not occur until after weaning. The desirability of 12-month interpar— turition intervals in mares dictates the extreme applied importance of the inter- val from parturition to the return to reproductive rhythmicity. Because main- tenance of a 12-month foaling interval demands that mares be bred soon after foaling, this area of equine reproduction deserves top research priority. Surveys on reproductive efficiency in England (1390), France (300), Ireland (123), and the United States (1810, 579), involving many breeds and >1,000 mares per survey, indicated that 51% to 62% of the mares in these breeding pro- grams were postpartum mares (means of percentages: postpartum, 57%; bar- ren, 30%; maiden, 13%). Most mares bred during a breeding season therefore are postpartum mares with the special problems associated with this reproduc— tive status. Yet, only a small portion (perhaps <20%) of mare reproduction research is done with postpartum mares. The apparent reasons for this paradox include the following: 1) Postpartum mares are not readily available to research laboratories; 2) A foal can severely complicate research procedures involving the mother; and 3) Although the equine industry needs research in this area, the limited influence of the industry on the selection of research areas is a reflection of the limited funds provided. 476 Chapter 1 1 11.5A. First Postpartum Estrus and Ovulation Estrus. The first estrus following par- turition is commonly called foal heat. Reported mean lengths of the interval from parturition to the start of the first postpartum estrus are generally given as 7 to 9 days, and the majority of mares (>90%) begin estrus within 5 to 12 days (review of early literature: 575). A distri— bution curve for the results of a represen- tative study is shown in Figure 11.11. It has been recommended that the term foal heat be used for only those estrous peri— ods that begin before 14 (448), 16 (961), or 19 (1035) days postpartum. Under such definitions, only a small minority ‘(<10%) of mares fail to exhibit foal heat. In Thoroughbreds, 8% did not show signs of estrus until 14 days or later (128). Length of foal heat conforms to the month in which it occurs (1396), as described elsewhere for nonparturient mares (pg. 174). Foal heats are generally assumed to be ovulatory. There is, how— Postpartum estrus & ovulation 0) O First day of estrus (n=428) N 01 M O . 9 First ovulation : 3'3/ (n=470) _l 01 Mares (%) 3 01 1 5 9 13 Number of days from parturition 17 21 25 29 FIGURE 11.11. Frequency distribution for first day of postpartum estrus and first postpartum ovula- tion. Estrus data adapted from studies in Thoroughbreds in Australia (1035) and ovulation data from a study in Thoroughbreds in the United States ( 989). ever, a lack of critically obtained compar- isons of the characteristics of postpartum estrus with those of contemporary estrus in barren mares. In a study involving six Thoroughbred mares, duration of estrus, day of ovulation in relation to estrus, and luteal life span for the postpartum estrus seemed similar to what would be expect- ed in nonparturient mares (985). Bain (128) stated that a large number (13%) of post- partum estrous periods were of short duration (2 or 3 days) and that many of these were low in fertility and accompa- nied by subestrus and the absence of folli- cles >15 mm; month of parturition was not given. Ovulation. The first postpartum ovula- tion occurs after nine days in the majority of mares (985). Time of occurrence of the first postpartum ovulation in the exten— sive study by Loy (989) is shown in Figure 11.11 (mean irSD for 456 mares: 10.2 4:24 days). Postpartum ovulation was arbi- trarily defined as occurring by 20 days; 456 of 470 (97%) mares ovulated by 20 days, and the earliest ovulation for the second ovulatory period was at 22 days. Seasonal effects and first postpartum ovulation. The hormonal events associat- ed with parturition are powerful stimuli for follicular development and ovulation, even in mares foaling during the anovula- tory season. In early studies, the length of the interval to first day of estrus was unaffected by month of parturition (1035, 1396). However, the study by Loy (989) detected an underlying effect of season on the interval from parturition to ovulation. The proportion of intervals as short or shorter than the mean (10 days) increased progressively by month of foal- ing (January to May; Table 11.3). All intervals longer than 30 days (overall incidence: 2%):occurred in mares foaling in January through March; none occurred in mares foaling in April and May. Some of the mares with a >30-day post— partum interval underwent a seasonal transitional phase, characterized by a prolonged period of estrus and growth TABLE 11.3. Percentage of Mares with a Parturition-to-Ovulation Interval as Short or Shorter than the Overall Mean (10 days) Parturition, Puerperium, and Puberty 477 TABLE 11.4. Effect of Month of Foaling on Postpartum Ovarian Activity in Ponies in France Percentage with a short parturition-to-ovulation Month of foaling interval (n=456) January, February 33% March 55% April 65% May 83% Adapted from Loy (989). and regression of large follicles (pg. 146). Most commonly, however, mares with a prolonged interval developed follicles at the expected time but failed to ovulate and then went into a period of ovarian inactivity. An abstract of a study in Hungary (921) stated that mares foaling in January and February (n=19) only exceptionally returned to estrus within 10 days; however it was noted that mares were in poor to moderate condition. Apparently, postpartum mares are sub- jected to the opposing pressures of parturi- tion (positive ovulatory stimulus) and anovulatory season (negative influence); in most horse mares (989) and a few pony mares (Table 11.4; 1210), the positive effects associated with parturition are suf- ficient to overcome the effects of season and consequently, the first postpartum ovulation occurs. A study of the effect of a 16-hour fixed photoperiod beginning on December 1 on postpartum events failed to show a reduction in the length of postpar- tum interval (755). However, the authors suggested that the foals may have been born too late in the year (March and April) to alter ovulation time. Seasonal effects and subsequent ovu- lations. Some mares revert to ovarian inactivity after the first postpartum ovu- lation, e’specially when foaling occurs dur- ing the anovulatory season (989, 1210). Incidence of this phenomenon has not been adequately defined but appears to be associated more with the influence of Number of mares Regular Inactivity Month of ovarian after first Immediate foaling activity ovulation inactivity J anuary 0 2 5 February 4 1 3 May 8 0 0 Adapted from Palmer and Driancourt (1210). season than with what has been called lactational anestrus (1210). In a limited study, a postpartum ovulation followed by ovarian inactivity occurred in 3 of 15 pony mares foaling in January and February but in none of eight mares foal- ing in May (Table 11.4). This phe— nomenon (ovulation followed by inactivi- ty) was less common than immediate postpartum ovarian inactivity. Use of an artificial lighting program influenced the incidence of postpartum ovarian inactivity (1210); two months of a 16-hour photoperiod before parturition decreased the incidence of inactivity for mares foaling in winter, whereas an eight- hour photoperiod increased the incidence for mares foaling in May. These authors recommended that a light regimen should begin two months before parturition for mares foaling in the winter (pg. 160). Apparently the positive effects of par- turition do not extend beyond the first ovulation. After the first ovulation, the influence of season becomes dominant and early—foaling mares (e.g., January, February) may become anovulatory (989, 1210). Thus, the ovulatory-inducing effect of foaling seems similar to induced follic- ular development and ovulation associ— ated with GnRH treatments during the anovulatory season in barren mares (pg. 168). That is, after the induced follicu- lar stimulation, usually followed by ovu- lation, the mares may revert to the anovulatory condition. 478 Chapter 11 Effect of body condition. Feeding mares to obesity during gestation did not alter gestation length, characteristics of partu— rition (919), or postpartum reproductive characteristics or performance (920, 716). In one experiment (716), mares in various groups were fed at a high or low nutritive level for three months before or one month after parturition. Pregnancy rate was reduced and embryo-loss rate was increased when mares were maintained in poor body condition throughout the four months. Poor body condition also interfered with a return to reproductive cyclicity postpartum, similar to what has been found for development of reproduc— tive cyclicity at the end of the anovulatory season (pg. 126). Apparently factors which are expressed as poor body condition (poor nutritive bal- ance, disease) can interfere with the repro- ductive response to seasonality changes, whether during the postpartum period or the spring transitional period. Mares that were thin at the end of gestation had a nine-day longer mean gestation length and lower circulating LH concentrations in association with the first ovulation (733). The reduced LH surge seemed simi- lar to those occurring during the onset of the ovulatory season, whereas the pat- terns in control mares seemed similar to those occurring in the middle of the ovu- latory season (pg. 254). The authors cited reports that undernutrition impairs GnRH secretion in other species. 11.5B. Effects of Nursing Lactational anestrus. A condition known as lactational anestrus may begin immediately after parturition or subse— quent to the postpartum estrus and may continue until the foal is weaned at 4 to 6 months. It has been stated that there are no follicles in the ovaries or that there may be only small follicles up to 2 cm (380). Early references indicated a high incidence (e.g., 22% and 74% in mares not conceiving during the foal heat) but provided little critical information (review: 575). The economic impact of such a condition would be considerable, and it seems unreasonable that it has been vir- tually ignored as a research problem. Critical studies are needed on its inci- dence. If it is found to be as prevalent as the reports cited in the review suggest, the nature of the condition must be char- acterized in detail. It is emphasized, how- ever, that so-called “lactational anestrus” may actually represent the effects of sea- son and body condition that are manifest- ed in postpartum mares, as discussed above. Body condition is an expression of nutritive balance and could be in an espe— cially precarious state if the mare is not nutritionally compensated for lactation requirements. Clearly, considerable criti- cal study is needed on the effects and interactions of parturition, lactation, nutrition, and season on the resumption of reproductive cyclicity. Inhibition associated with nursing. The effect of suckling on postpartum repro- ductive function has been the subject of limited study. It is paradoxical that the inhibitory effect of nursing on estrus and follicular development has been exten— sively studied in most common animals but not in mares—yet, postpartum mares are under the most pressure for rapid return to reproductive cyclicity. The effect of the foal on the return to estrus and development of follicles was studied in four mares in which the foal was present and in four in which the foal was removed on the day of parturition (623). Ovaries were removed and evaluated at six days. Foal removal hastened the onset of estrus (foal present: 0 of 4 in estrus by 6 days; foal removed: 3 of 4 in estrus) and the growth of a large follicle (foal present: 20.8 mm; foal removed: 33.2 mm). In another study (1645), removing the foal on the day of parturition reduced the inter— val to estrus (5.0 versus 7.3 days) and caused greater LH levels at 6 days post- partum (Figure 11.12). Concentrations of FSH were not different between mares Foal removal (n=4/group) Concentration (ng/ ml) 0 2 4 6 8 10 Number of days from parturition FIGURE 11.12. Effect of permanent removal of foal on concentrations of LH and FSH in pony mares. Foals were removed on the day of parturition. Day effect is significant for each hormone. Stars indicate means that are significantly different. Adapted from (1645). with foal present and those with foal absent for the first 10 days postpartum, but FSH levels averaged over the 10 days before the first ovulation were higher in the group with foal absent. Considerable ' additional study will be needed before these initial results can be integrated into an overall postulate on effect of foal on reproductive function before and after the first postpartum ovulation. The effects of weaning, either at the usual time or earlier, on the ovaries of the mare also need to be studied. In this regard, two pony mares were observed for 90 days after foaling; estrus was not seen and LH levels remained at the low prepartum concentrations (575). After removal of the foal from one of the mares at 90 days, there was an immediate increase in LH and a return to estrus and ovulation. Results of this preliminary trial indicate the need for a full-scale project in this area. Parturition, Puerperium, and Puberty 479 In a recent study (1392), foals were removed for 24 hours on the third or fourth day after parturition. Short-term foal removal had no effect on LH profiles, interval to the first estrus and ovulation, or the interval between the first and sec- ond ovulatory periods. Removal of foals for four hours did not alter LH or FSH in mares during one hour post-removal, but prolactin levels increased in 4 of 12 mares (1784). These negative findings on gonado- tropin levels are of comparative interest since removal of calves for 24 hours results in increased LH release. The effects of foal removal have applied as well as basic implications. At present, it is uncertain whether early weaning could have a bene- ficial effect on cyclicity in selected mares other than those attributable to improved nutritive balance. 11.5C. Postpartum Endocrinology Steroids. Levels of estrogen, progestins, and 11B-hydroxycorticosteroid were exam- ined in six Thoroughbreds by workers in California (Figure 11.7; 985). Estrogen changes were not detected, but the post— ovulatory progestin curve seemed similar to that of nonparturient mares. Cortico- steroids increased significantly between 15 and 22 days postpartum. In a Wash- ington study in six large saddle horses (762, 1141, 1142), estrogen concentration (expressed as estrone equivalent) during estrus was 25 to 50 pg/ml, with a subse— quent drop to 5 to 15 pg/ml. Estrogen peaks in 3 of the 4 mares that exhibited no foal heat seemed similar to those that did. The LH and progesterone curves, as well as the estrogen curves, seemed simi- lar to those of nonparturient mares, whether or not foal heat was detected. In summary, both studies indicated a simi- larity between postpartum and nonpar- turient mares in estrogen, progestin, and LH curves during the periovulatory peri- od. Noden and co-workers reached a simi- lar conclusion (1164). Others (994), howev- er, found that only those mares that had a 480 Chapter 11 postpartum estrus (n=4) showed estrogen peaks between 5 and 15 days; three mares which did not show estrus before three weeks had no postpartum peaks. Definitive conclusions in this regard require further experimentation with con- sideration of time of year. Luteinizing hormone. Concentrations of LH and FSH have been studied in intact and ovariectomized ponies during the periparturient period after foaling during the ovulatory season (May to September; 1645). During the 14 days before parturi- tion, daily LH concentrations were low, comparable to mid—diestrus, in both intact and ovariectomized mares (Figure 11.13). The low baseline LH levels are attrib- utable to the suppressing effect of pro- gestins (exogenous progesterone suppress- es LH in ovariectomized mares; pg. 248). Parturition 16 Ovariectomized ,’ ‘. . mares '4 . ’, ‘ 12 FSH : 8 i 4 " Concentration (ng/ml) -14 -7 0 5 10 0V Number of days from parturition FIGURE 11.13. Concentrations of LH and FSH in ovariectomized and intact ponies before and after parturition. Adapted from (1645). According to studies in other species (cited in 1147), the high levels of estrogens dur- ing late pregnancy inhibit the synthesis of LH and deplete the pituitary reserves. Illinois workers (1284) detected a signifi- cant transient rise in LH one-half hour after parturition using samples collected every 30 minutes. They attributed the surge to the removal of the negative feed- back effect of the steroids. In the study depicted in Figure 11.13, LH increased gradually in intact and ovariectomized groups for 10 days follow- ing parturition, indicating an effect due to positive photoperiodic influences rather than to the ovaries (1645). As the postpar- tum ovulation approached in the intact group, however, LH rose more rapidly. More rapid rate of LH increase is attributable to a rise in estrogens (pg. 248). Similar results have been reported in horse mares (1164, 1142, 807, 1392, 755, 511); plasma LH increased within 72 hours after the decrease in progestins associated with parturition, and the increase in LH was associated with an increase in estra— diol (1142, 1164). In mares exposed to an extended photoperiod beginning during the last few months of pregnancy, the LH levels during the postpartum interval were slightly, but significantly, elevated (755). This result likely relates to the posi— tive effect of seasonal or artificial photope— riod on LH levels (pg. 121). - The LH response to exogenous GnRH in late pregnant and postpartum mares was recently investigated in Colorado (1148, 1147, 685). Based on response to exogenous GnRH, pituitary concentra- tions of LH presumably were depressed in postpartum mares but were quickly restored. It was proposed that the appar— ent early LH replenishment of the pitu- itary was responsible for the short inter- val to postpartum estrus in mares in contrast to the long postpartum anestrus in sheep and cattle. These workers com- mented that the species differences could be due to a more rapid increase in pitu- itary GnRH receptors in mares. Follicle stimulating hormone. Mean concentrations of FSH in both intact and ovariectomized mares fluctuated nine- fold before parturition (1645). An appar- ent surge occurred in the ovarian-intact group on a mean of five days before par- turition (Figure 11.13). Other workers (807) have noted intermittent surges at a mean of 12 days before parturition. Prepartum FSH surges have not been adequately characterized. They require further study because their presence would occur despite the high levels of circulating estrogens. This estrogen-to— FSH relationship is contrary to what occurs during the estrous cycle and could reflect the associated high progestin lev- els in pregnant mares. Alternatively, this enigma may indirectly suggest that low FSH levels during estrus may be due to an inhibin—like substance as well as estradiol (pg. 251). A pronounced parturient surge of FSH occurred in all of 15 pony mares (ovarian-intact and ovariectomized) at the time of parturition (1645; Figure 11.13) and has been reported also in horse mares (807). The parturient surge began a few days before parturition, reached a peak just before (807) or on the day of parturition (1645), and declined significantly (1645) by the day after par- turition. The parturient FSH surge may be related to either the removal of inhibitory influences from the fetopla- cental unit or to the hormonal changes that initiated parturition. In this regard, however, an FSH surge was noted in association with the insertion of intravaginal sponges (pg. 165; 1605). Perhaps, therefore, physical pressures associated with the onset of parturition are primarily responsible for the par- turient FSH surge through neural sig- nals. The parturient FSH surge de- serves a major research thrust because it is likely responsible for the initiation of postpartum follicular development that leads to the all-important postpar- tum ovulation. Parturition, Puerperium, and Puberty 481 Following parturition, FSH remained high in the ovariectomized mares (Figure 11.13), probably due to positive environ- mental effects. In the intact mares, how- ever, FSH levels gradually decreased, indicating the inhibitory effect of ovarian factors. The postpartum FSH decline seemed similar to that occurring during late diestrus and estrus. The amount of FSH secreted in postpartum mares in response to exogenous GnRH tends to decrease from 3 to 6 days postpartum (685). The interval from the parturient FSH surge to ovulation seems similar to the interval from the last diestrus surge of FSH to ovulation in cycling mares. Pulses of LH and FSH. Simultaneous pulses of LH and FSH have been observed before foaling (732). Pulses of FSH, but not LH, were associated with an increase in mean levels. The pulses increased in frequency on the day of foal- ing to 2 to 4 pulses per day. Pulsatility of LH release postpartum has also been reported (511, 1392). 11.5D. Uterine Involution The biologic and practical aspects of uterine involution are tremendous, espe- cially since the uterus in this species must prepare to receive an embryo shortly after parturition in order to maintain a 12-month interval between foalings. Enormous morphologic changes in size and structure of the uterus and the associated suspensory ligaments occur gradually during the 11-month gestation. During the puerperium, how- ever, the uterus is restored to nearly the prepregnancy condition within approxi- mately three weeks. Rapid repair and return to a condition suitable for preg- nancy establishment in this species probably reflects, in part, the diffuse, epitheliochorial placentation. Ultra— structural studies indicate that in nor- mal parturition the villi separate cleanly at the maternal-fetal interface (1545). Uterine involution occurs at a time when 482 Chapter 1 1 the uterus is especially susceptible to bac- terial contamination. Despite the practi- cal importance of uterine involution, only sporadic studies were done on this subject during the 40 years following the pioneer- ing work of Andrews and McKenzie in 1941 (85). In the past decade, several important studies have been done, but many more are needed. A list of some reported end points associated with nor- mal involution is shown (Table 11.5). Size changes. Vandeplassche and asso- ciates (1685) have reported that the uterus and cervix weigh 7 to 9 kg immediately after parturition, and weight changes lit- tle during the first 2 days. These workers gave weights of 5 to 7 kg at 3 days and 2 kg at 8 days postpartum for a uterus that involutes at a normal rate. Due to involu— tion and elevation of the uterus associat- ed with contraction of the suspensory lig- aments, the uterus reportedly can be transrectally circumpalpated in horses at about 3 days (1685) and in ponies on the day after parturition (’96, 650). An enor- mous diameter reduction must occur over the first day or two. Available informa- tion is based on studies that did not begin until 1 day (650) and 3 days (1068) after parturition. Mean diameter of the midsec- tion of the gravid uterine horn was reported as approximately 60 mm at 1 day in ponies and 81 mm at 3 days in horses. These figures represent consider- able reduction over the diameter that must have been required to accommodate a fetus at term. According to the report (1685) that weight of the uterus changes very little during the first 2 days, these diameter reductions apparently represent retraction due to the loss of distention rather that a reduction in mass. Specific ultrasound, weight, and histologic studies are needed to characterize the nature of the immediate reduction in diameter. Furthermore, such studies should con- sider the myometrium as well as the endometrium. An ultrasound study indicated that the previously gravid horn was recognized by its larger size for a mean of 21 days, and uterine involution was complete (both horns of similar diameter) by 23 days (1068). In a more recent study (650), how— ever, a significant difference in dia- meter between the formerly gravid and TABLE 11.5. Reported Rapidity of Various Aspects of Uterine Involution Characteristic Rapidity of postpartum change Amount of vaginal exudate Extent of intraluminal fluid Progressively decreases through first ovulation ( 908) Decreases so that fluid is ultrasonically undetectable by 15 days (1068, 650) Uterine horn diameter Returns to pregravid size by 32 days (1855); Difference between gravid and nongravid horns ultrasonically indistinguishable by 23 days (1068); Gravid horn slightly, but significantly, greater in dia— meter at 35 days (650) Histology Resorption of microcaruncles Restoration of luminal epithelium Endometrium Endometrial gland dilation Glandular activity Composition of uterine flushings Lysosomal enzymes, plasmin, antitrypsin Total protein, acid phosphatase (914) Complete by 7 days ( 1855, 126) Intact by 7 days (1855, 126) Pregravid appearance by 14 days (1855) Corrected by 4 days (126) Increases to 12 days (126) Peak at 4 to 6 days and return to low values by 16 days Decreased levels between 4 and 8 days (941) nongravid horns was detected through the last day of the experiment (35 days; Figure 11.14). It appears, therefore, that there is a prolonged residual effect on uterine size that extends beyond 35 days. It is unknown how long the residual effect continues and whether there may be a cumulative effect of each succeeding preg- nancy. Diameter of the previously gravid horn did not differ between mares that became pregnant at the first postpartum estrus and those that did. not (908). Tone and contractility. It has been reported that uterine tone and tubularity increased for 5 days postpartum (1380). In a more recent study (650), however, the uterus was reported to be extremely firm for several days postpartum; the firmness gradually decreased over days 3 to 8 (Figure 11.15). The firmness was qualita— tively different from the characteristic turgidity of early pregnancy and may have represented postpartum edema. Contractility of the uterus, as deter- mined by daily one-minute ultrasonic observations, was negligible during the postpartum interval (Figure 11.15). This Postpartum uterine horns (n=10) 65 55 E, \ Formerly gravid horn E 45 H* a) i E \ .‘2 ‘ D i 35 We ++ i++ t + $+++ + . fl? ”Fri.“ 25 Formerly nongraVId horn 0 5 10 15 20 25 30 35 Number of days postpartum FIGURE 11.14. Ultrasonically determined dia- meters taken at middle of each uterine horn. The diameter of the formerly gravid horn was signifi- cantly greater than for the formerly nongravid horn on each day. Adapted from (650). Parturition, Puerperium, and Puberty 483 finding raises questions about the role of the myometrium in expelling intrauterine debris. Perhaps contractions occur in bursts with long intervening periods of quiescence and therefore were missed. In a limited electromyographic study on the day of parturition, no consistent pattern of electrical activity was detected (670). In cattle, contraction frequency decreased from 12 to 18 contractions to 0 to 6 con- tractions over the first 48 hours postpar- tum (review: 1588). Thereafter, the uterus was quiescent. Postpartum uterus (n=10) 4 r at \‘iab Tone 3 2 X} bc 0 . O ‘\ U) +c 2 \‘icd “\ng *de Contractility ‘Je Fluid collections Diameter (mm) 10 0 2 4 6 8 10 12 14 Number of days postpartum FIGURE 11.15. Postpartum tone, contractility of the uterus, and diameter of intraluminal fluid col— lections. Within each end point, means with no com- mon superscript letters are significantly different. Adapted from (650). 484 Chapter 1 1 Lochial contents and uterine secre- tions. Postpartum luminal fluids involve about 500 ml of mucus-like material at 3 days and are gone by 6 days if involution progresses without complications (1685). Some discharge was seen in the vagina in 29% of mares at 2 days, 56% at 5 days, and 24% shortly before the first ovula- tion; the presence of discharge did not influence the pregnancy rate (908). It has been noted that the cervix did not close until after the foal heat and the secretion of progesterone (1855). Postpartum lumi— nal fluids are lost presumably through the cervix. Ultrasonic monitoring indicated that the number of mares with detectable uterine fluid decreased after five days postpartum (1068); none had detectable fluid after 15 days. This observation was confirmed in a more recent ultrasound study (650); a decrease began at 5 days with a significant decrease by 7 days (Figure 11.15). Only small collections (2 to 10 mm) were detectable at 9 days, and none were detected after 16 days. More interesting, however, was a pro- found increase in extent of fluid collec- tions between 1 and 2 days postpartum (Figure 11.15). Five of 10 mares had detectable intrauterine fluid collections the day after parturition compared to 10 of 10 at 3 days. This finding indicates that the fluid does not represent residual fluids associated with pregnancy but, instead, is a result of the involution pro- cess. The intraluminal fluids decreased in temporal association with decreased uterine tone and diameter. Perhaps, therefore, some of the fluid represented an influx of fluid from the uterine wall into the lumen. One group of workers reported a decrease in ultrasonic echogenicity of uterine fluid as involution progressed (1068), whereas other workers found that the intrauterine fluid had minimal echogenicity, and a significant echogenic change did not occur (650). The echogeni— city presumably represents inflammatory tissue debris. Perhaps the mares used in the former study had a higher incidence of postpartum endometritis (age of mares not stated) than the mares in the latter study (age: 4 to 12 years). Apparently, the fluid that enters the uterus after parturi- tion remains clear (nonechogenic) if the involution process is uncomplicated. In the absence of endometritis, the fluid of a normal uterus at 3 days is reported to contain few cells and leuco- cytes (1685). When neutrophils were found in the lumen, they were also found in the tissue (853). More neutrophils were found in uterine swabs taken at 5 days than in those taken at 2 days (908). In a tissue culture study (941), the pattern of pro— teins secreted by endometrial tissues 4 days after parturition was similar to the pattern during estrus; total protein secretion, however, was greater after parturition. Composition of uterine flushings has been studied using lysosomal enzymes to measure cellular damage, plasmin to determine proteolytic activity, and anti- trypsin to monitor leakage of plasma pro- teins into the uterus (914). Peak values occurred on 4 to 6 days but declined rapidly to low values by 16 days. Concen- trations of all substances examined were already low during the first estrus and during the time the embryo would arrive in the uterus. These uterine flushing and endometrial culture studies indicate that the uterus rapidly returns to a receptive condition. History of histology. The classic and extensive studies of Andrews and McKenzie (85) included histologic exami— nations of the involuting uterus. It was stated that the mucosa was restored by the third or fifth day of the first postpar- tum estrus in some mares but not until about the fifth day after the end of estrus in others. More than 30 years elapsed before the various aspects of uterine invo- lution were given further consideration. The initial studies (994, 1623), however, were secondary to other goals. One study (994) concluded that endometrial regener- ation is incomplete at the time of foal heat as judged by biochemical criteria. In apparent contrast to this conclusion and those of Andrews and McKenzie, other workers (994) concluded that histologic indications of considerable repair of the endometrium occurred by 5 days, post- partum, with only minor additional repair between 5 and 15 days. A report of an in-depth study on postpartum histo- logic changes of the endometrium did not appear until 1979 (1855), 38 years after the original report. Pennsylvania work- ers concluded that the uterus attains many of the characteristics of a pre— gravid uterus by the end of the first estrus. This report included a series of photomicrographs depicting the regres- sion of the microcaruncles. The 1979 report on the histology of the endometri- um during uterine involution was soon followed by many additional reports from Canada (1423, 126), Finland (853), and Pennsylvania (1419). Histology of endometrial crypts. The microcaruncles (endometrial crypts that served as receptacles for the fetal micro— cotyledons) are clearly evident on the day after parturition (126, 853, 1855). Diameter of caruncles during the first 24 hours has been reported as 500 to 800 mm (853). The majority of the micro- caruncles were empty, although a few contained debris and leucocytes. The microcaruncles disappeared rapidly (1419, 126, 853, 1855), usually by a noninflamma- tory process involving cellular lysis or shrinkage (1855); eventually the contents condensed and the crypts collapsed, resulting in overall shrinkage. The lumi- na were no longer apparent by 3 days postpartum (126). The opening to the caruncles became covered with epitheli- Parturition, Puerperium, and Puberty 485 um by 4 days (126) but was identified by a depression in the surface of the epitheli- um (853). By 6 or 7 days, the caruncles were no longer evident or were apparent only as condensed stroma beneath the epithelium (1419, 1855). Even the condensed stroma virtually disappeared by 9 days (126), but former sites were detectable as late as 15 days by the presence of lympho- cytes and siderophages (macrophages that clear up cellular debris; 853). It has been noted (1855) that the microcaruncles are located within the endometrium rather that as projections into the lumen; there- fore, they are not sloughed or lysed and do not contribute to the lochia, unlike the sit- uation in cattle and other species with macrocaruncles. Histology of uterine glands. The uterine glands were numerous and dilated on the first day (126, 1419, 1855). By 4 days (126), 10 days (1419, 853) or 11 days (1855), the dis- tention was gone, except for an occasional cystic gland. Detailed histologic study of the glandular cells has been done (853). Histology of cellular infiltration. It has been concluded that the endometri- um is histologically normal by 14 days, except for the sporadic occurrence of inflammatory changes and the presence of foci of siderophages (1855). According to Katila (853), postpartum cellular infil- tration (neutrophils and lymphocytes) may be considered a normal phe- nomenon. In almost all mares, the uterus becomes contaminated with bac- teria immediately postpartum (cited in 853) and the leucocytes probably play a phagocytic role. Siderophages were found in large numbers, especially near the sites of the former microcaruncles (853, 1423). Siderophages may persist for weeks and sometimes for months (1855, 864). Focal and diffuse infiltrations of lym- phocytes may indicate an inflammatory process and were seen in varying intensi- ties during 7 to 21 days (1419); these resolved spontaneously. 486 Chapter 11 Histology of luminal epithelium. There are conflicting reports on the extent of desquamation of the luminal epithelium (review: 853). In a recent study (853), it was concluded that if loss of epithelium does occur, it is only in small areas above the microcaruncles and is restored rapidly. In this study, height of the epithelium increased significantly after 2 days. Recent studies have raised questions about the reliability of earlier work regarding appar- ent restoration of desquamated surface epithelium (cited in 853). It is not clear whether loss of epithelium is due to endometritis (1685) or artifacts associated with tissue preparation (864). In one study (126), electron microscopy did not substan- tiate histologic indications of damage to the luminal epithelium. It is concluded that loss and restoration of luminal epithe- lium (excluding the area overlying the microcaruncles) has not been convincingly demonstrated as a normal postpartum phenomenon. Cilia on the luminal epithelial cells have been studied by electron microscopy (126, 1423). Ciliated cells began to appear near the opening of the uterine glands at 3 days, and their number increased rapidly over the next two days. A few secretory cells could be seen by 12 days. Hormonal aspects. Little is known about the hormonal aspects of uterine involution. Comparisons of ovariectomized and ovari- an—intact mares failed to find a role of the ovaries in involution based on histologic criteria (1419). Administration of proges— terone (994) or a combination of estrogen and progesterone (1423) for ovulation con- trol purposes did not result in detectable histologic effects on the rate of uterine involution. However, the study was designed to examine the effects of steroids on delaying estrus rather than to study their role in involution. There is a sugges— tion that the uterus of mares that have an early postpartum estrus may involute more quickly than those that do not (1380). Uterine lavage. Uterine lavage has been advocated as an aid to rapid uterine involution (cited in 213). Lavage at 2 and 4 days (1041) and at 3, 4, and 5 days (213) did not produce a beneficial effect by 7 and 11 days, respectively. End points over the two studies included endometrial histology (various histologic characteris- tics, including inflammatory—cell score), diameter of uterus, presence of free fluid in the uterine lumen, number of mares ovulating, endometrial cytology and cul- ture, and pregnancy rates. Late entry. The timing of postbreeding uterine lavage may be crucial in regard to effects on fer- tility (1858). Lavage at four hours post- breeding did not adversely affect preg- nancy rate, whereas lavage at 0.5 hours or two hours resulted in reduced rates. It was suggested that lavage at four hours allowed adequate time for sperm to enter the oviducts. Myometrial stimulants. Stimulation of the myometrium is another suggested approach to hastening involution. The PGFZa analogues, prostalene and alfaprostol, caused an increase in intrauterine pressure (indicated by bal- loon—type pressure transducers), begin- ning in 7 to 15 minutes and extending to the end of the experiment at 60 minutes (958, 956). The PGan analogue, clo- prostenol, and exogenous oxytocin also have been shown to stimulate the myometrium (627). Prostalene given twice daily for 10 days improved the pregnancy rates, compared to untreated controls (first estrus: 77% versus 44%; second estrus: 67% versus 29%; 958, 956, 957). These interesting findings need to be confirmed and the effect on various indi- cators of uterine involution should be examined. The entire area of postpartum uterine contractions and their role needs investigation; as noted above, ultrasonic observations have suggested that the postpartum uterus may be quiescent after placental discharge (pg. 483). 11.5E. Artificial Control of Postpartum Ovulation Delaying the first postpartum ovula- tion or inducing a new ovulation after uterine involution is complete have been advocated for reduction of problems asso- ciated with breeding during the foal heat. The goal has been to improve pregnancy rates (pg. 505) and to reduce embryo-loss rates (pg. 541). Steroids. Results of an initial, but lim- ited, study (994) suggested that delaying the first postpartum ovulation by daily administration of progesterone may improve pregnancy rates. More recently, Loy and associates (992) demonstrated that a combined daily treatment regimen of progesterone and estradiol beginning within 12 hours of parturition, effective- ly delayed the first ovulation. Delaying the postpartum ovulation by an average of five days did not result in increased overall reproductive efficiency in com- parison with the results of a conven- tional management system (mating most mares at the first postpartum estrus and the remainder as soon as pos— sible thereafter). A similar conclusion has been reached by other workers (170). Bristol and asso- ciates (253) used the same combination of steroids to synchronize ovulation. Treat- ment was given for 1 to 10 days to mares that foaled over a 10-day period; treat- ment ceased on the same calendar day, and the mating program began seven days later. Ovulation occurred within 10 to 16 days after cessation of treat- ment in 95% of the mares. The pregnan— cy rate was 81%. McKinnon and associ- ates (1068) obtained improved pregnancy rates by administration of a progestin beginning on the day after parturition. More became pregnant when ovulation was delayed until 15 days than when ' ovulation occurred before 15 days (82% versus 50%) Parturition, Puerperium, and Puberty 487 Prostaglandin analogues. The luteo- lytic efficacy of PGFZoc or its analogues in postpartum mares has been demonstrat- ed (915). An analogue of PGFZoc was tested in a major field trial in Germany (1623). Forty-four mares that had an ovulatory first estrus were treated on Days 6 to 10 after the first postpartum ovulation or at 20 days postpartum. Approximately 95% responded to treatment, and the preg- nancy rate was 82%. Sixty—eight percent of 36 mares that did not show foal estrus also responded to treatment at 20 days; pregnancy rate was 79%. Overall preg- nancy rate for the treated mares exceed- ed that of 101 control mares. In addition, the rate of pregnancy loss was 8% for treated mares and 17% for control mares mated at the foal heat. Results of this field trial indicated that the postponement of mating for approxi- mately 18 days by the treatment pro- gram greatly increased the pregnancy rates and reduced the pregnancy losses. The pregnancy rate in treated mares was unusually high, however, exceeding that of control mares mated subsequent to the first estrus. Confirmatory study is need- ed. Other workers (266) concluded that administering PGFZOL during the first luteal phase did not improve reproduc- tive performance. The German workers also confirmed an earlier observation (931) that the PGan analogue induced estrus and ovulation, even in mares with plasma progesterone levels below 1 ng/ml. The explanation for this phe- nomenon is not. available but may be related to gonadotropin-stimulatory effects (pg. 282). 488 Chapter 11 11,6, Puberty The changing gross morphology of the ovaries during 4 to 10 months (estimated by emergence of teeth) is shown in Figure 11°6A' Morphology of Develop mg 11.16; photomicrographs are shown in Ovaries Figure 11.17. 4...}: EliliililmlliHmHill!HHHIHHIIEHHIEY l 2 3 4 FIGURE 11.16. Ovaries of pony fillies obtained from a slaughterhouse. Age was estimated by emergence of teeth. Months in which the ovaries were obtained and estimated age of ponies are as follows: A) October, 4—6 months, B) May, 6-9 months, C, D) March, 6-9 months before and after sectioning, E) August, 6 months, and F) June, 10 months (note corpus luteum). Specimens prepared by J. A. Wesson. The ovaries change from the large sym- metrical structures of the fetus and new- born (Figure 9.44, pg. 398) to kidney-shaped structures with a prominent ovulation fossa at puberty. The mechanisms involved in the dramatic ovarian morpho- genesis have been studied by California workers (1727). The cortex becomes local- ized beneath the infundibulum by approxi- mately three months, indicating the future site of the ovulation fossa. An indentation Parturition, Puerperi‘um, and Puberty 489 develops in the bulge of cortical tissue, and the indentation, but not the rest of the cor- tical surface, is covered with surface ger— minal epithelium. The invagination of the cortex (formation of ovulation fossa) into the medulla appears to be directed by con- nective tissue restrictions; the connective tissue is reorientated at the edge of the fossa (junction of mesothelium and cuboidal epithelium). During the invagina- tion, the two ovarian poles are drawn {m w INTERSTITIAL CEL S & PRIMARY FOLLICLES FIGURE 11.17. Some histologic features of an ovary from a four-month filly: alnormal, 1 mm follicle; germi- nal epithelium in the ovulation fossa; primary follicles located in a band tangentially to the ovulation fossa; and large cells, which are apparently interstitial cells remaining from the fetal ovary. 490 Chapter 11 toward one another to form a distinct ovula- tion fossa by the time of puberty. The ovula— tory fossa apparently can be distinct as early as 5 or 7 months (Figure 11.16; pg. 16). The cut surface of the midsagittally sec- tioned prepubertal ovary is dark brown at the greater curvature and pale yellow at the depressed border (Figure 11.16). Brown coloration is probably due to remaining interstitial cells or debris; large cells, apparently interstitial cells or macrophages, are abundant in this area (Figure 11.17). The brown color fades grad- ually over several years. Follicular and luteal data for fillies that were judged to have been born in the spring are shown (Figure 11.18; 1759). On the average, the follicles did not exceed 10 mm during the first summer and win- ter. Considerable follicular development occurred between the first winter and the second spring. Luteal bodies were found in 2/16 fillies during the first summer, although it could not be determined whether ovulation occurred. All the fillies had distinct luteal structures or remnants by the second summer. 11.6B. Age of Puberty Age of puberty is generally given as approximately 1.5 years (1362); however, as reviewed below, 1.5 years are required only when the fillies are born early in the year or approximately one-half year before natural onset of the ovulatory sea— son in adults. Three mares born during November to January reached sexual maturity during the second spring at 16 to 17 months of age (1574). In a study of February-born Welsh ponies (1210), puber— ty occurred on approximately May 1 when they were 14 and 15 months of age. The effect of anabolic steroids on ovarian activity in mares in Australia was recent- ly reported (1492); puberty, based on increase in progesterone concentrations to >25 ng/ml, occurred at 12 to 13 months in the control fillies and in most of the steroid-treated fillies. In a slaugh- Ovarian activity in fillies Percentage with ‘ac'; luteal tissue 3 40 d.) m 0 Paired ’5, ovarian bc :7 20 weight .I: .9 é’ 0 Diameter largest follicle ab Diameter (mm) 3 B 0 Follicles 2-10 mm 10 I 2 o E 3 Z Follicles >10 mm be (30) Sp Su Fa Wi Season Sp Su Fa Wi FIGURE 11.18. Seasonal changes in ovarian end points in fillies 4 to 21 months of age. Number of fil- lies is shown in parentheses. Sp = spring, Su = sum- mer, Fa = fall, Wi = winter. Means on the same line with different superscripts are different (P<0.05). Adapted from (1759). terhouse survey, puberty was estimated to occur at 12 to 15 months of age (1759). In a recent study in Cuba (1340), puberty was reported to occur at 10 to 12 months in 48% of riding-type horses. Incidentally, a study in Arabian mares indicated a sig- nificant association between the age of puberty and subsequent fertility, as mea- sured by the number of services required per pregnancy (463). Parturition, Puerperium, and Puberty 491 TABLE 11.6. Characteristics of Puberty and the First Breeding Season in Yearling Pony Fillies (Mean i SEM) Month of birth during the previous year Item Apr Jun/Jul Adults Onset of ovulatory season Date Jun 8 i6.0 Jun 1 i115 May 15 Age (days) 411a $5.5 34110 i165 — — — Weight (kg) 140 i118 127 i6.5 - - - Length of ovulatory season (days) 11281 i196 52b $1.0 152 No. ovulations / season 4.3 i0.9 3.0 i1.0 7.2 End of ovulatory season (date) Aug 31 Jul 21 Oct 11 Within each end point for fillies, means with different superscripts are different (P<0.05). Fillies and adults are not contemporary. Adapted from (1760; fillies) and (573; adults). In a study in pony fillies (1760), foals were born in April or during June to September; the first ovulation (puberty) for all ponies born in April occurred dur- ing late spring or early summer of the fol- lowing year (Figure 11.19; Table 11.6). Three ponies born during June or July also ovulated the following spring, where- as two that were born in August or September did not ovulate the following year. The mean date of the first ovulation (puberty) did not differ between the ponies born in April and those born in June and July and did not seem different First ovulatory season E Age in ' ' ' Group I-L Apr ovx .12MO ti It 4r Vi May Jun Jul Aug Sep OI Izl- r + . 2 = *2 ? *2 * la- ‘ *2 i = E- s z 5 r s *5 + I i * * i * W 3 i i s OI Oct .12Mo * + *§ *W W Anovulatory —v——-——> I Anovulatory ——_——> from what has been reported for adults. A nutrition—oriented study (464) indicated that the onset of puberty based on first estrus in pony foals was delayed when growth between 6 and 12 months of age was retarded by a restricted diet; 10 of 13 well-fed fillies showed first estrus in April (approximately 12 months of age), whereas 10 of 10 fillies fed a restricted diet showed first estrus during May to November. In conclusion, fillies that are born dur- ing the first half of the year and are well nourished can be expected to reach puber— ty (first ovulation) by spring of the next Nov Dec FIGURE 11.19. Occurrence of estrus (black bars) and ovulation (arrows) in six . , spring-born and five late- ; born yearling fillies and ' ' occurrence of estrus in three ovariectomized spring-born fillies. Arrows were placed on the last day of an ovulato- ry estrus or on the day of an ovulatory LH surge. OVX = ovariectomized; 01 = ovarian intact. Adapted from (1750). 492 Chapter 11 year at the beginning of the ovulatory season in adults. Health and puberty. An anecdotal account (600) suggests that at the time of seasonal transition fillies are reproductive- ly sensitive to events affecting health. Nineteen Quarter Horse fillies were pur- chased in March and April when they were 9 to 12 months old. Although maintained in good body condition, 13 fillies (68%) were recorded during June as having strangles (suppurative lymphadenitis in the sub- mandibular region). Ten of the 13 (77%) had a corpus luteum or a preovulatory- sized follicle in early June but were in an anovulatory condition (no corpus luteum or large follicle) in late July. Researchers intending to study fillies during puberty or the first ovulatory season, therefore, are advised to procure the animals well in advance of the anticipated experimental period and to maintain good body condition and health. Puberty in feral fillies. Direct studies on the age of puberty in feral mares apparent— ly have not been made. However, in a study of feral ponies, mares less than three years old did not foal, and the foaling rate of three—year—olds was only 23% (861). Similarly, in another study (1413), pregnan- cy rates (based on hormone levels) increased from 36% in two-year-olds (corre— sponds to foaling rates in three—year-olds) to about 85% in mares that were 6 to 15 years old. These indirect indicators of puberty assume that the fillies would have been mated during the season that they reached puberty. In addition, feral animals may not receive adequate nourishment. Furthermore, in contrast to feral fillies, most domestic fillies are born during the first half of the year and many before the natural onset of the ovulatory season. Therefore, domestic fillies would be more likely to ovulate as yearlings. Nature of first ovulatory season. The characteristics of the first ovulatory season apparently differ among spring— born fillies, summer-born fillies, and adults. As indicated above, the time of onset of the ovulatory season seemed simi- lar among these three groups (Table 11.6; Figure 11.19; 1760). However, the groups differed in length of the ovulatory season (significant difference between the two groups of fillies), number of ovulations per season, and date of ending of the season in the following decreasing progression: adults, spring-born fillies, summer-born fil- lies. The comparisons between spring-born and summer-born fillies were limited by small numbers, and the comparisons to adults were made by inference using data from a previous study (573). Therefore, these findings are considered preliminary. However, in a slaughterhouse survey (1759), fewer young mares (<5 years) were ovula- tory in the fall months (September to November), compared to mares 25 years (pg. 111). The observations from these stud- ies are rationale for the hypothesis that age has a pronounced effect on month of termi- nation of the ovulatory season but not on month of onset, provided fillies are approxi- mately 12—months old and in good health and body condition at the beginning of the ovulatory season in adults. 11.60. Gonadotropins Newborn. Concentrations of LH and FSH are low (<2 ng/ml) in the newborn (1758, 1756). However, concentrations of LH in females were significantly higher on the day of birth than on the following day (1758, 1756) and were higher in females than in males on the day of birth (Figure 11.20; 1758). Concentrations of FSH did not differ between genders or over the first nine days postpartum. It is not known whether LH in the fetal blood at birth is of fetal or mater- nal origin. However, maternal origin seems unlikely since concentration in maternal blood at parturition in other studies (1645) seems less than that found in the newborn. In addition, the half-life of LH is too short (pg. 45) to be compatible with maternal ori- gin. Nevertheless, maternal or placental steroids could have influenced the produc- tion of LH in the newborns. An effect of gender of the newborn on LH levels indi— cates a need for consideration of fetal gen- der in studies involving hormone levels in FSH & LH in newborn (n=5/group) 1.5 Male FSH 12 \ --""L 2 xi ---------- l E 08 I" E7 v 04 c \ .g LH S 0.0 E (D O C 8 1.2 0.8 0.4 0.0 1 2 5 9 Age (days) FIGURE 11.20. Plasma gonadotropin concentra- tions from birth to nine days of age for male and female foals. Mean LH concentration on the day of birth for the females was significantly higher than on any other day and was higher than on day of birth in males. Adapted from (1758). FSH & LH in fillies (n=4 to 9) Parturition, Puerperium, and Puberty 493 pregnant and periparturient mares, as well as in the late fetus and neonate. The role of fetal gender in differential development of fetal and placental steroidogenesis appar- ently has escaped research attention, except that it has been noted (669) that the production of estradiol-17B was not altered by fetal gender. LH prior to first ovulatory surge. Concentrations of LH in both intact and ovariectomized spring-born fillies remained low until the increase that presaged the ovulatory surge the following spring (Figure 11.21; 1756). This result contrasts with those for other farm species which have considerable LH during most of the prepubertal period. Similarly, in summer- born (July and August) Quarter Horse fil- lies, LH levels were undetectable for the first seven months (1130). However, a tran— sient increase occurred at 10 months (equivalent to the beginning of the ovulato- ry season in adults). FSH during the first year of life. Concentrations of FSH in fillies born in May increased during June and July, remained high in the summer, decreased in the fall, and increased again during late December to February (Figure 11.21, 1756). The decline in the fall is intriguing and apparently does not occur in other farm species. The FSH concentrations in E 2’ W. w W 8 ‘ .WX FSH , Xv; w FIGURE 1121. Concentration of .fi .‘ / [I illixy LH and FSH in pony fillies born 5 ‘, ,' in May. Four of the nine fillies : l wx . . . 8 \ Xyz z ’i yz were ovariectomized in August, 8 s‘ yz / ‘y ’ but data were combined since this 0 ‘|~ Z/lyz ‘yz procedure did not significantly May Jun Jul Aug Sep Oct Nov Dec affect the gonadotropin levels. H Within each isolated segment of data, means with no common let- ters are significantly different. Jan Feb Adapted from (1756). 494 Chapter 1 1 fillies seemed to follow a seasonal pattern similar to that of ovariectomized adults, whereas, as noted above, the LH concen- trations remained low and constant; these results suggested separate regula- tory mechanisms for the two gonado- tropins. It has not been determined whether the FSH changes are due to sea- sonal factors or to maturational processes of the hypothalamo-hypophyseal area. Ovariectomy in August (age: 4 months) did not alter the concentrations of either gonadotropin and indicated the absence of ovarian feedback effects at this time. In contrast, ovariectomy of adults in the summer is followed by an immediate increase in both gonadotropins (pg. 121) Colts versus fillies. Plasma gonado- tropin levels in male foals differed from female foals in the following ways (1758, 1761): 1. LE was lower on the day of birth; 2. FSH concentrations remained con- stant during the first summer and did not increase as in the fillies; 3. LH concentrations increased slightly during the first summer, whereas in fil— lies the concentrations remained low; 4. Gonadectomy in the fall at 4 months of age was followed by an increase in both FSH and LH, unlike the absence of an effect in fillies; and 5. LH response to GnRH given before 70 days of age was positively correlated with age in colts but not in fillies (1763). Apparently colts during the first sum— mer develop a gonadal feedback effect on the pituitary-hypothalamic axis, whereas fillies do not. The colts, in this regard, had changes in testosterone levels in con- junction with the LH changes. The pro— duction of steroids and proteinaceous con- trolling factors in the prepubertal filly needs to be investigated. First ovulatory season. Changes in plasma concentrations of FSH and LH in fillies preceding (Figures 11.22 and 11.23), during, and immediately after the first ovulatory season (1760) were similar to those reported for adult ponies. Concentrations of LH remained low in both intact and ovariectomized fillies until mid-March and then began to increase. A small but significant increase in LH concentrations began 24 to 32 days before the first ovulation. A similar increase has not been reported for adults. Although samples were taken only once per day, small apparent surges of LH were observed at sporadic intervals dur- ing approximately one month preceding the first ovulation. Similar LH surges, sometimes followed by a transient proges- terone surge, have been reported in other species (cited in 1760). Progesterone was not detected subsequent to the small LH surges in fillies, but this possibility was not intensively investigated. Studies are needed on the pulsatility of gonadotropin patterns preceding puberty. Because LH concentrations begin to change concurrently in both ovarian- intact and ovariectomized fillies, environ- mental factors likely are involved in the sequence of events leading to the first spontaneous ovulatory LH surge at puberty, as has been hypothesized for control of the onset of breeding season in adult mares (pg. 254). The ovulatory LH surge differed depending on whether it was the first, middle, or last surge of the ovulatory season, similar to what has been reported for adults. Retarding effect of artificial light. The effects of fixed 9-hour and 16-hour pho- toperiods on puberty have been studied (1762). Light treatments began on Dec— ember 17 in fillies 6 to 8 months old and extended to the following August. Hair shedding was hastened in the 16—hour group and delayed in the 9—hour group, compared to controls; in this regard, fil- lies responded as would adults. How- ever, the proportions of fillies with one or more ovulations (puberty) by the end of the project were 2 of 4, 2 of 4, and 5 of 5 in groups receiving 9 hours, 16 hours, and control lighting. The hypothesis that a fixed daily photoperiod would hasten puberty was not supported. Instead, the Gonadotropins preceding puberty _L co FSH —L A |—.._| U) Q. ‘s 3—— I "— ‘ + . ~ . . —-$ Ovariectomized I \,.I., fill, :11 E U) 5 7 C .9 E 3 l Ovarian intact E (n=5) fl.) 0 C o O Apr May FIGURE 11.22. Plasma gonadotropin concentra- tions in ovarian-intact and ovariectomized spring— born fillies during February to May of the following year. Concentrations of FSH increased earlier in ovariectomized fillies, indicating the presence of FSH-suppressing factors in the ovarian-intact fil- lies. The lsd bars indicate approximate differences required within days or between groups for a signif- icant difference. Adapted from (1760). 16-hour light treatment interfered with the attainment of puberty, as indicated by the reduced proportion of fillies ovu- lating; reduced numbers of ovulations, luteal bodies, and estrous periods per filly; extended intervals from the begin- ning of the project to the first ovulatory estrus; and shorter period of estrus. Puberty also appeared to be retarded in the 9—hour group, but the results were less pronounced than in the 16—hour group. Thus, the pineal—hypothalamic- pituitary-gonadal axis apparently did not develop normally in fillies that were denied normal changes in length of pho- toperiod during the winter preceding puberty. In contrast, in other long-day breeding species, constant long photope- riods stimulate onset of puberty (cited in 1762). Parturition, Puerperium, and Puberty 495 FSH & LH at puberty (n=8) Concentration (ng / ml) -68 -52 -36 -20 -4 12 Number of days from ovulation (puberty) FIGURE 11.23. Plasma gonadotropin concentra- tions at four—day intervals in fillies in association with the first ovulation of the year (puberty). Concentrations of LH began a gradual upswing 32 days before ovulation with a surge four days before ovulation. Data are normalized to the day of maxi- mum LH. Adapted from (1760). The phenomenon of puberty retardation when fillies are exposed prematurely to long photoperiods may account for failure of fillies born in late summer and fall to ovulate during the following summer. Such fillies would be exposed to increasing daylength when approximately only 6 months old. Early exposure may preclude ovulation at 12 months of age and thereby delay puberty until the following spring when they would be about 18 months old. Thus, a light—controlled puberty retarda— tion phenomenon may have evolved in late- born fillies so that puberty does not occur during the following year. It is not known whether disturbing the sexual maturation process by early light exposure (natural or artificial) has long-term effects that would carry over into the adult years. It seems prudent, in the absence of such informa- tion, to avoid exposing fillies to the light- stimulation programs used for adults. 496 Chapter 11 ‘ SUMMARY: Gonadotropins from Birth to Puberty, Luteinizing Hormone —— Ovarian intact Ovariectomized Follicle Stimulating Hormone ———- Ovarian intact Ovariectomized SOQOOOOOOOOOQ 0 May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May ‘Jun FIGURE 11.24. Working hypothesis on apparent FSH and LH interrelationships from birth to puberty for fillies born in the spring months and reaching puberty (first ovulation) the following spring. 0 Except for slightly higher levels at birth, LH levels remain low or unde- tectable until a gradual incline during the month preceding the first ovulatory LH surge. The low levels for the first 11 months of life do not involve the ovaries; levels are similar for ovarian-intact and ovariectomized fillies. 0 In contrast, FSH levels rise Within a month after birth and are high through- out the first summer and low during the Winter. The high summer levels are not influenced by the ovaries, nor do the ovaries appear to respond to the high lev— els; follicular activity is apparently mini- mal during the first summer. Thus, the first summer and Winter appear to be a period of pituitary maturation and devel- opment of responsiveness to photoperiod- ic influence Without ovarian modification. ' Concentrations of FSH during the interval between the Winter solstice and puberty are similar to those for the resur- gent phase of the anovulatory season in adults (summary: pg. 157). 10. 11. 12. 13. 14. Parturition, Puerperium, and Puberty 497 HIGHLIGHTS: Parturition, Puerperium, and Puberty Imminence of birth can be assessed by testing mammary secretions for calcium and magnesium using test strips developed for water hardness. Mares foal predominantly at night (e.g., 86%). The three stages of labor culminate in rupture of allantochorion (lst stage), expul- sion of foal (2nd stage), and expulsion of placenta (3rd stage). The allantochorion is usually discharged inside out (e.g., 80% of parturitions). Placenta expulsion should occur rapidly (mean: within one hour). Fetal adrenal glands may be involved in parturition, as indicated by higher levels of cortisol in the umbilical artery than in the umbilical vein. Massive doses of dexamethasone will induce parturition. PGFZa likely plays a role in myometrial contractions. Levels increase abruptly at parturition, and surges may occur on the following days. Limited study has been done on oxytocin concentrations at parturition. Exogenous oxytocin can be expected to induce parturition within one hour. Progestins are increasing and estrogens decreasing during the last two weeks and both decline precipitously at parturition. However, during the last day, progestins apparently decline, whereas estrogens do not, thereby increasing the estrogen:progestin ratio. Most mares (e.g., 57%) in breeding programs are of postpartum status. The first postpartum ovulation occurs after nine days in most mares (e.g., 82%). The mean for an extensive study was 10.2 days. Some mares do not have a postpartum ovulation or revert to ovarian inactivity after the first ovulation. This phenomenon seems more dependent on season than on the influence of lactation. A surge of FSH begins a few days before parturition and peaks just before or on the day of parturition, accounting for postpartum follicular development. The postpartum uterus involutes rapidly (e.g., luminal fluid nondetectable within 15 days and similar size of the uterine horns by 23 days). The first ovulation for spring-born fillies in good condition occurs during the next spring at the same time that adults first ovulate. However, the first breeding sea- son (interval between first and last ovulations) is longer in adults. 498 Chapter 1 1 MILESTONES: Parturition, Puerperium, and Puberty 1941 Early study on uterine involution (85). 1960s Documentation that foaling occurs primarily at night (cited in 1363). 1963 First inductions of parturition in mares With oxytocin (255). 1972 First study on the effects of removal of the foal on characteristics of the post- partum period (623). 1973 Measurement of peripheral levels of oxytocin during parturition (37). 1973-79 Reports from many laboratories on the concentrations of circulating hor- mones during the postpartum period (985, 762, 1141, 1142, 1164, 1645). 1975 Detailed account of morphology of terminal fetal placenta (1770). 1975 Several initial reports on hormonal interrelationships during parturition (1133, 985, 550, 156). 1976 Induction of parturition with a PGan analogue (1357). 1978 Discovery of the parturient FSH surge (807, 1650). 197 9-80 First studies on circulating LH and FSH in prepubertal fillies (1758, 1756). 1980 Extensive study of characteristics of the postpartum period (989). 1981-82 First studies of the ovulatory and gonadotropin patterns during the first ovulatory season in yearlings (1759, 1760) and the effects of light programs on onset of puberty (1762). Development of treatment regimen consisting of progestin and estrogen com- binations for delay and synchronization of the postpartum ovulation (992). 1984 Development of prepartum evaluation technique using test strips that mea- sure calcium and magnesium in mammary secretions (1191, 939). 1987-89 Initial studies on effects of body condition on parturition and postpartum period (733, 919, 920). 1987-90 Studies on role of GnRH in gonadotropin release during pregnancy and the postpartum period (1147, 685, 1148). —Cfiapter 12— REPRODUCTIVE EFFICIENCY This chapter will emphasize those aspects of reproductive efficiency that would interest reproductive biologists. Other reports and reviews can be con- sulted for detailed information on repro- ductive pathology (1056), infectious dis— eases (1289), and evaluating and treating infertility and endometritis (1004, 280, 544, 455, 109, 776, 108). The reputation of horses. Horses have a reputation for having the lowest repro- ductive efficiency among farm animals. A misleading but commonly used figure is a live annual foaling rate of 50% to 60%; a more reasonable figure is 80% (pg. 508). Reproductive patterns and mechanisms in mares differ greatly from those of other farm species, and there seems to be a tendency to attribute poor reproductive efficiency to these unusual reproductive traits (e.g., seasonal ovulatory pattern with prolonged estrus prior to the first ovulation, sporadic estrous behavior dur- ing the anovulatory season, a large ratio of estrus-to-diestrus length). Although mare reproductive patterns are unusual, they represent the evolution- ary effort of the species and are compati- ble with appropriate reproductive efficien- cy in the natural state—an efficiency that has counterbalanced all opposing pres- sures against species survival. The repro- ductive pattern of each species has evolved over millions of years under meaningful selection mechanisms. Dom- estication, however, has brought an array of negative influences on reproductive efficiency that was not involved in species evolution. These influences vary widely and profoundly among species, among breeds and types within a species, among farms within a breed, and among individu- al animals within a farm. Comparisons of reproductive efficiency among species and among breeds, therefore, would require consideration of the complex differences in domestication and husbandry. Artificially imposed negative pressures on the reproductive efficiency of horses include the following: 1) incompatibility between imposed and natural breeding seasons, 2) near absence of positive selec- tion of breeding stock on the basis of reproductive efficiency, and 3) the some- times great disparity between cost of maintaining an animal for a year versus the potential income of an offspring; thus, the efficiency records may include ani- mals that were mated despite known physical, medical, or geriatric problems. If the poor reproductive reputation of horses is deserved—and it probably isn’t—it would be more attributable to economic pressures of the horse industry than to species differences in reproduc— tive biology. Progress in improving reproductive efficiency. A German analysis of records for the Hanoveri‘an breed from 1815 to 1973 indicated that the foaling percent— ages remained fairly constant (50 to 60%) with no improvement in reproductive effi- ciency (1084). However, a recent examina- tion has been made of the annually reported data for Thoroughbreds in Britain (1326). The reported per season pregnancy rate and live-foaling rate increased about 10 percentage points, and the barren-mare rate decreased cor- respondingly over the 19 years of the 500 Chapter 12 study (Figure 12.1). Comparison of live- foaling rates from farm records reported in the first edition (71% to 73%) with those reported in the 1980s (76% to 84%; pg. 508) also suggests an increase in repro- ductive efficiency. It appears that there was no improvement in reproductive effi- ciency during the first 70 years of this century, followed by an impressive 10% improvement during the past two decades. Research in horse reproduction began a gradual upswing in the late 19608 and has involved an increasing number of research laboratories. In addition, dis— semination of knowledge and interest in equine reproductive biology has been expanding with the emergence of new equine journals and conferences for farm operators, veterinarians, and scientists. Presumably, the apparent improvements in reproductive efficiency are due, at least in part, to the increased research activity and reporting. Progress in reproductive efficiency 70 Pregnancy rate .9 ..-. . '.O-.“ 60 “(.1 o '7‘. P Q "e_ .‘-..:\ _ L .1 ‘ Foaling rate 0) I 2 g 50 Barren rate 1970 1974 1978 1982 Year 1 986 1 990 FIGURE 12.1. Increases in reproductive efficiency per year over a 19-year span in Thoroughbred mares in Britain based on examination of stud— books. Adapted from Ricketts and Young (1326). 12.1. Terminology Absence of standard measuring criteria and terminology complicates quantitating and communicating reproductive efficien- cy. The pitfalls of confounding factors and the communicability of terminology must be considered carefully in attempting to produce meaningful studies and reports. A glossary of terminology for reproductive efficiency is given in Table 12.1. Pregnancy establishment and foaling success. The paramount event in the establishment of an individual is fertiliza— tion—so much so that the fertilization phenomenon embodies the very essence of the word “reproduction” (pg. 347). Here, fertilization rate will be used to describe the frequency with which ova are fertil- ized or the probability that an ovum will be fertilized when the natural or opera- tional conditions exist for expected fertil- ization (number of ova fertilized divided by the number of ovulations x 100). This definition assumes that an oocyte will be released during each ovulation. Conception rate seems a synonym of fer— tilization rate but unfortunately is often used as a synonym for pregnancy rate at any defined or undefined time of pregnan- cy diagnosis. To minimize confusion, the term conception rate should be avoided and will not be used here. Pregnancy rate (the number of mares pregnant divided by the number mated x 100) is being used with apparent increasing frequency and requires a crite- rion for establishing that a conceptus is present. It is most important that the term be defined or accompanied by the day of pregnancy diagnosis (e.g., Day 15 pregnancy rate). To be especially in- formative, a definition of “day” is also needed (e.g., Day 0 = day of ovulation). Foaling rate (number of mares foaling divided by the number mated x 100) is usually in reference to a live foal and is a widely used overall criterion of reproduc- tive efficiency. A point of confusion that can be readily precluded by authors is Reproductive Efficiency 501 TABLE 12.1. Terminology for Describing Reproductive Efficiency Representative Terminology Definition farm rates 1. Fertilization rate No. ova fertilized + N o. ovulations 90% 2. Pregnancy rate No. pregnant on a specified day (e.g., Day 20) + No. mated a. On basis of an ovulatory period 55% b. On basis of operational season 85% 3. Conception rate Often used for pregnancy rate, but should be avoided 4. Foaling rate No. foaling + No. mated 80% a. Usually on basis of operational season b. Sometimes specified as live-foaling rate 5. Pregnancy-loss rate No. not foaling + No. pregnant on specified day (e.g., Day 20) 18% 6. Embryo-loss rate N 0. not pregnant at end of embryo stage (e.g., Day 40) + 6% No. pregnant on a specified day (e.g., Day 20) 7. Fetal-loss rate No. not foaling + No. pregnant on a specified day (e.g., Day 40) 12% a. May or may not include stillbirths 8. Stillbirth rate 9. Abortion 10. Embryo resorption Fetal loss rate near term (e.g., 2300 days) 2% Confusing term that should be defined in each context Misleading term that should be abandoned for routine use that pregnancy rate and foaling rate can be used in reference to either an ovulatory period or an operational breeding season. Here, unless otherwise specified, pregnan- cy rate will be used in reference to an ovu- latory period and foaling rate in reference to the breeding season. Pregnancy loss. Measuring and com- municating the rate of pregnancy loss also require care. Pregnancy loss implies that a positive pregnancy diagnosis was made, but the diagnosis was negative at a later examination, or a foal was not obtained. It is important to recognize that all losses occurring after successful fertil- ization are reflected in subsequent preg- nancy rates as well as in embryo-loss rates. That is, factors affecting either one of these measures of efficiency after fertil- ization automatically affects the other. Pregnancy-loss rate must be used in refer- ence to specified days of pregnancy diag- nosis; that is, to encompass a given period of pregnancy (e.g., Days 20 to 40 loss rate). Embryo-loss rate refers to losses occurring after fertilization (if the fertilization rate is known) or after a specified day of pregnan- cy diagnosis but before the end of the embryo stage (herein, Day 40; pg. 345). Fetal-loss rate refers to losses during the fetal stage and may or may not include stillbirths (losses occurring toward the end of gestation). The term, therefore, needs clarification in each communication. One investigator recommends the term still- births for fetal deaths after 299 days (2300 days; 1769). Embryo resorption is a term that has been used extensively, even in recent reports, but should be abandoned and not used as a synonym for embryo loss. Apparently most, if not all, of the embryon- ic vesicle or its debris is discharged through the cervix during the ensuing estrus (pg. 544). The term abortion is also confusing, and its use must be defined in each context. The term has been used for observed discharge of a conceptus late in pregnancy, observed or unobserved loss during the fetal stage but not the embryo stage, and loss of pregnancy at any time. 502 Chapter 12 Bias and confounding. The above mea- sures of reproductive efficiency are highly subject to bias; that is, the operator may be inclined (consciously or subconscious— ly) to push the rates in either direction. For example, the number of mares mated can be reduced and pregnancy rate there- by increased by deleting mares that were mated but failed to ovulate or later devel- oped endometritis. Researchers must be especially diligent and consistent in this regard. Blatant confounding occurs when attempts are made to compare groups that were not randomized (e.g., control group and treated group) or when groups (e.g., young group versus old group) are not handled the same except for the fac- tor (age) under study. For example, under farm conditions, old mares may be more rigidly culled or may be bred to less fer- tile stallions, giving the old group an advantage or disadvantage, respectively. Under farm conditions, confounding usu- ally cannot be eliminated or adequately minimized. Field reports should not, of course, be discounted on this basis. They represent important observations that encourage and provide rationale for hypothesis building and testing by con- trolled experimentation. In this regard, clinical studies and observations over the past few decades have provided a wealth of background material on reproductive efficiency—the danger is in the tendency to exaggerate the inherent reliability. 12.2 Measures of Efficiency 12.2A. Fertilization Rate Direct measurements. Estimates of in vivo fertilization rates have not been made in the strict definition of the term. Estimates of fertilization rate made at Day 2 or Day 4 on the basis of number of apparently cleaved ova, for example, rep- resent the fertilization rate minus the number of embryo losses before the indi- cated day. The sequential structural changes after death of fertilized and cleaved ova are not known; this factor complicates attempts to classify recovered structures. Furthermore, an investigator does not know whether failure to recover a structure is due to inadequate tech- nique or an aberrant biologic process. The only estimates now available on fer- tilization rates are the Day 2 and Day 4 pregnancy rates obtained by Woods and associates (146, 1255, 147). The oviducts of Standardbred and Thoroughbred mares were flushed after surgical excision. The mares were young (means: 4.9 and 5.7 years) and were judged to be reproductive- ly normal. The fertilization rates for recov— ered ova were 91% (10 of 11), 100% (21 of 21), and 96% (27 of 28) in three experi- ments (combined mean: 97%, 58 of 60; Table 12.2). Oocytes were not recovered in 8 of 68 mares and fertilization status was not determinable. The high fertilization rates were obtained under research condi- tions with an optimized protocol. The fer- tilization rates under farm conditions is unknown; differences between research and farm conditions are discussed below. Fertilization rates of >90% have also been reported in cattle (1528). The Day 2 pregnancy rates in mares with a history of subfertility were also high (92%, 12 of 13 recovered oocytes). As shown in the table, the subfertile mares were much older (mean: 17.4 years) than the mares judged to be normal. High fer- tilization rates in old mares are surpris— ing in view of the detrimental effects of old age on oocytes, as determined from studies in laboratory animals (pg. 541). Because of the fundamental nature of these findings, it is imperative that con- firmation is obtained, especially in regard to the effects of age. Calculated estimates. Some investiga- tors have used the distribution in length of the interovulatory intervals in mated mares to help distinguish between fertil— ization failure and embryo death. An interval of 30 days or less was used to indicate fertilization failure in one study (123). This approach does not distinguish Reproductive Efficiency 503 TABLE 12.2. Embryo Recovery Rates on Days 2 and 4 in Separate Experiments Ova-recovery rate Cleaved-ova rate Mean (No. recovered (No. cleaved Experiment Reproductive age No. ova + No. ova + No. (Reference) Day classification (years) mares ovulations) recovered ova) Exper. 1 2 Normal 4.9 14 11/15 (73%) 10/11 (91%) (146) Subfertile 17.4 14 13/17 (76%) 12/13 (92%) Exper. 2 4 Normal (3 to 13) 23 21/23 (91%) 21/21 (100%) (1255) Exper. 3 4 Normal 5.7 25 28/30 (93%)a 27/28 (96%)a (147) Subfertile 19.4 37 27/41 (66%)b 22/27 (81%)b Percentages with a different superscript within each end point are significantly different. between fertilization failure and embryo death occurring before the embryo or its remnants would block the uterine lute- olytic mechanism. That is, the length of the interovulatory interval presumably would be similar among nonmated mares, mares with fertilization failure, and mares with embryo loss before Day 11 and without other associated factors that would effect cycle length (pg. 522). Furthermore, an interovulatory interval of normal length (or reduced length in the event of endometritis) can occur despite the presence of a growing embryonic vesicle during late diestrus and estrus (pg. 544). The mathematical approach, based on length of interovulatory inter- vals, therefore, is inadequate for calculat- ing fertilization rate or, conversely, fertil- ization-failure rate. 12.23. Pregnancy Rate Pregnancy rate per ovulatory period can be used for monitoring reproductive efficiency during the breeding season. The rates will vary among farms, depending, for example, on the extent of selection against individual mares and stallions with poor histories or examination results. It is important to retain the concept that the pregnancy rate on a given day (e.g., Day 20) reflects previous loss as well as failure to establish pregnancy (fertiliza- tion failure). Nonpregnancy in reproduc- tively normal mares is attributable as much to embryo loss before the initial pregnancy diagnosis as to failure of fertil- ization, assuming adequate semen and operational conditions (pg. 296). Early reports. Reports on farm preg— nancy rates before 1979 were tabulated in the first edition (575). An important source of incongruity within and among reports was the inconsistency in the day of preg- nancy diagnosis. Pregnancy rates and subsequent pregnancy losses can be expected to be greater the earlier preg- nancy is diagnosed. Nevertheless, the pregnancy rate averaged over all tabulat- ed reports was calculated to obtain an overall estimate that included possible dif— ferences among breeds, operational condi- tions, countries, and other factors. The reported pregnancy rate per ovulatory period averaged over all surveys was 48%. Recent reports. A few large surveys of farm records were reported in the 19805. Pregnancy rates per ovulatory period were given as 53% (England, 1390), 54% (USA, 579, 1810), and 56% (USA, 607). Much higher pregnancy rates can be found in research reports, often exceed- ing 80% (e.g., 85%, 602; 84%, 1068). The higher rates can be attributed primarily to use of fertile stallions, early diagnosis (e.g., Day 11), and selection of young mares or mares with good fertility histo- ries or freedom from indications of repro- ductive problems such as endometritis 504 Chapter 12 (e.g., based on results of endometrial biopsy or ultrasonic scanning). Some research studies, for example, utilized ultrasound scanners to select day of mat- ing and to exclude mares with failure of ovulation and with collections of intrauterine fluid during the preceding diestrus (endometritis). In addition, stal- lions or semen of known high fertility may be selected; in some experiments, the semen from several stallions was mixed in an attempt to improve fertilization rates. In contrast, under farm conditions, attempts may be made to establish preg- nancy in mares that have a low likelihood of success or to use an economically valu- able stallion with known fertility prob- lems. It is recommended, therefore, that breeding farms should strive to exceed a 55% pregnancy rate per ovulatory period over all mares mated but should expect to exceed 80% for optimally mated mares with good reproductive histories or examination results, especially when the initial pregnancy determinations are done early. Differences among breeds. There are indications of different pregnancy rates among farms with different breeds. In one study (792), a higher pregnancy effi- ciency was found for Quarter Horse farms than for Thoroughbred and Standardbred farms. In another study (108), Quarter Horse farms (n=5) significantly exceeded Thoroughbred farms (n=7) in pregnancy rates per ovulatory period (51% and 43%, respectively) and in cumulative or season- al pregnancy rates after five ovulatory periods (85% and 77%, respectively). Although both groups of workers found breed differences in reproductive efficien- cy, they emphasized that the concept of “breed” encompassed management and environmental variables as well as intrin- sic factors. Unless real breed differences in fertility are demonstrated in controlled studies, it seems best to refer to differ- ences among farms of various breeds rather than to differences among breeds alone. Effect of reproductive status. An impor- tant factor affecting pregnancy rates is reproductive status of the mare (maiden, barren, lactating). Some reported findings are listed in Table 12.3. Pregnancy rate was diminished by approximately 10 per- centage points in barren mares as com- pared to lactating mares. As discussed in the next paragraph, pregnancy rates in lactating mares can depend on the proportion of mares mated at the first postpartum estrus. The barren-mare clas- sification includes mares that failed to become pregnant or aborted during the previous year in addition to mares that were not mated, so this classification will TABLE 12.3. Some Reported Effects of Reproductive Status on Pregnancy Rate per Ovulatory Period Maiden Barren Lactating Reference Breed No. % No. % No. % (1 5 78) Thoroughbreds 74 38% 2 17 34% 346 5 1% Quarter Horses 128 48% 233 46% 429 56% (1390) Thoroughbreds 543 53% 1255 48% 2855 56% (1810) Standardbreds 132 58% 293 47% 690 56% Mean percentage 49% 44% 55% Significant difference among statuses for all studies. likely have a disproportionate number of problem mares. It has been reported that the number of ovulatory periods required before pregnancy occurred was greater in barren and maiden mares (85). Pregnancy rates for maidens seemed low in two of the tabulated data sets but were equiva- lent to the rates in lactating mares in the two other sets (Table 12.3). Decreased pregancx rate for the first postpartum estrus. Fertility associated with the first postpartum estrus is a sub- ject of much importance. Failure of a mare to become pregnant at this time can mean considerable delay since the first postpar- tum estrus may be followed by a greatly prolonged interestrous interval. Many reports agree that pregnancy rates are lower for mares mated during the first postpartum estrus than for mares mated during a subsequent estrus. A signifi- cantly lower pregnancy rate was obtained for mating at first estrus versus a subse- quent estrus in 6 of 9 studies that were tabulated in the first edition (575), and no study showed a significant difference in the opposite direction; the significant dif- ferences ranged from 11% to 34%. The results of an extensive study are depicted in Figure 12.2; pregnancy rate for the first postpartum estrus was 17% lower than for the subsequent estrus. More recently, mares mated at the first estrus versus the second estrus had preg- nancy rates of 39% and 55% (962) and 47% and 67% (908) and foaling rates per ovulatory period of 29% and 49% (123). However, the foaling rates for the season were the same whether the mating program began at the first or second estrus (962). It is emphasized that most reports on the pregnancy rate for the postpartum estrus come from data accumulated in the field, rather than from controlled experi- mentation. It is often not possible to judge the extent of bias in the comparisons between the first and subsequent estrous periods. For example, mares in which problems were anticipated may have been Reproductive Efficiency 505 Postpartum estrus 70 (211) (150) I First mated at A 50 ist estrus s: *3 5o ; (335) O E . g, 40 First mated after (17)‘~ 2 ist estrus (6) n. . 30 1 2 3 4 Estrus sequence FIGURE 12.2. Pregnancy rates for mares mated at various times postpartum. Number in parentheses is total number of mares. From research data of American Breeders Service, DeForest, Wisconsin. Courtesy of J. Sullivan and W. Parker. mated subsequent to the postpartum estrus since several authors have recom- mended that the judged normality of par- turition and condition of the reproductive tract at the postpartum estrus should be considered in deciding when to mate (1068, 1849). The advantages and disadvantages of beginning the mating program at first estrus have been discussed (1849, 990, 948). Delaying the first ovulation with steroid regimens has been advocated for improve- ment of pregnancy rates (pg. 487). Decrease in pregnancy rate with advancing age. A decrease in reproductive efficiency (pregnancy rates, foaling rates) due to advancing age has been reported, and earlier work in this area has been reviewed (792, 929). In a recent study in Standardbreds (1689), pregnancy rate was significantly reduced in aged mares (30% versus 57%). In another study (1721), mares older than the mean age (11 years, n=192) had lower pregnancy and foaling rates and higher category scores for uter- ine biopsies, indicating more uterine inflammation. A controlled study (281) has been done that involved minimizing many of the factors that have confounded 506 Chapter 12 previously reported studies (e.g., breeding program inconstant among age groups). The Day 12 pregnancy rate was greater for young mares (9 of 9) than for old mares (15 to 30 years, 6 of 19), supporting the results of field surveys. Some findings on the association between age and pregnancy and foaling rates are shown in Figure 12.3. The fig- ure indicates an approximately linear reduction in performance as the mares aged, with a reduction ranging from 17 to 42 percentage points between young and old for the various studies and end points. These values, of course, were affected by all the age-related ramifications of the managerial programs used on the various farms. Note that these farms dealt with a minority of horses that were more than 14 years old (9%, 11%, 12%, and 26% for the four farms shown in Figure 12.4). It is not known whether depressed pregnancy and foaling rates are due to age or to multiparity. A recent study on the effects of age on uterine biopsy evalu- ations in maiden versus nonmaiden mares is currently available in an abstract (712). The authors concluded that histologic uterine abnormalities appeared to be age related regardless of whether mares were maiden or nonmaiden. Age— related deformities of the caudal repro— ductive tract are commonly thought to decrease pregnancy rates, presumably through increased access of contaminants into the uterus (review: 1241). Suturing of the vulva (Caslick’s operation) is there- fore a common practice on some farms. The extensive field study by Pascoe (1241) indicated that mares with a high Caslick index (based on length and angle of labia) had a significantly lower pregnancy rate and that the operation improved the rate. At present, attention seems to be directed toward the progressive effects of age on uterine pathologic processes (1325). It has been demonstrated that the inci- dence of endometritis increases with mare age, and the Day 11 pregnancy rate is adversely affected by endometritis Age & efficiency rates Rates / cycle 60 2212 '. ( ) (1268) Pregnancy / rate 50 (318) x \ Foaling (664) ‘x / rate 40 (803) x“ (if?) y ———— \ A (567) \ 2x: 30 (153) >. ‘o 8 (412) .9 20 .2 _ "“5 Rates / season .2 (1395) o 90 g (209) (858) o Q E. ‘2 “5 (D a) E o: 3 5 7 911131517 Yearsofage FIGURE 12.3. Associations between age and repro- ductive efficiency. Ages shown are the mean ages in various age-classification schemes, and oldest age includes ages greater than the indicated age. The total number of mares from which each percentage was derived is shown in parentheses. Adapted from tabulated data ( Z 390, 123). (pg. 520). Elucidation of the roles of vari- ous organs or structures (e.g., oocytes, oviducts, uterus) or various biologic and pathologic mechanisms in age-related reduced reproductive efficiency awaits a concerted research thrust. This will not occur until gerontology becomes a funded major research area. The detrimental affects of old age on pregnancy loss are discussed later (pg. 540). Pasture mating. Few critical studies have been done on pasture—mating effi- ciency in domestic equids contrasting with the extensive studies in other farm species Age of mares on farms 40 (205) England 00 O ._ (130) M Q Mares in mating program (%) 3 3 5 7 9 Years of age 11 13 and reflecting differences in management. Interest in this area was stimulated by the report by Bristol (250) involving mating with a single stallion placed for nine days with a group of 20 estrus-synchronized draft mares. Pregnancy was diagnosed in 85% of the mares when examined 38 days later. In subsequent studies, groups of 15 to 21 mares were placed with a stallion for 49 (251) and 70 (252) days. The mares had final pregnancy rates of 88% to 97% and foaling rates of 72% to 94%. The rates seem similar to those involving artificial insemination of research mares that had been preselected for good expected preg- nancy rates (e.g., 85%; pg. 503). Pasture mating was studied in ponies using 16 herds (one stallion and 20 or 60 mares per herd; 621). The reproductive histories of the mares were not known, and no attempt was made to eliminate mares with apparent reproductive prob- lems. There was no significant effect of herd size on the number of mares becom- ing pregnant per herd during 24 days (equivalent to one estrous cycle). Thus, the stallions produced an average of nine pregnancies (range, 3 to 18) for the first United States Reproductive Efficiency 507 FIGURE 12.4. Age of mares in the mating programs of farms in four countries. Ages shown are the mean ages in various age classification schemes, and old- est age includes ages greater than the indicated age. The total number of mares from which each percentage was derived is shown in parentheses. Adapted from tabulated data (123, 1810, 1273, 130). 24 days regardless of herd size (20 or 60 mares), excluding two herds in which the stallions were ineffective (no pregnancies). Perhaps the high pregnancy rates report- ed by Bristol (above) from pasture breed- ing are attributable to the use of highly fertile mares and stallions and should not be expected, generally. Considerable study will be needed on pasture mating pro- grams and on the number of mares a stal- lion can be expected to impregnate per breeding season under various schemes. Seasonal pregnancy rate in feral horses and donkeys in western United States has been estimated by serum concentrations of _ progesterone, eCG, and estradiol-17B and transrectal palpation (1806); rates for animals more than two years old were 57% to 81% for various groups of horses and 73% for don- keys. Pregnancy rates for horse yearlings did not differ from older mares; the yearling pregnancy rate in jennies was 25% compared to 73% for adults. Effects of other factors. The efi'ects of vari- ous aspects of the mating program, including postovulatory mating, on pregnancy rates are discussed in Chapter 8 (pg. 296). Irregularity in the expression of estrus and 508 Chapter 12 in the timing of ovulation could impair reproductive efficiency. These irregulari- ties include split and covert estrus (pg. 90), prolonged intervals from the end of estrus to ovulation (pg. 190), and prolonged estrus at the onset of the breeding season (pg. 174). Adequate information on fertility in association with such phenomena is not available, but if the mating program is based solely on the detection of estrus, lowered reproductive efficiency is likely. Although these conditions present obsta- cles to mating at the optimum time, there is no firm indication that they are other- wise associated with lowered fertility. In limited studies, pregnancy rates were good when such mares were mated at the proper time (85, 1680). There appears to be no difference in the pregnancy rates for ovulations that occur at various times during the year. According to a compilation of records from Thoroughbred farms (269), the preg- nancy rates for the first, second, and third ovulations of the season were 52%, 52%, and 46% (not significantly different; n=769). However, mares in which the first ovulation occurred late in the year had less chance of becoming pregnant for the season. This was attributable to fewer opportunities, rather than to a seasonal effect on fertility at the first ovulation. An abstract from the Soviet Union (523) stat— ed that pregnancy rate (per season?) was lower (78%) for a group of fast trotter mares than for a group of slower mares (86%). It could not be determined from the abstract whether other factors, such as age, confounded the results. End-of-season pregnancy rates. On some farms, fall pregnancy diagnoses are done in all mares mated during the oper- ational breeding season. The resulting figure has been called the final or end-of— season pregnancy rate. It is a measure of total mating success for the year and facilitates decisions on disposition of mares. Reported per—season pregnancy rates were reviewed in the first edition (575) and were reported as 76%, 77%, 80%, 83%, and 84%. A more recently reported rate from an extensive study on Thoroughbred farms in England was 87% (1390). It is recommended that farms should strive to exceed an 85% end-of- season pregnancy rate. 12.2C. Foaling Rate Reported foaling rates per breeding season in feral horses have ranged from 23% to 82%; the wide range has been attributed to differences in age, forage, and social equilibrium (cited in 251). As noted earlier, live—foaling rates in domestic mares over an operational sea- son are commonly stated to be 50% to 60%. According to a review by Osborne (1187), official foaling rates for Thoroughbred mares in four countries during the 1960s and 1970s ranged from 45% to 50%. Live—foal rates based on surveys involving many mares (575) ranged from 53% to 64% (mean of per- centages: 58%). The meaning of foaling- rate figures from official reports and questionnaires is not always clear. Osborne (1187) indicated that foaling fig- ures may be based not on the number of mares mated, but on the total number of mares registered in the studbooks of the various countries. Often the relationship between the stated foaling rate and peri- natal mortality is not clear. A live-foal rate of 53% of mated mares for North America was taken from the American Studbook (Thoroughbreds) for 1970 to 1973 (1072). The 47% that did not pro- duce foals included 29% for which a foal- ing report was not filed. Although foal- ing rates obtained from studbooks and questionnaires may have useful purpos- es, they should not be used to ascribe poor reproductive efficiency to horses. Estimates obtained directly from farm records are more meaningful. Estimates of foaling rates from Thoroughbred and Standardbred farms cited in the first edi- tion (575) were 71% and 73%. Subse- quently published reports gave foaling rates of 76% to 84% (mean percentage: 81%) for Thoroughbred, Standardbred, and Quarter Horse farms in several coun- tries (1390, 524, 240, 266, 1004). Although not directly comparable, there has been an approximately 9% increase in foaling rates over the past decade, which is con- sistent with the conclusion of improved reproductive efficiency (pg. 499). As a con- venient figure, operators of farms should strive to exceed an 80% live-foaling rate. 12.3. Pregnancy Loss Overall measurement of reproductive efficiency must, of course, consider the incidence of pregnancy loss as well as establishment of pregnancy since the goal is birth of a healthy foal. Recent reviews on equine pregnancy loss are available (590, 138, 152, 145, 1809, 588). A high-priority research area. Con- sidering the economic setback and anguish associated with loss of a diag- nosed pregnancy, this area of investiga- tion has been deplorably underfunded. A recent evaluation of annually reported data by Thoroughbred owners in Britain suggested that the positive progress made in the incidence of pregnancy establish- ment over the past two decades (Figure 12.1) has not been matched by progress in the reduction of the incidence of pregnan- cy loss (1326). Considering currently avail- able technology and the economic hard- ships traceable to pregnancy loss, it is anticipated that the pathogenesis of the various forms of pregnancy loss will undergo a major research thrust in the 1990s. Technology. A past technologic problem in studying early loss has been the lack of a nonterminal method for detecting early pregnancy, monitoring embryo progress, and pinpointing the time of loss. Transrectal palpation has permitted diag- nosis of pregnancy by approximately Day 20. Earlier losses, however, could not be differentiated from fertilization failure, except by inference when the mare had a Reproductive Efficiency 509 prolonged interovulatory interval or became pseudopregnant. In addition, the day of death cannot be determined ade- quately by transrectal palpation, since the vesicle can remain intact for many days after cessation of embryo heartbeat (pg. 544). Ultrasonography has halved the formidable interval from Day 1 to Day 20; an embryo of normal size can now be detected and monitored, beginning as early as Day 9 or 10 and consistently by Day 11 (590). As a result, several experi- ments on losses over Days 11 to 20 were reported in the 1980s, and more progress can be expected in the near future. The ability to detect and monitor the embryo by ultrasonic imaging beginning on Day 10 is fortuitous because Day 10 precedes the time that the embryo must block the uterine luteolytic mechanism. In addi- tion, the precise time of death in embryos with a heartbeat (e.g., >Day 24) can be determined by heartbeat cessation. Other technologic breakthroughs in- clude oocyte in-vitro maturation, assisted fertilization, and embryo transfer (pg. 335), with the resulting ability to study embryo loss that involves the oocytes, fertiliza- tion, and exposure to the oviductal and early uterine environment. Emphasis on pathology of the tubular genitalia. In mares with healthy tubular genitalia, some failure of pregnancy establishment (negative initial pregnan- cy diagnosis) and some pregnancy loss (positive diagnosis followed by a nega- tive diagnosis) can be accepted as inevitable. The failures and losses, in part, can be considered a selection pro- cess to compensate for defective gametes or embryos. Losses that occur early in pregnancy afford an early opportunity for re-establishing pregnancy. Individuals trained in physiology with little exposure to pathology tend to attribute pregnancy loss to spontaneous deviations in physiologic mechanisms or function. Research conferences and jour- nal reviews have purported to explore the 510 Chapter 12 suspected factors associated with preg— nancy loss in farm animals with no men— tion in the entire research report of the role of pathology of the tubular genitalia. Three chapters in this book have been devoted to an array of complex, interre- lated physiologic mechanisms underlying pregnancy. Yet, most pregnancy failures and pregnancy losses under equine farm conditions are attributable to pathologic processes, as indicated by macroscopic or microscopic lesions in the tubular geni— talia, rather than to aberrations in physi- ology (function) unaccompanied by overt pathologic change. For example, a touted cause of embryo loss in large domestic species (especially in grant proposals) is aberrations in the embryo-uterine mecha- nism for the luteal response to pregnancy (Days 11 to 15 in mares; pg. 438). There are no firm data implicating deviations of this mechanism in embryo loss in normal mares (free of pathology); however, embryo development or the luteolytic mechanism can be profoundly and detri- mentally altered by oviductal or uterine pathology, respectively, causing embryo loss through failure to block luteolysis or through active induction of luteolysis. In the following sections, current knowledge of the nature and causes of pregnancy loss will be reviewed. Many of the considered factors are involved in low pregnancy rates (fertilization failure or embryo loss before the first pregnancy diagnosis), as well as in loss of pregnancy after a positive pregnancy diagnosis. 12.3A. Overall Pregnancy-Loss Rate Attempting a unifying interpretation of literature reports on pregnancy loss car— ries with it the hazards of coalescing con- clusions that originated from divergent examination programs and reference points among studies and within studies. Another source of befuddlement in attempting to estimate loss rate from the literature concerns the manner and extent, if any, of consideration of twins. Twins bring the added dimension of pos— sible conceptus loss without pregnancy loss (embryo reduction or elimination of one member of a twin set). Here, the twinning phenomenon will be given sepa- rate consideration (pg. 546). The estimates of pregnancy loss per ovulatory period by various field investi— gations prior to 1980 were approximately 12% (tabulated in the first edition; 575). These studies were based on transrectal palpation, and many of the initial preg- nancy diagnoses were made on approxi— mately Day 40 and only occasionally as early as Day 20. Studies done in the 1980s began to use ultrasonic pregnancy diagnoses (review: 590). Because reliable diagnosis of pregnancy with this technolo— gy can begin much earlier in pregnancy, the detected incidence of pregnancy loss is expected to be greater. In a study on Thoroughbred farms in the United Kingdom (1390), mares were examined initially as early as 16 days after mating (more precise information on day of the initial diagnosis not given); the overall loss rate was 11%. In another recent study (524) in Thoroughbreds in Ireland, pregnancy loss from approxi- mately Day 18 to foaling was given as 10% and 12% in two years. In a study in Thoroughbreds and Standardbreds in the United States (240), an initial pregnancy diagnosis was done on Days 12 to 20, and the loss rate was 13%. A recent extensive field study by ultrasound in France found an incidence of 18% pregnancy loss (300). The initial diagnosis was done at a mean of 22 days after service (standard devia- tion: i5 days) and the last diagnosis at 310 days, therefore excluding most still— births. A large number of mares of sever- al breeds was involved. In conclusion, convenient numbers for expected pregnancy-loss rate are 18% when initial diagnoses are done at approximately Day 20 and 12% when done at Day 40. For a given farm, of course, pregnancy loss rate will be influ— enced by many factors (e.g., proportion of old mares, proportion of mares with a his— tory of subfertility, postpartum breeding program). 12.3B. Time of Occurrence Information on the incidence of preg- nancy loss at various times during gesta- tion is important in the design of manage- ment programs (e.g., optimal days of pregnancy determination) and for provid- ing temporal indications of the underly- ing causes. Increments in the days of pregnancy in the following account were selected so that time of occurrence could be related to reproductive events and diagnostic limitations. The time of occur- rence and the incidence of loss are pro- foundly related to health of the reproduc— tive tract. Separate consideration, therefore, will be given to normal mares (e.g., young, no history of reproductive problems, no ultrasonic or biopsy indica- tions of reproductive pathology) and sub- fertile mares (e.g., old, pregnancy failure for the past one or two years, ultrasoni- cally detected intrauterine fluid collec- tions, histopathology indicative of endometritis). Days 1 to 5. Direct critical studies of the rate of embryo loss during the oviduc- tal sojourn have not been reported. Problems associated with such investiga- tions or their reporting include: 1) confu- sion between number of mares as opposed to number of ovulations per mare, 2) extent to which failure to recov- er an embryo can be attributed to embryo loss, 3) inadequate consideration of age or reproductive histories and evalua- tions, 4) difficulties in classifying all ova into fertilized and unfertilized, and 5) difficulties in comparing results among laboratories because of divergent protocols and judgments. Spontaneous parthenogenetic development (cleavage of an unfertilized ovum) apparently is not as serious a diagnostic problem as once believed (146). Parthenogenic division appears to be rare both in vitro and Reproductive Efficiency 51 1 in vivo (1851). Flushing oviducts and classifying the recovered ova has yielded cleaved-ova rates of 75% (n=20, Days 2 to 5, 3— to 8-year-old Thoroughbreds, 524), 71% or 76% depending on classification considera- tions (n=28 or 33, Days 1 to 5, ponies, 198), and 82% (n=28, Days 2 to 6, 2— to 6-year- old Welsh ponies, 67). The studies were not designed to examine loss rates over time, however. The studies of Woods and asso- ciates (original reports: 146, 1255, 147; reviews: 1813, 151, 1812) are of special interest because they were done on speci- fied days and mares were classified as either reproductively normal or subfer- tile. The results of their studies are sum- marized in Table 12.2 (pg. 503). The follow- ing conclusions can be drawn from the table: 1. On Day 2, recovery rates (number of recovered ova/number of ovulations) and the percentage of recovered ova that were cleaved (embryos) were not different between normal and subfertile mares, and the percentage of cleaved ova for both groups was high (>90%); and 2. On Day 4, the frequencies of ovum/embryo recoveries (mean over 2 experiments: 92%) and the proportion of recovered structures classified as embryos (mean: 98%) were high in nor- mal mares; however, both end points were significantly reduced in subfertile mares (66% and 81%). A high embryo-loss rate in subfertile mares was indicated by reduced frequen— cies for both recovery and cleavage at Day 4 (significant difference between normal versus subfertile mares) but not at Day 2. The percentage of wastage up to Day 4 in the subfertile mares in these studies would have been approximately 40% (dif- ference in rate of ova recovery between normal and subfertile mares, 27%; differ- ence in proportion of cleaved ova, 15%). If the loss continued at the same rate, the total would have been approximately 50% by Day 5. These estimates assume that failure to find ova, as well as reduced 512 Chapter 12 frequency of cleaved ova at Day 4, repre- sented loss of fertilized ova. Although the Day 2 and Day 4 collections were done in separate experiments, the data provide rationale for the hypothesis that embryo loss between Days 2 and 4 is negligible in normal mares and great in subfertile mares. The above conclusions seem rea- sonable for young mares, since three sep- arate experiments indicated high (>90%) pregnancy rates on Day 2 or Day 4. The data for subfertile or old mares are more tenuous since the conclusions are depen- dent on the results of only one study at Day 2. As noted earlier (pg. 502), high fer- tilization rates (pregnant at Day 2) in old subfertile mares are unexpected and in need of further study. In an embryo transfer study by the same New York investigators (147), embryos were recovered on Day 4 from the oviducts of normal and subfertile mares and transferred to the uterus of normal mares. The Day 10 recipient preg- nancy rates for embryos collected from normal and subfertile mares were 15 of 27 «(56%) and 6 of 22 (27%; significant dif- ference). Thus, considerable embryo loss occurred in the uterus of normal mares on Days 4 to 10 when the Day 4 embryos were transferred from the oviducts of sub— fertile mares. Apparently, a defect or irre— vocable damage was already present before Day 4. A previous study had shown that autotransfer of Day 4 embryos to the uterus was as effective as transfer to the opposite oviduct (1255). Still required, in addition to confirma- tion of high fertilization rates, is an extensive critical investigation in which the fertilization rate and the Day 5 embryo-survival rate are compared in a single study. Also, the sequential mor- phology of fertilized ova after death and the fate of such structures have not been determined. It is not known, for example, whether death of cleaved ova at Day 2 would result in structures similar to unfertilized retained ova (passive degen- erative process; pg. 303) or whether such structures rapidly disappear or become unrecognizable (active degenerative pro- cess or other form of loss such as dis- charge into the abdomen). Knowledge of the degenerative changes occurring after loss of viability is crucial to meaningful evaluation of recovered structures. Days 6 to 10. This early period of intrauterine life of the embryo is given special attention because it begins on the day of embryo arrival into the uterus and ends the day before the embryonic vesicle is almost always detectable by ultra- sonography. Because the vesicle is not detectable, except for a minority of instances on Days 9 and 10, embryo loss is difficult to estimate, and there have been no direct studies of the loss rate dur— ing this time. For a mathematical approach in young mares judged to be reproductively nor- mal, one could assume a fertilization rate of 94% (pg. 502) and a Day 11 pregnancy rate of 86% (pg. 503). The losses occurring after fertilization and before Day 11 under these assumptions would be 8.5% (94% minus 86% = 8%; 8% divided by 94% = 8.5%). Assuming negligible losses in the oviduct, the 8.5% loss-rate would have occurred over Days 6 to 10. Similar speculation for old mares seems unwar- ranted, pending clarification of the extent of involvement of defective oocytes, defec- tive fertilization, and defective oviductal environment. However, since Day 11 pregnancy rates are low in old and sub- fertile mares (e.g., 82%; pg. 521), it can be concluded that a high incidence of preg- nancy failures, whether fertilization or survival failures, occurs before Day 11. Embryo collection and transfer studies have provided most of the information 011 losses on Days 6 to 10. Embryo recovery rates from normal and subfertile mares are shown in Table 12.4. For all studies, embryo recovery rate was much lower for subfertile mares (15% to 40%) than mares that did not have a recent history of subfer- tility (39% to 80%). Older mares, which have a higher incidence of subfertility Reproductive Efficiency 513 TABLE 12.4. Embryo Recovery and Survival Rates in Embryo Transfer Studies Embryo survival in recipients Location Day of Donor mare Embryo (Reference) flush classification recovery rate Day Rate Kentucky Days 6 to 10 Controls 56/100 (56%)a 100 14/28 (50%) (423) Barren 22 yr 12/35 (34%)b 100 4/8 (50%) Kentucky ..... Barren 22 yr 41/146 (28%) 100 22/41 (54%) ( 424) Colorado Days 8 & 9 Normal, cyclic 128/160 (80%)a 35 15/23 (65%) (1510) Infertile, 21 yr 10/36 (28%)b 35 8/10 (80%) California Day 8 Maiden 21/54 (39%)a (1237) Barren, >2 yr 5/34 (15%)]? New York Days 7 to 9 Maiden 29/42 (69%)a (1812) Subfertile, 2 yr 17/42 (40%)b New York Days 7 & 8 Normal 44/83 (53%) (144) Texas ..... Maiden 40/66 (61%)a . . . 34/69 (69%)a (1712) Postpartum 146/276 (53%)a 87/144 (60%)ab Subfertile 81/282 (29%)ID 38/78 (49%)b $— 2 to 8 years 80/132 (61%)a 59/84 (70%)a 9 to 17 years 18 to 28 years 50/97 (52%)b 50/90 (56%)b 94/183 (51%)a 93/309 (30%)b Percentages Within each set with no common superscripts are significantly different (pg. 505 and pg. 540), had lower recovery rates. These findings demonstrated that sub- stantial embryo loss occurred in old mares before the collection attempts or that embryos of old mares were technically more difficult to recover. At least a portion of the failures to recover can be attributed to embryo loss, however, because Day 7 to Day 9 embryos from subfertile mares are more often morphologically defective. In one study (1812), 9 of 17 embryos collected on Days 7 to 9 from subfertile mares were classified as abnormal, compared to 1 of 29 from maiden mares; in addition, mean diameter of embryos from subfertile mares was only 50% of the diameter of embryos from maiden mares. In a histo- logic and ultrastructural study (1405), fewer embryos from subfertile mares were normal; 4 of 10 (40%) from subfertile mares were classified as having good mor- phology, compared to 26 of 32 (85%) from maiden mares. Embryos recovered from subfertile and old donors also were less likely to develop after transfer to normal recipients (1712, Table 12.4). Furthermore, embryos from subfertile mares that established detectable pregnancies in normal recipi— ents were more likely to undergo subse— quent loss (losses after a positive preg- nancy diagnosis in recipients: 13 of 38 for subfertile donors versus 4 of 34 for maid- en donors); the days of pregnancy diagno— sis were not stated (1712). Other workers (1510) obtained similar results (50% versus 13% loss between Days 35 and 50 for sub- fertile versus maiden mares). 514 Chapter 12 In conclusion, embryos collected on Days 6 to 10 from subfertile donors had an increased incidence of morphologic defects (indicated by diameter and struc- ture), reduced recovery rates, reduced pregnancy—establishment rates in normal recipients, and increased loss rates for established recipient pregnancies. Days 11 to 40. During this period, the embryonic vesicle can be monitored directly by ultrasonic imaging. An exami- nation of the time of occurrence of embryo loss during this period was done (600) using data from investigations that were reported or conducted between 1985 and 1990 (604, 1824, 186, 182, 603). The data were from mares that were judged by ultra- sound to be free of extensive uterine cysts and intrauterine fluid collections during diestrus and were pregnant during the previous year (defined as normal mares). The results from these mares were com- pared to those from a herd with a high proportion of subfertile mares (604), including mares with diestrous fluid col- lections, short estrous cycles, early embryo loss, or failure to establish preg- nancies during the previous year. Mares in both the normal and subfertile groups were mated before ovulation using the same insemination protocol. Only mares with a single ovulation were used. None of the mares were given exogenous hor- mones or other substances or experimen- tal challenges, except that some received hCG or GnRH to induce ovulation. The mares were examined every 1 to 3 days after pregnancy diagnosis on Day 11 or 12. Before the detection of an embryo proper, the day of embryo loss was deter- mined by disappearance or collapse of the embryonic vesicle. After detection of an embryo proper (Days 20 to 24), the day of loss was defined as the first day that a heartbeat was no longer detected. The days were grouped into five-day incre— ments, and the day of loss was assigned to the increment in which the embryo was present on the first day of the increment and absent or dead on the last day. Embryo loss rates 20 | l I | —L 01 \ Subfertile mares _L O | Normal mares .~~.\ / We ‘v ‘ ‘0 - - - -o -(113)-(108) - (94)--(92)--(54)--(54) )-(144) -(139)—(120)—(90)—(57) indicated day increment (%) v a Rate of embryo loss during the 15-20 Number of days from ovualtion 25-30 35-40 45-50 FIGURE 12.5. Embryo-loss rates for five-day incre- ments extending from Days 11 to 50. Total number of mares from which each percentage was derived is shown at the bottom. From analysis (600) of data over several surveys (see text). In the groups of apparently normal mares, the loss rate for each of the five- day increments extending from Day 11 to Day 40 ranged from 1.1% to 2.1%, and there were no significant differences among increments (Figure 12.5). The con- stant (no significant differences) five—day loss rate continued into the early fetal stage (to Day 55). In the herd with a large proportion of subfertile mares, the loss rate on Days 11 to 15 was 18.2% which was significantly greater than in the nor- mal mares (1.3%). The loss rates were greater on Days 15 to 20 in the subfertile mares than in the normal mares (3.3% versus 2.0%), but not significantly. On Days 20 to 40, the loss rates were similar for the two groups; there were no signifi- cant differences between groups for any five-day increment or totaled over Days 20 to 40 (normal, 6.6%; subfertile, 4.8%). An examination was also done (600) using data from two groups (2, 4) of prese- lected subfertile mares. The mares were classified subfertile as described above. The Day 11 or 12 pregnancy rate was low (32%), and the subsequent embryo loss rates were high between Days 11 to 15 (23%) and Days 15 to 20 (13%; Table 12.5). In conclusion, the two analyses indicated that loss rates in subfertile mares were very high between Days 11 to 15 (e.g., 20%), intermediate on Days 15 to 20 (e.g., 8%), and were similar to those of normal mares on Days 20 to 40 (e.g., 7%). Other reports have involved day incre- ments approximating some of those in the above studies. Two losses occurred in 17 mares between Days 10 and 14 (146). In an embryo transfer study, embryo loss occurred in 1 of 40 mares during this time (Day 13; 144). One study examined five- day increments extending over Days 15 to 50 (1703); the total loss rate was 17.3%. Several extensive field studies have been done with ultrasonography for a period approximating the last half of the embryo stage (Days 20 to 40; Table 12.6) The loss rates were approximately 6%. This seems to be a convenient guideline figure for the expected loss rate between Days 20 to 40; the unknown proportion of subfertile mares in 4 of 5 of these studies probably did not greatly alter the estimates because in other studies (described above) the main loss rate in such mares occurred before this period. In other studies, the loss rate was greater between Days 14 Reproductive Efficiency 515 TABLE 12.5. Pregnancy Rates and Embryo Loss Rates for Subfertile Mares Pregnancy rates Day 11 30/93 (32.3%) Day 40 18/93 (19.4%) Embryo-loss rates Days 11 to 15 7/30 (23.3%) Days 15 to 20 3/23 (13.0%) Days 20 to 40 2/20 (10.0%) Days 11 to 40 12/30 (40.0%) From an analysis (600) of data obtained primarily from previous reports (4, 2). and 28 than between Days 28 and 42 (1810), greater between Days 18 and 25 than between Days 25 and 45 (808), and greater between Days 14 and 21 than between Days 21 and 42 (170). In conclusion, for all reports with appropriate data, the loss rates during approximately the first half of the inter- val extending from Day 11 to Day 40 greatly exceeded those for the last half. Based on Figure 12.5, the increased losses during the first half are attributable to subfertile mares. After Day 40. The day-to-day probabil— ity of conceptus loss during the fetal stage (>Day 40) is considerably less than during the embryo stage. However, the embryo-loss rate over Days 20 to 40 TABLE 12.6. Embryo-loss Rate During Approximately Days 20 to 40 When a Normal- appearing (Ultrasound) Singleton was Present at the Initial Diagnosis Day of Rate of pregnancy Location Principle pregnancy Day of loss loss during the (Reference) breed diagnosis determination given time span England Thoroughbreds Approx. Day 20 Approx. Day 45 16/326 (5%) (1483) France Mixed Mean: Day 23 Mean: Day 43 69/1298 (5%) (299) France Mixed Mean: Day 22 Mean: Day 44 186/3070 (6%) (300) New Zealand Standardbreds Day 17 or 18 Days 42 to 45 17/179 (10%) (808) From Ponies and Day 20 Day 40 . . . (6%) Figure 12.5 riding-type horses 516 Chapter 12 (1.6% per five-day increment, Figure 12.5) seemed to extend into the early fetal stage (last day studied: Day 55). In this regard, it was stated in a trans- rectal palpation report that rate of loss did not appear to diminish until after 60 days (130). These conclusions seem com- patible with the daily risk of loss calcu- lated from a field ultrasound study (300) and with the finding of an equivalent number of losses between Days 21 to 42 versus Days 42 to 63 (170). In conclusion, the daily rate of loss apparently is constant over Days 11 to 60 in normal mares and over Days 20 to 60 in subfertile mares; thereafter, the daily rate of loss declines. As noted above, a per season pregnancy rate of 85% and a foaling crop of 80% seem to be reasonable estimates of expected reproductive efficiency on equine farms. These figures imply that a farm that mates 100 mares can expect that 85 will be diagnosed pregnant after the end of the breeding season, 80 will foal, and 5 (6%) will lose their pregnan- cies after the fall pregnancy diagnosis. Fetal-loss rates in extensive field stud- ies based on transrectal palpation were 7.0% after 40 to 50 days (810), 3.8% after 5 months (130), and 1.9% after 6 months (128). These early reports are difficult to evaluate and are beclouded by the condi- tions underlying the data gathering pro- cess. They are, nevertheless, noteworthy contributions in this difficult area of field investigation. In the recent French study (300), losses were calculated for a period extending from 44 days (mean) after last service to 310 days. The mean day of the initial examinations approximated the beginning of the fetal stage as defined herein; however, the variation around the mean of 44 days was considerable (standard deviation: i12 days). The loss rate between means of 44 and 310 days was 9.1% for approximately 3,000 mares of several breeds. Considering all of the above reports, a fetal-loss rate (Day 40 to term) of 12% seems a reasonable guideline figure. In a recent study in feral horses (998), loss rates between 120 days and term were evaluated by measuring fecal estro- gen concentration to determine pregnan- cy. The loss rates over four years were 10%, 40%, 25%, and 26% (n=62 to 80 mares per year). These findings indicated very high loss rates after 120 days for some years in this feral population. Days of increased peril. Many remark- able events and physiologic transitions occur during pregnancy, such that one or more hypotheses can be offered for what— ever period is considered to be perilous. However, reports of narrow perilous periods were not adequately documented or have not been confirmed. Suggested periods include Days 25 to 31 in mal- nourished mares (1856), Days 30 to 35 (1703), and approximately Day 150 (1153). The most extensive early attempt to determine whether pregnancy loss occurs more frequently at certain times during pregnancy was that of Bain (130). The study was based on examination of breeding and palpation records on Thoroughbred farms in Australia and involved 2,562 mare—years. Pregnancy diagnoses were done on Days 20 to 40, and many diagnoses were repeated after 40 days. The author concluded that preg- nancy-loss rate diminished later in preg— nancy, but no critical period was appar- ent. Recent field studies in France (299, 300) and the study depicted in Figure 12.5 failed to find a period that was characterized by a surge of pregnancy losses. The loss rate of oviductal embryos in normal mares appears to be very low (pg. 511), and a representative loss rate on Days 6 to 10 was calculated to be 8.5% (pg. 512). If these assumptions are correct, a period of great peril is the first few days after entry of the embryos into the uterus. Reproductive Efficiency 517 SUMMARY: Time of Occurrence of Pregnancy Failures 100 (95%)) Cumulative pregnancy successes (%) ’o‘ o\ v m m L 2 "E *- >- o c m c a) w L a. m > 1: .E 3 E 3 0 20 Subfertile mares 30 Pregnancy successes Subfertile mares I Pregnancy ' failures (36%) (36%)/ 40 50 60 Number of days after ovulation FIGURE 12.6. Schematic presentation of pregnancy-loss rates at various intervals in mares with an apparently healthy reproduc- tive tract (defined as normal) and mares with a history of reproductive failures (defined as subfertile). Mares in the latter category were often 01d mares with histologic or ultrasonic indications of endometrial pathology. ' Available data indicate that the fertiliza- tion rate is high in normal mares and that losses between Days 2 and 4 are negligible. If that is correct, the rate of loss between Day 4 and Day 11 may be greater than at any other time, as shown. 0 The conceptual presentation of a much greater loss rate over Days 1 to 20 for subfertile maresthan for normal mares is documented. However, adequate information is not'avail- able on the time and nature of losses before Day 11 in subfertile mares \' ' The loss rate for Days 20 to 60 is depicted as equivalent (parallel lines) for the twp groups of mares. This assumption [is based on results of a single study (Figure 12.5) and requires confirmation. 0 The expected rate of loss after Day 60 in subfertile mares is not clear. According to field investigations (Figure 12.16, pg. 540), old mares have a higher rate of both embryo and fetal losses. The confounding between age classifica- tions and normal/subfertile classifications is great and precludes a satisfizing interpretation. 518 Chapter 12 12.30. Role of Salpingitis in Pregnancy Loss Salpingitis. The equine oviducts have been nearly ignored as organs for clinical consideration. Earlier work on oviductal pathology has been cited and discussed (723, 694, 1382). The oviducts of mares, in contrast to those of other farm species, have been considered less prone to salpin— gitis (inflammation) because of the dorsal position relative to the uterus and the nature of the uterotubal junction. Belgian workers (723, 1687), however, made an extensive study of slaughterhouse speci- mens. Histologic evidence of inflamma- tion of the oviducts, especially the infundibulum, was high and exceeded the incidence of uterine inflammation (Table 12.7). Both endometritis and infundibuli- tis were more frequent in mares that exceeded 15 years of age than in younger mares. There was a positive correlation between the presence of salpingitis and endometritis. The incidence of infundibu- lar adhesions was 49%. A recent study (1382) also found a high incidence of oviductal pathology (e.g., adhesions, fibrous bands, lymphocytic infiltration). The incidence of slight lymphocytic infil- tration in the infundibular—ampullary region and the incidence of intraepithelial cysts were greater in nonpregnant mares than in pregnant mares (5% versus 23% and 0% versus 48%). These findings are compatible with a role of oviductal TABLE 12.7. Incidence of Histologic Indications of Inflammation in the Uterus and Various Segments of the Oviducts in Slaughterhouse Specimens Segment Incidence Uterus 47/272 (17%) Utero—tubal junction 16/13 1 (12%) Isthmus 10/332 (3%) Ampulla 37/392 (9%) Infundibulum 9 1/336 (27%) Adapted from ( 723). pathology in subfertility. The data were not partitioned according to age, and the specimens were from a slaughterhouse (unknown reproductive histories). These studies and earlier studies cited by these workers indicate a need for careful con- sideration of a possible role of salpingitis and other oviductal pathology in subfer- tility. No cases of total oviduct obstruction, hydrosalpinx, or congenital abnormalities were found by the Belgian workers, although a few cases of hydrosalpinx have been reported by other workers (616, 694, 742, 1382). Methods for clinically evalu- ating occlusions of the equine oviduct have been assessed (39); injection of a starch suspension onto the ovary before ovulation was followed by starch recovery at the cervix in 4 to 7 days. The salpingitis hypothesis. There is now adequate rationale for the hypothesis that salpingitis is a cause of embryo loss. The following considerations constitute the rationale or are compatible with the salpingitis hypothesis: 1. Ova-recovery rate and cleaved-ova rate in mares inseminated under research conditions were high at Day 2 in both subfertile and normal mares but were reduced at Day 4 in subfertile mares (Table 12.2, pg. 502); 2. In many embryo transfer and mor- phologic studies, embryos collected from the uterus of subfertile mares were more likely to be defective than those from normal mares (pg. 512); 3. Day 4 oviductal embryos from sub- fertile mares had lower survival rates when transferred to the uterus of normal mares than did oviductal embryos from normal mares (Day 14 survival rates: 23% versus 52%; 147); 4. Transfers of uterine embryos on Days 7 or 8 from normal donors were equally successful when embryos were inserted into either normal or subfertile recipients (IO/20 versus 8/10 Day 28 preg- nancy rates, 144); 5. Oviductal embryos are not covered by a capsule (pg. 352) and therefore may be less resistant to an adverse environment; 6. Inflammation of the oviducts has been reported (described above); and 7. Salpingitis could account for failure of some subfertile mares to respond to uterine therapy. As indicated in Point 2, the oviductal embryos of subfertile mares are either intrinsically defective (pg. 502) or are detri- mentally affected by the oviductal envi- ronment. Distinguishing between intrin- sic defects inherent in the embryo and the effects of an adverse tubal environment requires specific study (e.g., involving transfer of in vitro fertilized follicular oocytes; early oviductal collection and transfer between young and old mares). It is emphasized that the salpingitis hypothesis has not been tested—only that sufficient observations are available to indicate a need for testing. The pathogenesis of salpingitis in embryo loss could involve: 1) direct and active inflammatory toxic effects on the embryo, 2) premature expulsion of the embryos into the uterus (hastened trans- port), and 3) loss into the abdomen (reverse transport); any of these possi- bilities would account for a reduction in number of embryos between Days 2 and 4. If the salpingitis hypothesis is sup- ported by critical testing, techniques pos- sibly could be developed for insertion of therapeutic substances directly into the oviducts (e.g., by Visualization of the uterotubal orifice with an endoscope). 12.3D. Role of Uterine Inflammation and Fibrosis Most studies on the role of pathologic processes in pregnancy loss have been directed toward the uterus. This is to be expected because of the role of this organ as the site of conceptus development com- bined with its accessibility for sampling and experimentation. On the other hand, the ready accessibility of the endometri- um, as apposed to the myometrium, has Reproductive Efficiency 519 focused virtually all of the research atten— tion to the endometrial layer. Recent find- ings concerning reduced contractility and defective clearance of the uterus in older mares (pg. 540) should serve to direct some attention to the myometrial layer. Biopsy evaluation of endometrial health. Endometrial histopathology from biopsy (1661) and endometrial cytology from uterine swabs or flushes (1366, 410) have been advocated for assessing uterine health. Endometrial biopsy results are commonly categorized to reflect the asso— ciations between pathology and reproduc- tive efficiency. A recent review (1661) can be consulted for information on biopsy techniques and the classification systems being used as predictors of reproductive efficiency. As an example, foaling rates of 70% to 90%, 50% to 70%, and <10% have been reported for biopsy Categories of 1, 2, and 3, respectively (864); the increasing category designations are based on increasing severity of inflammation and fibrosis. In a recent study (1721), pregnan- cy rates in four categories of increasing severity of endometritis were 79%, 49%, 33% and 0%; mares older than the mean age (11 years) had higher category scores and lower pregnancy rates. The associa- tions of biopsy scores with pregnancy rates, embryo-loss rates (described below), and foaling rates (review: 1661) seem well-documented, but the associa- tions with fetal-loss rates are not. It has been stated in recent reviews that the association of extensive periglandular fibrosis with pregnancy loss between 35 and 60 days is well docu- mented. However, the cited reference (864) states only that fibrotic change appears to be the most frequent cause of embryo and early fetal death. Thus, this appropriately hedged original observa- tion, unaccompanied by data, seems to have grown to a prestigious acceptance level without the benefit of critical test- ing. However, it was concluded in a recent report, currently available only as an abstract (712), that Category 1 and 2 520 Chapter 12 mares had similar initial pregnancy rates, but pregnancy loss after 150 days was greater in Category 2 mares. The number of layers of connective tissue (fibrosis) surrounding the glands has been reported to be correlated with the number of years barren, and greater number of layers was associated with a reduced foaling rate (945). Ultrasonic evaluation of uterine health. Initial studies of the uterine lumen by transrectal ultrasonography disclosed occasional pockets of free fluid, and these were termed intrauterine fluid collections (614). Sizes ranged from barely perceptible pockets to large collections of purulent exudate (e.g., 40 mm height, pyometra). Subsequent studies (described below) have shown clearly that even the small collections (e.g., 4 x 10 mm), especially when seen during mid- to late diestrus, are indicators of endometritis. The uter- ine fluid can be considered an inflamma— tory exudate or a response to uterine irri- tants. Ranking scores or grades have been used to indicate the extent of fluid volume (4, 1068, 1065) and the extent of echogenicity (1068, 1502, 1065). Mares with fluid collections were significantly older (means: 1’7 versus 8 years), and fluctua- tions of fluid quantity in affected mares during the estrous cycle have been char- acterized (4). A recent study (1023) indicated that physical stimulation of the mucosa of the cervix or uterus (e.g., infusion of saline solution) results in an influx of proteins and neutrophils. It was suggested that this influx may counteract infectious agents. This report also reviewed earlier work involving the influx of substances into the lumen following, for example, uterine contamination. In this regard, fluid flushed from the uterus of infected mares was higher in proteins (216). Fluid collections, histopathology, and embryo loss. A series of studies has been done on the associations among ultrason- ically detected free intrauterine fluid col- lections, biopsy scores for endometrial histopathology, pregnancy rate, and embryo-loss rate. In the initial study (610), mares were selected from the herd that had the high incidence (18%) of embryo loss described above. Mares were assigned to 1 of 2 groups depending upon the detection, at least once during the breeding season, of either small free col- lections of intrauterine fluid during diestrus or embryo loss on Days 11 to 15. Results indicated that mares with a his- tory of intrauterine fluid collections were similar to mares with a history of embryo loss during Days 11 to 15 in the following ways: 1) Day 11 pregnancy rate was reduced; 2) Mean progesterone concen- trations on Days 7 and 11 were reduced; 3) Mean length of the interovulatory interval was reduced; and 4) The condi- tion was measurably repeatable within individuals. These similarities suggested that the factors responsible for intra- luminal collections of fluid were the same as those responsible for embryo loss on Days 11 to 15. Indications of uterine inflammation in these mares were found on Day 16 in uterine biopsies in 5 of 5 mares that lost the embryo on Days 11 to 15 (590). The hypothesis offered was that the fluid collections represented an inflammatory exudate and the observed embryo loss was due to luteolysis induced by the inflammatory process (discussed below). The hypothesis was tested in a subse- quent study (4). An endometrial biopsy was taken on Day 8, and mares were mated during the following estrus. Blood samples were taken, beginning on Day 2 after the postmating ovulation, as an aid for distinguishing luteal developmental defects from induced early regression of a normal corpus luteum. A herd was used that had a high proportion of mares with embryo loss and small intrauterine fluid collections, excluding mares with frank pyometra. The extent of intrauterine fluid collections during diestrus was positively correlated to biopsy score, indicating that the fluid was indicative of inflammation. Furthermore, higher biopsy scores and higher fluid scores were both associated with reduced pregnancy rate on Day 11, higher embryo-loss rate after Day 11 (Table 12.8), shorter interovulatory inter- vals, and an early reduction in previously normal progesterone levels. The proges- terone levels were not significantly reduced until after Day 5 (Figure 12.7), indicating that the corpus luteum was not developmentally or initially defective (further discussion: pg. 522 and pg. 525). Accumulations of free fluid and endometrial edema due to infusion of a pathogen also have been reported in asso- ciation with early embryo loss (150). Recent reports (1502, 1068) have confirmed the association between intrauterine fluid collections and subfertility. Small amounts of echogenic uterine fluid mixed With semen extender were detrimental to fertility (1502). The uterine fluid also sup— pressed sperm motility, and the suppres— sion was reduced when the uterine fluid was diluted with a semen extender. Fluid accumulations during the first postpar- tum ovulatory period were associated with reduced pregnancy rates (1068), and apparently the extent of fluid collections reflected the degree of uterine involution. Reproductive Efficiency 521 Progesterone & biopsy scores _A N G) Progesterone (ng/ ml) 2 5 8 11 15 20 Number of days from ovulation FIGURE 12.7. Progesterone concentrations in mares in relation to biopsy score (increasing score represents increasing inflammatory change). Within each day, values with different superscripts are different (P<0.05). Adapted from (4). Information on assessing and managing postpartum mares by ultrasonic assess- ment of uterine fluid was also presented. These authors suggested the use of a grading system to record the degree of fluid echogenicity in postpartum mares (pg. 484). TABLE 12.8. Pregnancy Rates and Embryo Loss Rates According to Scores for Biopsies and Ultrasonic Intrauterine Fluid Collections Pregnancy rate Embryo-loss Technique Score Non—pregnant at Days 11-12 rate by Day 40 Biopsy 1 3/8 (38%) 5/8 (62%) 0/0 (0%) 2A 26/31 (84%) 5/31 (16%) , 3/5 (60%) 23 8/10 (80%) 2/10 (20%) 2/2 (100%) 3 3/4 (75%) 1/4 (25%) 1/1 (100%) Fluid 0 13/23 (56%) 10/23 (44%) 3/10 (30%) collections 1 14/15 (93%) 1/15 (7%) 1/1 (100%) 2 6/8 (75%) 2/8 (25%) 2/2 (100%) 3 7/7 (100%) 0/7 (0%) 0/0 (0%) Biopsy scores are ranked according to increasing extent of inflammation based on the classifications of Kenney and Doig (866). Within each technique and for each end point, biopsy score 1 or fluid score 0 is different (P<0.05) from the combined values for the other three scores. Adapted from ( 4). 522 Chapter 12 Pathogenesis of observed embryo loss (Days 10 to 20) in mares with endometri- tfi. The above described studies showed that uterine inflammation (biopsy and fluid scores) was positively associated with subfertility and that the pathogene- sis of observed losses involved early induced luteal regression. Thus, a patho— logic process (endometritis) altered a physiologic process (the uterine luteolytic mechanism) and thereby led to embryo loss. In this regard, monitoring of endogenous PGFZa levels has demon— strated that PGonc can be released pre- maturely in mares with endometritis (1136, 1537). The ability of endometrial tis- sue from mares with chronic endometri- tis to release PGFZa and other prosta- glandins has been studied in vitro (1736). The inflamed tissue released more PGFZoc than PGE, whereas there was no differ- ence between the prostaglandins in nor- mal tissue. Ovarian steroids influenced the synthesis of prostaglandins in response to an inflammatory stimulus (1742). Release of PGFZoc by endotoxins. Infu- sion of a bacterial endotoxin has been shown to cause embryo loss, due to release of endogenous PGan and associ— ated decrease in progesterone levels (371, 878, 369, 368; review: 370). Such losses were prevented by administration of a pro— gestin or a PGFZoc blocker (flunixin meg- lumine), indicating that the loss was due to induced luteolysis rather than to a direct pathogenic effect of either endotox- in or PGonc on the conceptus (367). The sequence of events associated with embryo loss due to endometritis- induced luteolysis for embryos that sur- vive to an ultrasonically detectable diameter is shown in Figure 12.8. Other studies. The relationships be- tween uterine histopathology and subfer— tility also have been demonstrated by studying the associations among results of uterine biopsy, bacterial culture, and uterine flushing of maiden mares (n=14) and subfertile mares (n=14) on Days 7 to 9 (1812, 1820). Maiden mares more often had normal—appearing embryos and had no evidence of moderate-to—severe endometritis (periglandular fibrosis) or positive bacterial cultures. Eight subfer- tile mares had fibrosis, including six which also had endometritis, and four of the six had positive bacterial cultures. In another study (1237), endometrial patholo— gy and prolonged infertility were associ- ated with a marked reduction in the recovery of embryos at Day 8. In addition, recipient mares with a normal endometri- um had a higher pregnancy rate than recipients with moderate endometrial pathology. Results of all of these studies demonstrated the association between uterine pathology (endometritis, fibrosis) and early embryo retardation and loss. Pyometra. Hughes and associates (782) have reported on large collections of intrauterine purulent material (pyome- tra). Unlike the small fluid collections described above, pyometra represents severe inflammatory change commonly associated with extensive loss of surface epithelium. Pyometra, therefore, is often associated with luteal maintenance due to absence of release of PGFZoc (782), whereas the small collections of fluid are more likely associated with premature release of PGFZa and associated luteoly- sis (described above). Deficient release of PGFZoc by endometria from mares with pyometra also has been demonstrated in vitro (1736). Day 11 pregnancies probably would not occur in the presence of pyometra; most likely, such massive quantities of purulent material would be toxic to sperm. Even if embryos did survive for a time in such material, they would escape casual detection techniques. Specific investigations on the toxicity of inflam- matory exudate to sperm and embryos are needed. In this regard, echogenic fluid flushed from mares at estrus caused reduced sperm motility (1502). The clinical aspects of pyometra were reviewed recently (1129). Reproductive Efficiency 523 SUMMARY: Embryo Loss Following ‘Lut‘eolysis NORMAL PREGNANCY EMBRYONIC LOSS Junction 0' left horn end body a Vesicle mobility Vesicle mobility Inadequate @progesterone inede uete uter ne tone Increased uterine tone Vesicle fixation Fixation ieiiure 6E Thickening ot Edoreel well Continued ex snelon Vesicle orientation end mobi ity 69 OM”... @ uterus Orientation complete Expulsion through cervix DAY 21 FIGURE 12.8. Events associated with to uterine-induced luteolysis, in associa— embryo loss due to progesterone reduc- tion with endometritis and reieaSe of tion before the expected time of fixation. PGFZoc. Although most embryos in mares For each given day, compare the events With endometritis are'lost before Day 11 of a normal pregnancy with the postu- those that survive are eliminated as ' lated events associated with embryo shown From (590) ' loss. The inadequate progesterone is due 524 Chapter 12 Defense mechanisms. An area that is receiving major research attention con- cerns uterine defense mechanisms (recent reviews: 971, 969, 1690, 1735, 107, 1635, 967). According to the reviews, there is a differ- ence among mares in susceptibility to chronic uterine infection. Resistant mares are capable of spontaneously eliminating bacteria or contaminants from the uterus. Such mares are usually young and have a healthy uterus as determined, for exam— ple, by endometrial biopsy. Susceptible mares are unable to eliminate bacteria and frequently develop a purulent dis- charge. Susceptible mares are usually older and subfertile. In this regard, older (489) and susceptible (1636) mares were less efficient at clearing material from the uterus, and older mares had reduced uterine contractility (281). Circulating concentrations of ovarian steroids also are related to uterine resis- tance. During progesterone dominance, the uterus is more susceptible to invading organisms, whereas during estrogen dom- inance, pathogens are more readily elimi— nated (1734). Mechanisms of uterine defense include the role of immunoglobu- lins, neutrophils, opsonins (substances that enhance phagocytosis by neu- trophils), physical clearance, and hormon- al status. For further consideration of the pathogenesis of uterine infections, recent reviews, such as those cited above, can be consulted. Uterine therapy. Treatment of endometritis will not be considered here, except to note that many studies on ther- apy have been done but seldom on a sci- entific basis. The results of most trials cannot be validly assessed because of the absence of randomly selected controls—a common problem in clinical settings. For example, a beneficial effect of uterine infusion of blood plasma on endometritis and reproductive efficiency has been highly touted. However, the hypothesis of a beneficial effect was not supported when tested by a conventional research protocol (2). Real progress, it seems, is being hampered by a flooding of the lit- erature with reports of trials that vio- late fundamental research principles. Approaches that may help resolve some of the current confusion in etiologic, patho- logic, and therapeutic aspects of endo- metritis involve the use of experimental models (e.g., 1734, 1298, 150, 316, 1297, 315, 489), pregnant mares (150, 149), and mares that have not been previously subjected to a myriad of therapeutic attempts (2). Bacterial culturing. Culturing of bacte- ria from the reproductive tract is a com- mon practice. For information on methodology, value, and interpretation of results of bacterial culturing, and for lead-ins to the literature, recent reports can be consulted (e.g., 736, 737). Uterine cysts. Uterine cysts are receiv- ing more attention now that transrectal ultrasonic scanning is used widely. With these instruments, cysts are readily detected (614) and questions are being raised about their relationship to subfer- tility. The incidence of ultrasonically detected cysts in a group of 73 horse mares acquired from slaughter sale barns was 15% (4). Cysts were scored 0, 1, or 2 to represent increasing severity. The pregnancy rate at Day 40 tended (P<0.1) to be reduced in the group with the high- est score. In the study of Chevalier- Clément (300), cysts were associated with a greater embryo-loss rate between means of 22 and 44 days (embryo loss of 24% in mares with cysts versus 6% in mares without cysts), but no significant effect of cysts on fetal loss (44 to 310 days) was found. A recent study, currently available only in abstract form (452), indicated that the occurrence of cysts was correlated with higher biopsy categories. Mares with cysts were significantly older (mean: 13 years) than those without cysts (mean: 7 years; 4). Older age of mares with cysts has been confirmed (281, 300); in addition, cysts were more fre— quently found in foaling mares. The direct role of cysts in subfertility is not known and will be difficult to assess because other forms of uterine pathology, as well as cysts, are more common in older mares. The role of cysts has not been partitioned from the role of other factors that also are associated with age. 12.3E. Luteal Progesterone and Pregnancy Loss Essentiality of progesterone. There is no question that luteal progesterone is essential for various mechanisms associ- ated with early pregnancy; the embryo cannot survive without a progesterone source (Figure 12.8; pg. 437). Exogenous progesterone is commonly given, some- times to all mares on a farm, and proges- terone monitoring schemes are being promoted to reduce the incidence of preg— nancy loss. The efficacy of routine proges- terone programs, however, has not been evaluated. Allen (50) and other authors have questioned the widespread use of progesterone regimens for prevention of pregnancy loss. Many reports and reviews are available on progestin prophylaxis, including discussion of products and regi— mens (e.g., 1512, 1453, 1523, 1518, 813, 35, 1134). Primary luteal deficiency in non- eguine species. Luteal insufficiency lead- ing to infertility has been described in women but apparently is not fully accepted (review: 588). The condition is said to be characterized by a short luteal phase, sometimes occurring repeatedly in an individual or by repeated early pregnancy loss. A report in sheep also indicated an association between proges- terone profiles and embryo survival; embryo loss was associated with a later rise in progesterone and lower mid- luteal concentrations. In cattle, a posi- tive relationship between luteal—phase progesterone concentration and pregnan- cy rate has been found by some workers but not by others. Lower concentrations of circulating progesterone were found on Day 6 in repeat-breeders. In several Reproductive Efficiency 525 studies, progesterone supplementation increased the pregnancy rates in cattle, but the pregnancy rates in control groups were low. A recent study (1446) in subfertile cattle found indications that luteal inadequacy, due to diminished response to luteotropic hormones, may contribute to embryo loss. Primary luteal defects versus induced luteolysis. As noted above, defective physiologic mechanisms, unassociated with microscopic or macroscopic patho- logic processes, have not been implicated convincingly in embryo loss in mares. This includes defects in function of the primary corpus luteum and defects in the uterine luteolytic mechanism. The terms luteal insufficiency, inadequacy, and deficiency have been used to refer to hypofunction of the corpus luteum. These terms do not clarify whether the primary defect in the corpus luteum occurs during development (full function not attained) or after development (full function attained but not maintained). As a further complication to terminology in this area, failure to maintain function could result from induced luteal regres- sion (luteolysis) of a normal corpus luteum (activation of uterine luteolytic mechanism) or from a primary defect in a fully developed corpus luteum. Herein, the term primary luteal defect will be used to refer to a basic or primary prob- lem inherent in the corpus luteum or the ‘ pituitary-luteal axis. The defect can involve a developing or fully developed corpus luteum; the defect can be within the corpus lute11m or can involve a mech- anism required for luteal development or maintenance (e.g., inadequate LH). Primary luteal defect from failure of adequate development. The profound role of early activation of uterine-induced luteolysis in embryo loss traceable to endometritis was discussed above (pg. 522). Some of the same studies that have impli- cated induced luteolysis have failed to implicate a primary developmental luteal defect. In mares that were subfertile or 526 Chapter 12 had high biopsy scores indicative of endometritis, progesterone concentrations were significantly reduced on Day 8 and thereafter but not on Days 2 and 5 (Figure 12.7; 4). These results supported the hypothesis that subfertility was due to early regression of the corpus luteum (secondary to uterine inflammation) and was not due to a primary developmental luteal defect. Another study (524) failed to find a difference in progesterone levels between pregnant mares and mated non- pregnant mares before Days 9 to 11. Monitoring progesterone levels of 16 mares that lost their pregnancies 2 to 5 times over 6 years also apparently did not indicate a primary luteal insufficiency (374). A recent report (372) concluded that primary luteal insufficiency was not observed in several habitual aborters, although one mare repeatedly had an apparent slow and small postovulatory rise in progesterone. Additional study is needed, especially in view of the contin- ued pressure to support pregnancies by exogenous progestins. In conclusion, a reduction in proges- terone concentrations is clearly part of the pathogenesis of embryo loss due to endometritis, but primary luteal insuffi- ciency from an undeveloped corpus luteum has not been shown to be involved in embryo loss before Day 25 during the normal ovulatory season. However, a primary developmental luteal insuffi- ciency can be associated with ovulations induced during seasonal ovarian inactiv- ity (pg. 168). Classifying mated nonpreg- nant mares as having luteal insufficiency when progesterone levels are reduced on Day 12 (424) does not seem appropriate since progesterone levels decline by this time during some normal estrous cycles. Can progesterone therapy override luteolysis due to endometritis? It is not known whether exogenous progesterone would salvage some ultrasonically observed embryos in the face of activation of luteolysis by endometritis, and this question deserves investigation. It has been shown that pregnancies can be res- cued several days after experimental withdrawal of endogenous progesterone on Day 12 (PGFZoc injection or ovariecto- my; 850), but adequate studies have not been done in the known presence of endometritis. An attempt was made to reduce the incidence of pregnancy loss with an injection of synthetic progestin after impending loss was suspected on Days 20 to 60 (661). Impending loss was based on a decrease in size of the concep- tus as determined by transrectal palpa- tion. No effect of the progestin was found. Perhaps embryo death (cessation of heartbeat) occurred before the rescue attempt. In a preliminary study (373), flu- nixin meglumine (PGFZOL synthesis inhibitor) or a progestin (altrenogest) was given to two mares with indications of endometritis and classified as habitual aborters; pregnancy was maintained in both mares. Endogenous progesterone declined, and the primary corpus luteum regressed in the mares treated with the progestin. Adequate rationale now seems available to encourage critical testing of the hypothesis that progesterone supple- mentation may help prevent some cases of embryo loss due to endometritis. In evaluating this potential approach, it also should be considered that a progesterone milieu may exacerbate a uterine infec- tious process (24). , Occurrence of primary luteal insuffi; ciency in a fully developed corpus luteum. Embryo loss that is associated with endometritis and early activation of luteolysis will already have occurred by Day 20, or the embryo, if not yet lost, will be mobile (failure of fixation) and will probably be expelled through the open cervix during estrus (pg. 544). The inci— dence of embryo loss during Days 20 to 40 was not different between mares with endometritis and normal mares (pg. 514). Apparently, endometritis is not a major cause of embryo loss on Days 20 to 40; the embryos are usually eliminated before Day 20. The characteristics of 21 embryo losses were described (183). Thirteen (62%) of the losses occurred before Day 20 and 3 of the 13 (23%) were associated with mainte- nance of the corpus luteum (Figure 12.9). Eight (38%) losses occurred after Day 20; 4 of 8 mares (N, O, P, Q; Figure 12.9) with embryo death on Days 20 to 40 had a maintained corpus luteum with an interovulatory interval of approximately 60 days. In one of the four remaining mares (Mare S), an influx of intrauterine fluid (probably inflammatory exudate) occurred suddenly on Day 35 and was accompanied by both embryo death and luteal regression; apparently this mare developed acute endometritis. In another mare (Mare U), the output of progesterone was continuously low (2 to 4 ng/ml) but was adequate to delay ovulation, resulting in an interovulatory interval of 114 days. The mare was 17 years old, and the uterus was chronically flaccid (estrus- like). Perhaps this was an example of luteal insufficiency, but progesterone was not assayed until Day 12; therefore, it is not known whether the defect in the cor- pus luteum was present initially or occurred later. In 2 of 8 mares (25%) with embryo loss after Day 20, an apparent decline in progesterone occurred prior to cessation of heartbeat (Mares R and T). In Mare T, the corpus luteum regressed, and the embryonic vesicle became dislodged while the embryonic heart was still beat— ing. Cessation of heartbeat occurred on the following day. Dislodgement before cessation of heartbeat also has been noted in another report (281) and in mares treat- ed on Day 30 with PGan (589). Apparently dislodgement is a sensitive indicator of inadequate progesterone. In conclusion, these data suggested that primary luteal insufficiency can occur on Days 20 to 40 and is responsible for embryo loss in a minority of mares (appar- ent incidence: 2 of 8 losses on Days 20 to 40; 25%); however, the involvement of nonluteal factors (e.g., defective embryo or uterus) was not eliminated. Reproductive Efficiency 527 Characteristics of 21 embryo losses N o. 101 mares (days) Loss Day 20 CL maintained (Mares N, O, P, Q) 4 62—68 CL regressed Mare R . . 66 Mare S . . 54 Mare T Anov Chronic low progesterone Mare U . . 114 Progesterone & embryo death Pregnancy maintained (n=52) ,1 i2SD 16 Mean \ Concentration (ng/ ml) ca 420730 O (I) Z | L T'.-- —.._. -.._. —~.—..'_¥. Heartbeatlcessation (Mean, Day 31) -12-9-6-3036912 Days in relation to cessation of embryo heartbeat FIGURE 12.9. Characteristics of 21 embryo losses including length of interovulatory intervals (IOI), progesterone concentrations in reference to cessa- tion of embryo heartbeat, and word descriptions for individual mares. Adapted from (183). Mares N,O,P Q: Maintenance of progesterone production; probably pseudopregnant. Mare R: Progesterone appeared to begin a decline before cessation of heartbeat and reached nondetectable levels 12 days after embryo death. Mare S: Precipitous decline in progesterone, heartbeat cessation, and large collection of echogenic intrauterine fluid on the same day (acute endometritis). Mare T: Gradual decline in progesterone before heartbeat cessation. Vesicle dislodged and became mobile the day before embryo died. Mare U: Uterus remained flaccid (estrus-like) and progesterone remained low (2 to 5 ng/ml) over Days 12 to 112. Embryonic vesicle was oversized (e.g., 40 mm on Day 27), did not develop a detectable embryo proper, and ruptured on Day 42. 528 Chapter 12 In another study (808), progesterone monitoring was done in a herd of 179 mares beginning on the day of pregnancy diagnosis (Days 17 or 18) and continuing to Days 42 to 45. The authors concluded that an apparent decline in progesterone did not precede pregnancy loss in any of 15 mares, with one possible exception. In another study (1408), 2 of 4 mares with an apparent decline in progesterone lev- els before loss was diagnosed and the other two did not. However, the method of determining day of loss was not stat- ed; perhaps the day of embryo death (cessation of heartbeat) preceded the day of diagnosed death, leading to the impression of a preceding progesterone decline. 12.3F. Pseudopregnancy Definition and cause. Pseudopreg- nancy in the mare has been defined as the development and maintenance of uterine turgidity and maintenance of the corpus luteum (pg. 228). It has not been convincingly demonstrated in non- mated mares. Pseudopregnancy can be considered an expression of embryo death after the embryo successfully blocks the uterine luteolytic mechanism and initiates development of uterine turgidity in the absence of other factors that would actively initiate luteolysis (e.g., endometritis). Blockage of luteoly- sis appears to be initiated by Day 11 and is completed by Day 15 in pregnant mares (pg. 438) and presumably in pseu- dopregnant mares. The embryonic vesi- cle can disappear, presumably from collapsing, before Day 15, and yet pseu- dopregnancy sometimes occurs. Perhaps the products or fragments of the vesicle can linger long enough to complete the blockage of luteolysis. A similar phe- nomenon has been reported in ewes (457). Manual rupture of the embryonic vesicle on Days 12 to 14 resulted in pseudopregnancy in 7 of 7 mares (583). Insertion of cultured trophoblastic vesi- cles on Day 10 resulted in luteal mainte- nance even though the trophoblastic vesicles did not grow to a diameter detectable by ultrasound (142, 139). Surgical removal of the embryonic vesi- cle on Day 24 also was followed by pseu— dopregnancy (906), which is consistent with the occurrence of pseudopregnancy in most mares following embryo loss after Day 20. Progesterone concentrations and diameter of ultrasonic images of the cor- pus luteum were initially similar between pregnant mares and mares with a prolonged interovulatory interval after embryonic loss (183). After Day 35, however, a resurgence in growth and output of the primary corpus luteum occurred in pregnant mares but did not occur in the mares with embryo loss. Instead, the circulating levels of proges- terone gradually decreased during this time (Figure 12.10). Anecdotal observa— tions indicate that the blockage of lute- olysis can be partial or complete (pro- gesterone levels similar to those of pregnant mares). When apparent par— tial blockage occurs, the progesterone levels decline (e.g., 50%). Reported inci- dences of partial blockage of luteOlysis are 1 of 4 in pseudopregnant mares (590) and 1 of 6 in mares with a maintained corpus luteum (uterine tone not exam- ined; 183). Incidence. The frequency of pseudo— pregnancy after embryo loss on Days 11 to 20 has been reported (604) as 29% (12 of 41). Similarly, maintenance of the corpus luteum (interovulatory interval >30 days) occurred in 3 of 13 mares (23%) with losses on Days 11 to 20 (Figure 12.9; 183); however, uterine tone Resurgence of primary corpus luteum 27 Corpus luteum Pregnancy maintained (n=21 to 52) N 01 Diameter (mm) M w 21 Embryo loss [ i (n=7 to 9) | 19 12 Progesterone _L O 0') Concentration (ng/ml) as 21 24 27 30 33 36 39 42 45 48 Number of days from ovulation FIGURE 12.10. Resurgence in diameter and func- tion (progesterone concentrations) of the primary corpus luteum in pregnant mares and absence of resurgence in mares with embryo loss and main- tained corpus luteum. The group-by-day interaction is significant for both end points. Stars indicate sig- nificant difference between groups for the indicated days. Adapted from (183). was not determined, and it cannot be stated unequivocally that the mares were pseudopregnant. Most losses before Day 20 are not accompanied by pseudopregnancy or a maintained cor- pus luteum; such losses are usually attributable to endometritis which would likely induce luteolysis. Appar- ently (not directly studied), if the uterus Reproductive Efficiency 529 is healthy, losses before Day 20 can be associated with pseudopregnancy. Pseu- dopregnancy occurs in association with most losses after Day 20 since blockage of luteolysis will already have been com- pleted. In one study (604), 8 of 8 losses after Day 20 involved pseudopregnancy. In a recent study (183), definite mainte- nance of the corpus luteum occurred in 4 of 8 losses (Mares N, O, P, Q; Figure 12.9). Research needs. A maintained corpus luteum can result either from an absence of luteolysis associated with severe endometritis (e.g., pyometra) or from failure of an embryo to block the luteolytic mechanism in mares with a healthy uterus. Studies on the condition defined as pseudopregnancy are there- fore challenging since it is necessary to demonstrate not only that the corpus luteum is maintained but also that uterine tone is greater, on the average, than during diestrus and is equivalent to that of early pregnancy. Development of an objective method of measuring uterine tone is needed. This remains the primary technologic obstacle to confirm- ing and further elucidating the pseudo- pregnancy phenomenon and studying the relationships of embryo loss to uter- ine turgidity. Until such techniques are developed, uterine turgidity is best assessed without knowledge by the oper- ator of experimental groups or days. 530 Chapter 12 PHOTOGRAPHIC PLATES The various forms of uterine pathology 12.13), and ultrasonograms of various that effect reproductive efficiency will pathologic processes (Figure 12.14). not be further discussed here. Reviews Figures 12.11 and 12.12 were prepared noted above (pg. 499) can be consulted. by G. P. Adams. However, the following illustrations are A diagrammatic summary of the pos- presented, especially for the interest of tulated associations among inflamma— biologists: Videoendoscopic views (Figure tion of the oviducts and uterus, life of 12.11), photomicrographs (Figure 12.12), the corpus luteum, and pregnancy loss is a diagram of uterine pathology (Figure given in Figure 12.15. FIGURE 12.11. Facing page. Videoendoscopic Views of various forms of uterine (A to H) and cervical (I to K) pathology. Uteri were filled with air except in views D and E. A. Slightly cloudy exudate in the uterine lumen. The fluid collection was charac- terized by a thin nonechogenic line on ultrasonic examination. Mucopurulent exudate. An intrauterine fluid collection (diameter, 10 mm) was detected ultrasonically. Mucopurulent exudate following fetal loss. A ring of endometrial cups are visi- ble along the rim of the exudate pool. Exudate clinging to endometrium after flushing the uterus with saline. Film of mucopurulent exudate that was present throughout the uterus. The structure on the left is the intercornual septum. Calcified endometrial cups following fetal loss. Two cystic compartments 4 mm and 5 mm in diameter. Large cystic complex several centimeters in diameter. The complex protruded into the uterine lumen during several minutes of continuous viewing, as Shown, and then receded from the lumen so that it was no longer Visible by the Videoendoscope. Deviated cervix adherent to the wall of the vagina by fibrous bands. The cervi- cal os is to the right. The adhesive bands were dense and cast shadows when viewed ultrasonically. Tear in the cervix at seven o’clock. The tear was readily detectable by digital examination. Fibrous finger-sized cervical mass at two o’clock. Finger is in the cervical 0s. The mass produced shadows during ultrasonic examination. 531 Reproductive Efficiency Chapter 12 532 Reproductive Efficiency 533 FIGURE 12.12. Facing page. Photomicrographs of various forms of endometrial pathologic processes. A. Low-power view of local infiltration of mononuclear leucocytes in the stratum spongiosum (chronic inflammation). B. Same as A. High-power View. C. Mononuclear leucocyte infiltration in the stratum compactum. The luminal epithe- lium is pleomorphic. D. Acute endometritis with infiltration of polymorphonuclear leucocytes and erosion of the luminal epithelium. E. Glandular nest with cystic dilation and severe periglandular fibrosis. F. Severe cystic dilation of fibrotic glandular nests. G. Dilated lymphatics. H. Low power View of a calcified endometrial cup following fetal loss. The calcifica- tion was confirmed by a calcium-specific stain. The mineralized cups were evident ultrasonically by discrete areas of echogenicity and shadowing. The endometrial glands beneath the cup are dilated. Chronic Epithelial inflammation pleomorphism Pregnancy Thickened ‘ bosemenl fin”: " ‘um a u membrane ‘nl‘fli‘l'uln _ !!!‘...E_.; :"'”.f!!.'!a §Ffl$£L5 I '3 L - .w» ‘ ’a'n~~: . _ ’.‘ . 1. ~f' g" .: '1‘, 9." ’a'.(:' 1" =j‘7‘)x .‘ w: . . .. _ LymphOPy'e i . . . ‘ ”if.” :9 E; . ’ . ' . . stGSIs ' :37." .35 5; . ’ _ r, 7‘ Morgmahan ' ~ 0 "3‘; ,1’) E g) “7‘“ i, ck \XJ \\ r of PMNS Lymphatic ‘ _,\_ ,’ ”ll/.1; 52 :5; . N j v\ - A ‘ '1 ‘ '5‘ k; . > lacuna ,_. 3 :Y‘ ”2/. /“. .9,‘ > . JA 5' a /‘$“.I'& _. " 2 Chrome -\ , W Q _ g I ‘ ~ .9 ~/ . ‘ ¢,. ,. : reaction :( t 7/ :5 ’M‘kil‘” [,5 E3, ’ r’ ‘ ‘ ‘ .- ’ ~ .~ 3 ‘iv// 2?} E”: N i i; 5' / w’ 3'. :: . a g: . . F“ t; . Penglandular "D 91' Gym: fibrosis \ f; E. dilatation 53" =2 ,2 .: :r .~ 5 ES . 7‘ g '3‘. A, Ll _ " t? a; Penglandular 5; :2 . fibrosis p g, ‘3 7 \‘-(_P) . I .15 FIGURE 12.13. Dia- gram prepared by Kenney (review: 1661) showing pathologic changes in the endo- metrium. ‘-.VVO“)U y kiwi-V3333}; . 1" ' p. €03»? '0 ll '9‘:3 D" ,3 _ , 534 Chapter 12 FIGURE 12.14. Ultrasonograms of various pathologic processes of uterus and cervix (width of each ultrasonogram: 50 mm). Small intrauterine fluid collection in a uterine horn. Similar to Figure 12.11,A. Larger intrauterine fluid collection. Same as in Figure 12.11,B. Pyometra. The two circumscribed structures are uterine cysts. Same as in Figure 12.11,D,E,I. Degenerating, calcified endometrial cups surrounded by 20 mm fluid collection. Distinct shadows beneath calcified cups. Same as in Figure 12.11,F. Fetal bone fragment following fetal death. Note the distinct shadow. Small uterine cysts. Same as in Figure 12.11,G. Uterine cysts in the uterine body. Shadows resulting from cervical adhesions. Same as in Figure 12.11,I. HEQWEU CPU?” Reproductive Efficiency 535 l. Defect in embryo or its support system A. Return to estrus B. Pseudopregnancy CORPUS LUTEUM 2. Regresses at normal time Maintained OVIDUCT 1 Dies before Dies after blockage ' blockage of uterine Iuteolytic of uterine mechanism and Iuteolytic stimulation Cdf ' uterine turgi ity mechanism EMBRYO (—— Turgid UTERUS ll. Diseased oviducts & uterus A. Salpingitis & endometritis B. Pyometra 2. Regresses due to activation of uterine iuteolytic mechanism by endometritis Maintained due to loss of PGF- producing uterine cells 1. Dies or retarded due to inflammatory toxins or poor nutrition Purulent exudate (probably toxic to sperm) l II‘ 3. Survivors and remnants discharged at estrus ..O.......o......o...... FIGURE 12.15. Postulated interrelationships among corpus luteum, oviduct, uterus, and embryo depending on whether the primary defect is in the embryo or oviduct and uterus. 536 Chapter 12 12.3G. Role of Embryo Defects Theoretically a defective embryo could originate from chromosomal or other anomalies, involving either of the gametes or resulting from aberrations associated with fertilization or cleavage. Age-related or other factors (e.g., temper- ature) also could have a detrimental effect on the gametes, fertilization, or early development (see also pg. 540). Age effects may be from old animals (sire or dam) or gametes that aged before fertil- ization. A long interval from insemination to ovulation would result in aging of sperm, and postovulatory breeding would result in aging of ova. Although detailed studies on this subject have been done in laboratory animals, only limited consider— ation has been given to horses. It is doubtful that these factors would play a major role on a herd basis under farm conditions unless an unusual mating pro- gram was used that detrimentally affect- ed age or condition of gametes. The stage at which intrinsic or acquired primary embryo problems cause a reduction in reproductive efficiency has received little attention. Presumably, extreme defects would preclude normal fertilization. Pregnancy loss after a successful, but defective, fertilization has not been demonstrated in horses. Losses possibly due to primary embryo defects. It is difficult to document losses due to defects in embryo development that are not attributable to oviductal or uterine abnormalities. It is well docu- mented that embryonic vesicles that are undersized during the mobility phase have a high probability of later loss (pg. 542), but it has not been established whether any of these losses are due to inherent defects. The following case descriptions of embryo loss seem compati- ble with intrinsic defects; 1. An embryo that resulted from in vitro fertilization was first detected at Day 17 (7 mm), grew to 22 mm at Day 27, and remained at this approximate size without development of a detectable embryo proper before disappearing at Day 46—the mare was given exogenous progesterone in an attempt to maintain the embryo (281); 2. An embryonic vesicle was first detect- ed at Day 15 (3 mm), grew to 13 mm, and remained mobile before loss at Day 28 (281 ; similar losses described in 590); 3. An embryonic vesicle with an abnor- mally dark inner cell mass grew slowly after embryo transfer, a detectable embryo proper did not develop, and the vesicle was gone by Day 26 (1064); 4. Four embryos that had been subject- ed to a cooling procedure had a post- transfer history similar to the description in Point 3 (284); 5. An embryonic vesicle was oversized (40 mm at Day 27), did not develop a detectable embryo proper, and collapsed on Day 42 (183); and 6. A high rate of embryo loss occurred on Days 15 to 20 (19%; 1824) and on Days 16 to 25 (38%; 912) in mares inseminated after ovulation (pg. 537). Except for the spontaneous losses (Points 2 and 5), the embryos resulted from or were subjected to artificial proce- dures (in vitro fertilization, transfer, or cooling) or postovulatory insemination; these factors could have played a role in the development of a defect, perhaps in the inner cell mass or embryonic disc. In this regard, the freeze-thaw process is more detrimental to the cells of the inner cell mass than to trophoblastic cells (158, 1799); perhaps, therefore, embryo defects are more likely to involve the inner cell mass. Although the losses described above are compatible with a primary embryo defect, the possibility of damage imposed by an adverse oviductal or uter- ine environment was not eliminated. Defective chromosomes. Flagrant chro— mosomal abnormalities in horses have been described. Genetic gender (XX or XY) is established at fertilization, which then leads to gonadal and somatic (body) gender. The following aberrations have been described in horses: 1. KO embryos that become small infer- tile females; 2. XY embryos with defective testes— determining genes that become pheno- typic females with dysgenic gonads; 3. XY embryos with defective androgen metabolism that become intersexes or females with intra—abdominal testes; and 4. XY embryos that undergo sex-rever- sal syndrome, leading to phenotypic mares with the karyotype or chromoso— mal makeup of a stallion. These syndromes will not be further discussed; recent reviews and lead-ins to the literature are available (e.g., 869, 1321, 1407, 870, 1564, 979, 667, 1290, 898, 1457, 1637, 239). Whether or not embryos with these karyotypes have a higher risk of loss has not been established. The incidence of chromosomal abnormal- ities is unusually high (50 to 60%) in spon- taneously aborted human conceptuses and especially in the conceptuses of older women (review: 1791). In nonequine domes- tic species, the frequency of chromosomal abnormalities in aborted fetuses and still- bOrns was 12% in one study (cited in 1791). Early embryos (Days 2 to 16) had a fre- quency of anomalies of 8% to 15% in vari- ous species, but it is unknown what pro- portion would have resulted in pregnancy loss. These data cannot, of course, be extrapolated to horses. Chromosomal abnormalities were not found in 22 and 122 equine embryos subjected to karyotyp- ing (cited in 138) or in 26 aborted fetuses (217). In another study (cited in 1769), only one case of chromosomal abnormalities was found in 30 aborted horse fetuses (35 days to term). Gamete aging. A prolonged interval from mating to ovulation (>3 days) or from ovulation to mating (>12 hours) resulted in reduced Day 11 pregnancy rates in mares (pg. 299). However, it was not determined whether this reduction was due to failure of fertilization or to embryo loss between the day of fertiliza- tion and Day 11. A significant increase in Reproductive Efficiency 537 embryo loss occurred in mares mated after ovulation compared to before ovula- tion (1824). The increased loss rate occurred on Days 15 to 20 (loss rates: 7 of 36 versus 1 of 42). Similarly, in another study (912), embryo loss occurred between Days 16 and 25 in 5 of 13 pregnant mares bred after Ovulation and in 0 of 13 bred before ovulation. The relationship, if any, between these observations and the age- related effects is not known. If the prob- lem began in association with fertiliza- tion, it was not expressed continuously since loss rates on Days 11 to 15 were not altered (1824). Hunter (786) has dis- cussed the effects of gamete aging on the mechanisms of oocyte penetration and fertilization in horses based on extrapo— lation from studies in other species. Embryos from postovulatory insemina— tion were significantly reduced in diameter (pg. 300). Postovulatory mating causes asynchrony between hormonally con— trolled stage of the endometrium and age of the embryo. The asynchrony expected in this study (1824) was well within the wide limits of acceptable asynchrony of uterus and embryo found in embryo transfer studies (pg. 339). The time of occurrence of loss (Days 15 to 20) is com- patible with failure of the embryonic vesi- cle to block uterine-induced luteolysis, whether due to fertilization-related defects, asynchrony, or other factors. Breed and Sire. No effects of breed or sire on pregnancy loss were found in the study of Chevalier-Clement (300). The study involved five breeds or types (Thoroughbreds and Trotters, saddle, draft, and pony types) and 261 stallions. Sire effects were reported for individual stallions in earlier field studies wherein use of certain individuals resulted in a higher pregnancy-loss rate (cited in 138, 300). Field reports are difficult to evalu- ate; random selection of animals for vari— ous test groups is not compatible with field conditions. Venereal transmission of contagious diseases or contaminants could be grouped under sire effects (138). 538 Chapter 12 Immunologic aspects: Results of recent studies have suggested that the coeffi- cient of inbreeding (894) and specific dam alleles (1002) could affect foaling rates. Abstracts from the Soviet Union state that immunogenetic factors are involved in fertility. Fertility decreased as the blood type differences of mating pairs increased (1286). For example, in immuno- logically compatible matings (stallions and mares of same blood type), the foal— ing rates were 73% to 74% and abortion rates were 10% to 11%; for immunologi— cally incompatible matings, the corre— sponding rates were 62% to 70% and 13% to 16% (significant difference between compatible and incompatible matings; 1447). The increased abortion rates would account for the decreased foaling rates. Selective matings were used in another study (449). Out of 42 mares bred for a homozygous fetus, the pregnancy rate, abortion rate, and foaling rate were 79%, 17%, and 62%. For 52 heterozygous pair— ings, the corresponding figures were 94%, 6%, and 80%. These interesting results from the Soviet Union indicate an associ- ation between dam and sire blood types and infertility, and the relevance of these findings to breeding programs in other countries should be investigated. Allen and associates (59, 60, 926) have utilized between-species transfer of horse and donkey embryos as a research tool for study of fetal-maternal interactions and the immunologic aspects of fetal loss. In horses with donkey fetuses, the allanto— chorion (donkey) is attacked by a mater— nal (horse) cell-mediated reaction. It was proposed that the donkey cells do not pro- vide adequate antigenic stimulation of the horse immune system. Another recent report (1854) discusses the causes of steril- ity with a gradual progression to fertility in hybrids of the horse and donkey. The immunologic aspects of pregnancy in mules is being studied by Antczak and associates (86). In recent case histories (1852, 1224), anti- sperm antibodies were found in two stal- lions following testicular trauma. It was suggested (1852) that trauma may have compromised the blood-testis barrier, resulting in high titers of antisperm auto— antibodies. This report also presented data that the antibodies may have caused infertility based on pregnancy rates; the authors considered their results to be cir- cumstantial, however, and a link between infertility and autoimmunity has not been established. In man, however, the pres- ence of antisperm antibodies in the gener- al circulation or genital tract of either sex has been accepted as being associated with infertility (review, including discus- sion of the two stallions reports: 242). 12.3H. Other Aspects of Pregnancy Loss Stress. Transportation, confinement, extremes in temperature, and social sepa- ration have been reported to be stressful in domestic animals, leading to hormonal imbalances and embryo loss (cited in 162). The effect of transportation during the third and fifth week of pregnancy on embryo loss in mares was examined in a controlled study by Colorado workers (162). Mares were hauled for nine hours. Transportation caused elevated cortisol and progesterone levels and other plasma changes indicative of acute stress but did not alter the incidence of embryo loss. Post—transportation progesterone levels decreased but not below those of control mares. Additional studies are needed involving longer transport and mares that are later in pregnancy. Short-term stress (e.g., restraint with a twitch for 5 minutes; anesthesia) caused an in- crease in cortisol and PRL concentrations but did not alter LH and FSH concentra- tions (1597). Progesterone concentrations in most mares begin to increase in the early fetal stage due to resurgence of the primary corpus luteum and the development of secondary and accessory corpora lutea. Placental progestins appear in the maternal circulation by approximately Day 60 (pg. 445). Critical information on fetal losses (>Day 40) due to inadequate progesterone or progestins is not avail- able, but there are anecdotal accounts and indications that stress can reduce circulating progestins to dangerous lev- els. In an initial monitoring study during the fetal stage (1117), plasma progestin concentrations prior to fetal loss (inter- val from sampling to loss not clear) were lower than in control mares, and loss could not be attributed to infectious causes. In fetal-loss mares in which infectious agents were isolated, the plas- ma progestin concentrations were within one standard deviation of those for con- trol mares. Ten of 13 losses that were associated with low progestin concentra- tions occurred before 135 days, and 7 of 8 that were not attributable to reduced progestins occurred on 180 to 270 days. Other studies by the same workers have raised the question whether fetal loss could result from progestin depression in association with stressful conditions (pain, disease, weaning, withdrawal of the concentrate portion of a ration; 1675). A dramatic drop in plasma progestins was reported to occur in mares with colic, navicular disease, and acute babesiosis. The depressive effect of stress on pro- gestin concentrations may be mediated by cortisol. A transient drop in progestins occurred in each of two pregnant mares treated with prednisolone (1672). An anec- dotal account (1118) concluded that nurs— ing can have a detrimental effect on maintenance of adequate progesterone levels, resulting in pregnancy loss. A recent abstract (1391) further suggested an association between stress (colic) and loss of fetus; stress was associated with increased circulating cortisol and a possi- ble increase in fetal loss (fetal-loss rate: 4 of 16; controls not available). Progestin concentrations in mares with fetal loss dropped to low levels, but the temporal relationship between progestin decrease Reproductive Efficiency 539 and fetal loss was not clear. Experimen- tal models are needed to test the hypoth- esis, suggested by these observations, that stressful conditions during the fetal stage, especially after the fetus becomes dependent on fetoplacental progestins, can reduce progestin levels and thereby lead to fetal loss. Progesterone deficiency during fetal stage not related to stress. An anecdotal report (1119) states that pregnancy losses in several mares with singletons were due to inadequate luteal function, evi- dently compounded by an inability of the placenta to adequately complement'the luteal output. In addition, progesterone (progestin) deficiency was the suggested cause of pregnancy loss in three mares carrying twins; concentrations were not at the expected high levels characteristic of twins. These two reports are currently available only in abstract form and can- not yet be adequately evaluated. Mares with an energy-deprived diet (mean loss in body weight: 8 kg/day) tended (P<0.07) to have increased fetal loss (1288); how- ever, the losses were not clearly attribu- table to progesterone deficiency. It also has been hypothesized that light-treated mares mated before the nat- ural mating season may undergo abortion when put on lush pasture in the spring, due to stimulation of estrus with accom- panying cervical relaxation (1580); accord- ing to this hypothesis the relaxed cervix allows pathogens to enter the uterus. This hypothesis apparently has not been tested. Yearlings. A high rate of pregnancy loss has been reported for 137 yearling mares run in small groups with stallions (1105). Pregnancy was diagnosed in 69% of mated mares, but 44% of the pregnant mares lost the pregnancy by Day 160. The losses were evenly distributed over Days 60 to 140. The reason for the high loss rate in this group of yearling mares was not determined, and confirmation of a high loss rate in yearlings is needed. In this regard, in a recent study of feral 540 Chapter 12 mares (998), the estimated fetal loss rate (120 days to term) in mares in their third, fourth, fifth, and sixth summers were 70% (7 of 10), 46% (19 of 41), 17% (5 of 30), and 6% (1 of 18), providing further indication that loss-rate may be high in young mares. Whether the apparent age- effect is a reflection of nutrition or other factors is unknown. Old age. It is well documented that preg- nancy loss is frequent in old mares. In recent studies, pregnancy loss was greater in Standardbreds and Trotters that were older than 15 years (1627), greater in Standardbreds aged 20 to 26 years (26%) than in mares aged 10 to 11 years (10%; 1689), and greater in ponies that were >15 years (62%) than in mares that were 5 to 7 years (11%; 281). Rates of embryo loss (beginning on Days 14 to 22 for the vari- ous studies) and fetal loss for some field studies, with data partitioned according to age, are shown (Figure 12.16). Study of the figure indicates that the rate of both embryo loss and fetal loss increased after approximately 15 years of age. As a conve- nient number, the loss rate after about 15 years was approximately doubled. The age factor is confounded by other factors (e.g, multiparity, endometritis, salpingi- tis, uterine cysts, vulvar conformation, and dental and body condition). The con- tribution of each age—related factor would be difficult to determine in uncontrolled field settings and expensive to study in controlled research settings. Confounding between age and uterine pathology is strong (1721), and likely it would be diffi- cult to assemble a large group of old mares with healthy uteri. Old age was associated with increased uterine inflammation (based on histo- pathology and intrauterine fluid collec— tions), reduced uterine contractility and tone, later fixation of the embryonic vesi- cle, reduced Day 12 pregnancy rate, and increased embryo-loss rate (281). Uterine impairment (reduced contractility and tone) is consistent with the reports (489, 1636) that physical clearance of material Age & pregnancy losses 3'0 Embryo losses I. 20 _L O O 03 0 Rate of loss (%) Fetal losses I 20 10 Years of age FIGURE 12.16. Reported relationships between age of mare and pregnancy loss. Ages shown are the mean ages in various age classification groups, and the oldest age includes ages greater than the indi- cated age. Adapted from tabulated data (A, cited in 138; B, 1810; C, 300; D, cited in 138; E, 453; F, 300). from the uterus is deficient in old or endometritis—susceptible mares. Perhaps the pathogenesis of low pregnancy rate and high embryo-loss rate in old mares involves, in part, the following cyclic pro- gression: 1) reduced uterine contractility and tone, 2) reduced clearance of bacteria and foreign material, and 3) increased incidence of endometritis (Figure 12.17). In a review of biopsy records (419), it was found that increasing biopsy scores (increasing degree of endometrial fibrosis) was associated with increasing average age of mares. A study currently available as an abstract (1754) has confirmed that ~~~~~ —— ‘~ Reduced , contractility ‘ & tone /\ ‘ Increased Reduced \ incidence <—* clearance , of metritis of bacteria ,1 ~ — ———————— Reduced pregnancy rate Increased pregnancy-loss rate FIGURE 12.17. Hypothetical manner in which cyclic or self-perpetuating uterine events may con- tribute to reduced reproductive efficiency: young mares usually have histologically normal endometria (only 6 of 45 mares <6 years were Category 2 or worse) and old mares usually have deteriorating endometria (only 2 of 51 mares aged 20 to 25 were Category 1 or high 2). Late entry. The effect of age and parity on the development of chronic endo‘met'rial degeneration, based on uterine biopsy, has been studied (1869); mares aged 17 years or older were more likely to have severe degeneration. One investigator (300) has examined the parity question using mathematical mod- els. Embryo-loss rate appeared to increase with parity and was significantly greater after 6 to 10 pregnancies (10%) than after 1, 2, or 3 to 5 pregnancies (3 to 6%). However, fetal-loss rate increased significantly only between the first and second pregnancies (5% and 9%) and appeared to remain unaltered, thereafter. The proportion of old mares in farm mat- ing programs is relatively small (e.g., 10%; Figure 12.4,pg. 507). It is not known whether aging in mares has an effect on pregnancy loss, distinct from the loss due to pathology of Reproductive Efficiency 541 the tubular genitalia. Delayed and abnor— mal development of the blastocyst occurs in rats in association with aging and is believed to reflect impaired oocyte quality or altered oviduct environment. One studyy(385) showed a reduction in number of blastocysts in old rats (6/rat) compared to young rats (ll/rat) without a decline in ovulation or fertilization rates. In another study (386), middle-aged rats had a high incidence (24%) of abnormal embryos at Day 3 compared to young rats (5%); the problem was attributed to defective oocytes, rather than to hormonal deficien- cies. A similar age-related reduction in reproductive efficiency occurs in other laboratory animals. On a comparative basis, therefore, old age in mares may increase the incidence of defective oocytes, leading to problems in fertiliza- tion or embryo survival. The only available study in mares found a high fertilization rate (Day 2) in old subfertile mares (pg. 502). The compar- ative indicators are compelling, however, and the question of fertilization rates in old mares cannot be considered resolved. Therefore, the reported reduction in num- ber of embryos between Days 2 and 4 in old subfertile horses (pg. 511) could reflect defective embryos, presumably from defective oocytes, as well as defective oviduct physiology (reduced function) or oviduct pathology (salpingitis). Studies in this exciting and important area have begun, and hopefully adequate research funding will be generated and sustained. Reproductive status. Conflicting con- clusions have been made about the effect of reproductive status at the time of con- ception on pregnancy loss. Some studies found no differences in rate of embryo loss (299, 1812, 1811, 1810) or fetal loss (130, 824) among reproductive statuses (maid- en, barren, and lactating or nonlactating versus lactating). An early study in Thoroughbreds (1273), however, indicated that lactating mares were more suscepti- ble to fetal loss after 42 days. Results of the survey of Chevalier-Clement (300) are 542 Chapter 12 shown (Table 12.9). Embryo-loss rates between means of 22 and 44 days were not significantly different among status- es. However, fetal loss rates (means: 44 and 310 days) were significantly higher in lactating mares, but only when the mares were mated at the first postpar— tum estrus (<17 days). In lactating mares, other workers found (1287) or failed to find (1810) an effect of time of postpartum estrus on embryo-loss rate and found (1273, 506) or failed to find (989) an effect on fetal-loss rate. Pregnancy loss was higher (26%) for mares mated at the postpartum estrus, compared to a later estrus (16%) or to mares that were nonlactating (8%); the highest proportion of losses (50%) occurred on Days 14 to 21, 78% by Day 42, and 100% by Day 63 (end of study; 170). The absence of comparable examination schedules among and within studies beclouds conclusions on the effects of reproductive status on preg- nancy loss. In conclusion, mating at the first post- partum estrus significantly increased the embryo-loss rate and fetal-loss rate in some studies but not in others. The most extensive study, using ultrasonic pregnancy evaluation, did not find a sig- nificant effect on embryo loss but did find a significant increase in fetal loss in mares conceiving at the first postpartum estrus. Undersize of embryonic vesicle. Collec- tion of embryos on Days 7 or 8 has shown that blastocysts from old subfertile mares are smaller than those from young nor- mal mares (pg. 513). The undersize phe— nomenon continues to Days 11 to 15 so that the embryos may not be ultrasoni- cally detected until 1 or 2 days (604) after detection of normal-sized vesicles. In a detailed study of the undersize phenomenon (604), the average diameter of embryonic vesicles that were lost dur— ing Days 11 to 15 was reduced signifi— cantly during the examinations preced— ing loss. Although the average diameter was reduced, many of the vesicles were well within the range of sizes found in the controls. Most of the vesicles (84%), whether undersized or not, appearedto grow at a normal daily rate until the day of loss. Loss on Days 11 to 15 was diag- nosed on the basis of failure to find a vesicle in a mare in which a vesicle was previously detected. A reduction in size, collapse, or fragmentation of a vesicle prior to loss was not detected directly during this time. Undersized vesicles (greater than two standard deviations below the mean) were found during 13/415 (3%) examinations during Days 11 to 20 in mares that subsequently maintained the embryo and in 21/206 (10%) in mares that lost the embryo. In retrospect, the probability that undersize indicated eventual loss was therefore 62% (21/34). Undersize of doomed embry- onic vesicles on Days 11 to 15, despite normal growth rate, has been confirmed; 1824, 4, 183). In another study (170), the mean diameter at Day 14 was signifi- cantly less for mares that lost the embryo on Days 14 to 21 (18 mm versus 22 mm). TABLE 12.9. Effects of Reproductive Status on Loss of Pregnancies in an Ultrasound Survey in France Reproductive status Embryo-loss rate Fetal—loss rate M ‘d a Loss rates determined between a1 en 22/491 (45%) 21/427 (49%) means of 22 to 44 days for embryos Barren 42/782 (5.4%) 40/738 (5.4%)a and 44 to 310 days for fetuses. Lactating 84/1299 (6.5%) 120/1206 (10.0%) Perflentages with different SUP??- V b scripts are Significantly dif— Bred 316 days 50/670 (7.5%) 72/576 (12.5%) ferent. Adapted from Chevalier- Bred >16 days 34/629 (5.4%) 48/630 (7 .6%)a Clement (300). The undersized phenomenon, along with normal growth rate, suggested that a retarding effect occurred early in preg— nancy. It can be speculated that the retarding effect occurred in the oviduct or immediately upon entry into the uterus before encapsulation of the blastocyst. Some vesicles were able to continue development but lagged behind normal vesicles and were eventually lost. Interestingly, embryos that were lost late (>Day 20), as well as those lost early (30 days (n=3 to 9) ....... IOIg30 days (n=9 to 10) 80 .X : Uterine body location ..'.x A 60 V ‘ o\° > O C a) 3 o- 2 u. 12 13 14 15 18 Number of days from ovulation were eventually lost were significantly small- er (indicated by different superscripts within days). Lower panel: Doomed embryos spent more time in the uterine body during the mobility phase (indicated by different super— scripts within days). The increase in body location at Day 18 (interovulatory interval $30 days) is consistent with failure of fixation or a return to mobility after fixation for embryos that were still present at Day 18. Adapted from (183). 544 Chapter 12 The extent of mobility or length of time in the uterine body are important considerations because of the postulated role of embryo mobility in the crucial blocking of the uterine luteolytic mecha- nism (pg. 305). Habitual abortion. A mare can lose pregnancies over consecutive years because of a chronic condition or by chance. Practitioners commonly refer to such mares as habitual aborters. In regard to chance, the probability of ran- dom pregnancy loss in consecutive years is the product of the probabilities for each year. Thus, in a group of mares with a 10% probability of loss in one year, the probability that a given mare will lose pregnancies in two consecutive years by chance alone is 1% (10% times 10%). The chance factor must be kept in mind when judging the likelihood that consecutive pregnancy losses in an indi- vidual mare represent a chronic underly- ing problem. Chance could contribute to the failure of presumed habitual aborters to abort after being assigned (noted below) to an intensive study. A group of 16 mares was selected that lost pregnancies 2 to 5 times over six years (374, 375). The mares were then studied for four breeding seasons; 10 mares had normal pregnancies, four lost their pregnancies, but only once or twice, and two did not develop a detectable pregnancy. There is no ques- tion, however, that pregnancy loss does involve a degree of repeatability, indicat- ing a chronic problem for some mares. In a study (610) of a herd with a high embryo—loss rate, the loss rate for 18 mares with re-established pregnan- cies (50%) was significantly greater than the loss rate for all pregnancies (38 of 154, 25%). In addition, mares that were not pregnant at the first diagnosis (Days 11 or 12) and had ultrasonically detectable intrauterine fluid collections tended to have reoccurrences of short (<16 days) estrous cycles. The recurring problem in this herd was attributable to chronic uterine inflammation in many of the mares. The condition did not sponta— neously resolve between estrous cycles or pregnancies in a significant propor- tion of the mares. Other studies have also shown (808, 1811) or have failed to show (1810) repeatability of embryo loss. Fate of dead embryos. The ultrasonic appearance of embryonic vesicles before and after death has been described in detail (604, 589, 590). Embryonic vesicles that were lost during Days 11 to 15 usu- ally disappeared without previous indi- cations, except in a few mares in which the vesicle was floating in a small collec- tion of fluid (604). In mares with cycles of normal length, vesicles lost between Day 15 and the day of reovulation failed to become fixed but continued to grow and move inside the uterus until appar- ent expulsion at the time of return to estrus (604). Passage of an embryonic vesicle through the cervix and into the vagina was observed in one mare during embryo mobility trials. In a recent report (750), a degenerating 15 mm vesicle from a previous ovulation was recovered by uterine flushing, along with a Day 7 vesicle from the most recent ovulation. Ultrasonic indications of loss or impending loss at later stages included failure of fixation, dislodgement after fix- ation, an echogenic ring (vesicle) or mass floating in a collection of fluid, an echogenic area in the dead embryo, absence of a heartbeat, and disorgani— zation of the conceptus (604, 589). Dislodgement of a formerly fixed vesicle appears to be a sensitive indicator of progesterone deficiency and can occur even before cessation of heartbeat (pg. 527); this has been observed for both induced (589) and spontaneous (183, 281) losses. The solid remnants and at least some of the fluids resulting from late embryo and early fetal death were retained sometimes for weeks or months until the debris apparently was expelled through an open cervix at the next estrus. It is not clear whether the apparent decrease in volume of the placental fluids reported by one group of workers (589, 604) represented resorption of the fluids or dispersal throughout the uterus. It was clear, however, that at least the solid debris did not disappear until the cervix opened, usually in association with a return to estrus. In an anecdotal account (604), the fetus seemed normal on Day 55, but on Day 56 a heartbeat was not detected, and the height of the allan- toic fluid was reduced approximately 50%, presumably from dispersal within the uterus. Over the following 50 days, the volume of fluid and fetal remnants remained constant and echogenic spots appeared in the fluid (probably frag- ments of placental membranes and fetus). The fetal mass and associated flu- ids and debris were mobile, sometimes located in a horn and at other times in the uterine body. All of the ultrasonical- ly detectable fetal debris and fluids was gone on Day 117, two months after death, and the mare was beginning to show estrous signs. The mare ovulated on Day 124. The fate of the dead embryonic vesicle was studied in seven mares with sponta- neous cessation of heartbeat at Days 29 to 35 (183). The mean interval from cessa— tion of heartbeat t0 degenerative rupture of the embryonic vesicle and spillage of its contents was 12 days. Measurement of the quantity of fluid after rupture was not attempted, but embryo debris was visible until the corpus luteum regressed, usually after an extended period (e.g., one month). Studies in cattle have failed to support the dogma that a dead conceptus is removed by resorption. For example, immediate embryo death (cessation of heartbeat) occurred when the amnion Reproductive Efficiency 545 was manually ruptured on Day 42 (851). The corpus luteum and volume of uter- ine contents were maintained for an extended period (mean: 27 days), and uterine volume did not decrease until a few days before ultrasonic detection of cervical opening in association with a return to estrus. In heifers in which luteolysis preceded spontaneous embryo death, the conceptus was lost rapidly with minimal degeneration (852). However, when death occurred in the presence of a functioning corpus luteum, the uterine fluid volume and the corpus luteum were maintained for up to 42 days after death. A similar project, including careful ultrasonic measure— ment of uterine contents after embryo death, is needed in mares. For the rea- sons discussed in this section, the term resorption should not be used routinely to describe embryo loss (pg. 501). 12.3I. Etiology of Late Fetal Loss Information is available elsewhere on diagnosis and management of infectious causes of late fetal loss (e.g., 1769, 105). Whitwell (1769) has reviewed the causes and pathology of fetal and neonatal losses in horses based on material sub- mitted to a diagnostic laboratory. Condi- tions discussed» included umbilical—cord abnormalities (e.g., twisting), underde- velopment of chorionic horns with con- finement of the fetus to the uterine body, developmental malformations (2 to 3% of aborted fetuses), stillbirths (fetal loss after 299 days; 26% of losses), fetal diarrhea, and infectious diseases (bacterial or fungal, 13%; rhinoneumoni- tis). Deaths from 1 hour to 7 days post- partum are also discussed. Other reports and reviews are avail- able on the causes of late fetal death (e.g., 105) and the causes and pathogene— sis of stillbirths and premature births (1545, 343). 546 Chapter 12 12.4. Twins Twinning has been one of the most emotionally charged issues in equine reproduction because of the likely out— come of twin fetuses—abortion or birth of dead or weak foals. During the 19808, the use of ultrasound scanners as a research tool resulted in a wealth of information on the nature and outcome of twin embryos. In addition, scanners have provided veterinarians with superb diagnostic and management approaches. Reviews have been published recently on twin equine embryos (590, 1663, 953), including origin and early development (595, 583), the nature of natural embryo reduction (elimination of one member of a twin set; 596, 594), and the designing of twin—prevention programs (593, 597). Much less information is available on the des- tiny of twins that enter the fetal stage (>Day 40) intact. The factors affecting double ovulation rate were discussed pre- viously (pg. 218). 12.4A. Incidence The incidence of twin foals based on examination of Thoroughbred studbooks is 1 or 2% (823, 1187). These observations, plus the consideration that the double ovulation rate is high (e.g., 20%; pg. 218), indicated that many twin sets are lost or that a natural process allows for elimina- tion of one member of a twin set. As a further example, double ovulations were diagnosed in 10% of 947 Quarter Horses, but externally observed twin conceptuses were seen in only two mares (607). The close associations between number of ovulations and number of embryos (described below) indicate that the num- ber of twin sets would be affected by the factors that increase the double ovulation rate (breed, reproductive status, repeata- bility; pg. 218). Factors that have been shown directly TABLE 12.10. Examples of Reports on Factors Influencing the Incidence of Twin Conceptuses or Foals Factor Reported results Breed Externally observed Thoroughbreds, 3.3% (cited in 404) Arabians, 0.8% (cited in 404) Quarter Horses, 0.2% (607) Early ultrasonic detection Thoroughbreds, 15.4% (240) Standardbreds, 6.1% (240) 2.8% 3.4% 3.6% 6.8% Age 4 to 7 years (404) 8 to 11 years 12 to 15 years 15 to 20 years Reproduc— tive status (579) Lactating 1.4% Barren 6.0% Maiden 1.7% (404) 2.1% 6.0% 3.4% to increase the twin-conceptus rate include breed, age, and reproductive status (Table 12.10). In addition to the tabulated reports, first diagnosis of twins on Days 12 to 25 in a large survey on Thoroughbred and Standardbred farms in Australia yielded fewer twins in lac— tating mares (8.8%) than in maiden (15.3%) or barren mares (14.0%; 1244). Based on this finding and those given in the table, twinning incidence would presumably be highest in old nonlactat- ing Thoroughbreds. The high incidence in Thoroughbreds and draft mares seem similar, although survival rates are said to be higher in draft breeds (1688). Twinning is rare in ponies (823, 404, 1582) and primitive horse breeds (404). 12.43. Origin of Twins Identical twins. Theoretically, twin embryos could originate from oocytes dis- charged from multiple follicles, from a single polyovular follicle, or from splitting of a single fertilized ovum or early con- ceptus (identical twins). A study of farm transrectal palpation records (608) showed that the majority of diagnosed twin embryos and twin fetuses were from mares with only one recorded ovulation. These data initially indicated that many double ovulations were missed or that twins sometimes occurred after one ovu- lation (identical twins or two ova per folli- cle). Only one case of identical equine twins has been reported (823). In this regard, however, 63% of 111 twin sets were of the same sex (cited in 1085). The observed ratio of same-sexzopposite—sex twins is a significant departure from equality, indicating that factors may be involved that tilt the ratio toward more twins of the same sex (e.g., splitting of fertilized ova) or fewer twins of the oppo- site sex (e.g., embryo reduction more com- mon when twins are of different sex). Confirmation of the sex ratio of twin foals or aborted fetuses is needed. Polyovular follicles. Multiple oocytes per follicle have been described in some species (e.g., juvenile monkeys; 1122) but not in mares. Equine oocyte recovery experiments have failed to yield more than one oocyte per follicle (1174). Two follicles per twin set. An ultrasonic study was made of the associations between detection of double ovulations and the subsequent detection of twin embryos on Days 11 to 15. Double ovula- tions were recorded in 38 of 41 (93%) mares with twin embryos. In the remain- ing three mares, two corpora lutea were clearly discernible on the day of twin detection, and therefore these three exceptions were attributable to errors in the detection of double ovulations. These data not only demonstrated a close associ- ation between double ovulations and twins but also emphasized that error in detection of multiple ovulations can occur, even with ultrasonic scanning. It is likely, therefore, that the transrectal pal— pation studies of the association between number of ovulations and number of Reproductive Efficiency 547 embryos per mare (described above) were flawed. The difficulties associated with detection of double ovulations by trans- rectal palpation have been described (578). In a uterine flushing experiment (1513), embryo recovery per mare on Day 7 was 53% for single ovulators and 106% for double ovulators, further demonstrating the close relationship between number of ovulations and number of embryos. Asynchronous ovulations. Earlier stud- ies (review: 591) of breeding records based on transrectal palpation suggested that twins are more likely to originate from asynchronous than from synchronous ovulations. This concept, however, seemed inconsistent with later reports of the frequent ultrasonic detection of twin vesicles on farms, even though many of the double ovulations apparently occurred on the same day. One practi- tioner, for example, detected 32 sets of twins (15%) in 214 Thoroughbreds (240). Others noted that twin vesicles were often similar in diameter (1244), and another (591) reported that the average number of embryonic vesicles per preg- nant mare for O, 1, and 2 days between ovulations was 1.7 (n=37) 1.6 (n=22) and 1.7 (n=6), respectively. In a controlled ultrasound study (602), no significant dif- ference was found between synchronous and asynchronous ovulatory patterns in the number of mares with 0, 1, or 2 embryos on Days 11 or 12 after each ovu- lation. In conclusion, recent results from stud- ies using the most critical available tech- nology (transrectal ultrasonography) for the in vivo detection of double ovulations and twin conceptuses have refuted the earlier concept (developed from transrec- tal palpation records) that twins are more likely to originate from asyn- chronous ovulations. 548 Chapter 12 1240. Early Development of Twins Growth rate. The mean growth rate between Days 11 and 16 was not signifi- cantly different between single and multi- ple embryos (591). Similarly, diameter of Day 14 embryonic vesicles was not signifi- cantly different between single (16.6 mm, n=34) and synchronous multiple ovulators (15.9 mm, n=74 embryos). In mares with asynchronous double ovulations, however, the diameters of the smaller and larger vesicles on the day of first detection of both were attributable to the number of days between ovulations. That is, the presence of two vesicles did not appear to have a direct effect on diameter of vesi- cles, other than the effect related to age of vesicles. This is of practical importance in selecting the earliest day when one is like— ly to find both vesicles in mares with asynchronous ovulations. Mobility. Early equine twin conceptus- es interact constantly with the uterus in dynamic physical fashion, similar to the interaction of the uterus with singletons (pg. 305). Twin conceptuses traverse the length of the uterus many times per day from the time they are first detectable by ultrasound (Days 9 to 11) until the day of fixation (Figure 12.19). In one study (585), twin conceptuses spent more than 50% of the time in the uterine body during Days 9 to 12. In twins of dissimilar size, the preference for the uterine body seemed to be an independent function of each vesi— cle based on its size or age; during two- hour mobility trials, the smaller member of twin sets was found in the uterine body three times more often than the larger member. After approximately Day 12, or when the diameter of vesicles exceeded 9 mm, the number of entries into the uterine horn increased, and the vesicles began a maximum mobility phase that continued until fixation. During maximum mobility, each mem- ber of a twin set moved from one horn to another an average of 0.9 times per two- hour trial (equivalent to 11 times per Mobility of twins FIGURE 12.19. Example of the sequential loca— tions of each embryonic vesicle in a twin set during the mobility phase. Location determinations were made every five minutes for two hours. The solid arrows are for one vesicle, and the dashed arrows are for the other vesicle. The numbers are the num- ber of minutes a vesicle spent in each segment. The series starts at the star. Initially, the two vesicles moved together, spending five minutes in the left horn/middle segment, five minutes in the anterior body, and 20 minutes in the right horn/posterior segment. One vesicle then returned to the anterior body and the other vesicle remained in the posteri- or right horn for 25 additional minutes. Note that one embryonic vesicle was traversing the left horn at the time the other was traversing the right horn. This example illustrates that members of a twin set sometimes move independently. Adapted from (585). day) and from horn to body or vice versa an average of 2.5 times (equivalent to 29 times per day). The relative locations of twins during the maximum mobility phase were as follows: both in one horn, 25%; both in the body, 12%; one in each horn, 30%; one in a horn and one in the body, 33%. The propulsive force for con- ceptus mobility is uterine contraction, apparently in response to the production of a stimulant by the conceptus (pg. 307). The role of the conceptus in stimulating contractions is also indicated by the independent movement of each member of a twin set. In one study (585), frequen- cy of both embryos in a given uterine segment (28%) was significantly greater than the expected frequency (18%) if each embryo had moved independent- ly of the other. Thus, 64% of the loca- tion changes occurred independently (18% divided by 28% x 100). Fixation. Fixation is defined as cessa- tion of mobility and is postulated to occur when an embryonic vesicle becomes so large, and uterine tone so great, that the vesicle no longer can move despite con- tinued uterine contractions (pg. 309). The day of fixation is similar for singletons and twins, and in one study (585) 97% of twin vesicles were fixed by Day 16. The role of vesicle diameter in determining when fixation occurred was indicated by the following observations: 1) Mean diameter of the largest vesicle on Day 15 was significantly greater for mares with fixation on Day 15 (24 mm) than for mares with later fixation (19 mm); and 2) A mare in which a location change was not detected after Day 13 had the largest Day 13 vesicles (18 and 20 mm). Movement of the smaller member of asynchronous twin sets apparently tends to continue after fixation of the larger member, but this phenomenon has not been documented adequately. Similar size Dissimilar size (0-3 mm) (:4 mm) Prefixation (Days 1 1-15) 0 o O No. mares 26 629/:9\ WWW? No. mares 26 4 Day of fixatlon (Mean: Day16) Reproductive Efficiency 549 Preference for unilateral fixation. Fix- ation of twins occurs unilaterally (both in one horn) more often than would be expected by chance. Fixation of each vesi- cle independently of the other would, by expectancy calculations, result in 50% unilateral fixations and 50% bilateral fix- ations. Combined data from several stud- ies, however, showed that unilateral fixa- tion occurred in 70% (48 of 68) of mares with twins (Figure 12.20). Similarly, in Thoroughbreds a 66% (69 of 105 mares) incidence of unilateral fixation was reported (240). It is important to note that these data are in reference to the mobility phase or before embryo reduction would occur; after embryo reduction, bilateral distributions will be observed more fre- quently due to embryo reduction in the unilaterally fixed twins. Dissimilarity in diameter of vesicles or asynchrony of ovulations profoundly increased the frequency of unilateral fix— ation (Figure 12.20; 595). The preference for unilateral fixation may be related, in part, to the tendency for twins to be in the same location during mobility more often than would be expected. The favor- able effect of size dissimilarity on unilat- eral fixation may be a result of one fixed vesicle acting as an impediment to move- ment of the other so that both vesicles fix in one location. In the 68 sets of twins depicted in Figure 12.20, all of the uni- lateral fixations involved close apposi- Size difference ignored 68 FIGURE 12.20. Effect of differ- ences in diameter between twin vesicles on the incidence of uni- lateral versus bilateral fixation. Note that the preference for uni- lateral fixation was especially pronounced when the twins dif- fered in diameter by 4 mm or more. Adapted from a review 48 20 (595). 550 Chapter 12 tion between the two vesicles in the cau— dal portion of one of the uterine horns. Fixation is postulated to occur in the caudal portion of a horn because the flexure in that area results in the great- est impediment to continued mobility of the expanding conceptus (pg. 309). Fixations in the uterine body occur only occasionally, and it is sometimes difficult to determine ultrasonically if a given vesicle is in the extreme caudal portion of a horn or in the cranial portion of the uterine body. In addition to the 68 cor- nual fixations depicted in Figure 12.20, one or both members of three twin sets (3 of 71; 4%) were judged to be in the uterine body. 12.4D. Embryo Reduction Embryo reduction is defined as the nat- ural elimination of one member of a twin set so that only one vesicle enters the fetal stage (608). Reduction before Day 11. There are conflicting reports on the existence and incidence of reduction on Days 0 to 11. The results of a uterine flushing experi- ment in superovulated mares with syn- chronous multiple ovulations suggested that reduction occurred between Days 7 and 11 (1816). However, a retrospective study (591) of the relationships between number of multiple ovulations (sponta- neous and induced) and number of con— ceptuses detected ultrasonically on Days 11 to 16 indicated that a conceptus developed in association with 74% of the detected ovulations in pregnant multiple ovulators. Furthermore, the high inci- dence of twins detected prior to fixation in Thoroughbreds, Standardbreds (240, 1244), and Quarter Horses (591) seems inconsistent with an efficient early reduc— tion mechanism. As a critical test of the hypothesis that embryo reduction occurs before Day 11, the pregnancy rate per ovum was com- pared between single and double ovula- tors (602). The results indicated that each ovum in bilateral ovulators had the same chance of developing into a Day 11 con- ceptus as an ovum in single ovulators. In the unilateral double ovulators, the dif- ference between observed and expected frequencies was significant but was attributable to a greater frequency of mares with no embryonic vesicles, rather than to embryo reduction. Reduction before Day 11 would have been indicated by a greater frequency of double ovulators with only one embryonic vesicle. Thus, the hypothesis that some embryo reduc— tions occur before Day 11 was not sup— ported for either unilateral or bilateral double ovulations. The increased propor- tion of unilateral double ovulators with no embryonic vesicles is consistent with an earlier report that fewer twin concep- tuses were recovered by uterine flushing on Days 6 or 7 from unilateral double ovulators than from bilateral double ovu- lators (1511). The cause of this phe— nomenon is not known and would be an interesting research area. Reduction on Days 11 to 16. Loss of both members of a twin set between Days 11 and 16 is minimal and similar to that expected for singletons (586). Com— parisons of the number of mares with two conceptuses between the first day of detection and the day of fixation indi- cated that embryo reduction during Days 11 to 16 is negligible (591). These findings indicated that embryo reduction prior to fixation of the vesicles or on the day of fixation is not an important aspect of the natural correction of twins. Natural elimination of one member of a twin set can, therefore, be attributed to postfixa- tion reduction (586). Reduction on Days 17 to 40. The inci- dence of postfixation embryo reduction, as used herein, is in relationship to the number of twin sets during the mobility phase or on the day of fixation. The embryo reduction rate summed over uni- lateral and bilateral pregnancies was 60%, relative to twins detected during the mobility phase (596). A field survey Reproductive Efficiency 551 TABLE 12.11. Incidence of Embryo Reduction and Survival in Mares with Unilateral (n=48) and Bilateral (n=20) Fixation Incidence of reduction Incidence of survival Diameter Unilateral Bilateral Unilateral Bilateral difference fixation fixation fixation fixation (mm) (No. mares) (No. mares) (No. mares) (N0. mares) 0-3 19 (45%) 0 (0%) 7 (17%) 16 (38%) 24 22 (87%) 0 (0%) 0 (0%) 4 (13%) Total 41 (60%) 0 (0%) 7 (10%) 20 (30%) Incidence is based on all twin sets detected during the mobility phase. Adapted from a review ( 596). found a 62% reduction rate relative to an initial pregnancy diagnosis on a mean of 22 days after service (300). The incidence of postfixation embryo reduction was significantly influenced by type of fixation (unilateral versus bilat- eral) and by inequalities in diameter of the two embryonic vesicles. In a series of 68 twin sets, all reductions were associ— ated with unilateral fixation, and the incidence of reduction for twins that fixed unilaterally was 85% (Table 12.11). The effect of unequal diameters of the two vesicles on the incidence of postfixa- tion reduction is shown (Figure 12.21). Reduction occurred in all of 22 mares with unilaterally fixed vesicles of dissim- ilar size (4 mm or more) but in only 19 of 26 mares (73%) with vesicles of similar size. A diameter difference of 4 mm or more during the mobility phase is Similar size Dissimilar size (0-3 mm) (24 mm) )1 W mm 0M Defined end of embryo stage (Day 40) 7 19 O 22 No. mares Day of fixation (Mean: Day 16) No. mares attributable to asynchronous ovulations because the diameter of each member of a twin set is dependent on its age (591), and the mean growth rate of vesicles in sever- al studies (review: 590) was 3 to 4 mm per day. Loss of both embryos of a twin set on Days 17 to 40 was not different from the embryo-loss rate for singletons (10%; 586); this observation has been confirmed (300). Days of occurrence of postfixation reduction. The day of completion of embryo reduction is defined herein as the first day that one of the embryonic vesicles is no longer distinguishable by ultrasound. As a generalization, as the number of days after fixation increases, the probability of subsequent reduction decreases, and the time required for completion of reduction increases. Most reductions occur by Day 20. Therefore, if the first pregnancy examination is not performed by Day 20, Size difference ignored 8 “9/:5") if if FIGURE 12.21. Effect of diame- ter differences during the mobil- ity phase on the incidence of embryo reduction for twins that fixed unilaterally. Adapted from a review (596). 552 Chapter 12 there will be no indication that these twins had been present. In the data set shown (Figure 12.22), 59% of the embryo reductions occurred by Day 20. The remaining embryo reductions were com- plete by Days 21 to 30 in 27% and by Days 31 to 38 in 14% of the mares. Reductions that occurred by Day 20 usually were manifest within one day (80% of 14 mares; 594). That is, two normal—appearing vesi- cles were present on one day and only one was detectable on the next. In the remain— ing mares (20%), a vesicle of reduced size was noted the day before disappearance. The reductions that occurred after Day 20 usually were preceded by a gradual decrease in size of the vesicle undergoing reduction (e.g., over 7 days). Diameter differences between embryon- ic vesicles had a significant effect on day of occurrence of embryo reduction. Mean day of reduction was later for vesicles sim- ilar in diameter (0 to 3 mm difference) than for those dissimilar in diameter (4 mm or more; Figure 12.23). When twins Day of completion of embryo reduction 59% 27% 1 4% 10 Number of mares 15 19 23 27 31 35 39 Number of days from ovulation FIGURE 12.22. Number of mares in which embryo reduction of unilaterally fixed vesicles was complete (one vesicle no longer detectable by ultrasound) on various days. Reduction occurred most frequently by Day 20. Adapted from a review (596). were similar in diameter, they were more likely to survive; if reduction did occur, it was more likely to do so after Day 20. In conclusion, asynchrony of ovulations or dissimilarity in vesicle diameter increased the likelihood of unilateral fixa- tion, increased the incidence of reduction for unilaterally fixed vesicles, hastened the day of occurrence of reduction, and shortened the interval from initiation to completion of reduction. The mechanism of postfixation embryo reduction. It has been proposed (594) that embryo reduction occurs when a major portion of the three-walled area (ecto- derm, mesoderm, and endoderm; pg. 364) or the vascularized wall of one of the embryonic vesicles is in apposition with the wall of the adjacent vesicle rather than with the endometrium (deprivation hypothesis; Figure 12.24). Size differences & day of embryo reduction <12 3 4 5 6 7 8 9 :10 Difference in diameter (mm) FIGURE 12.23. Effect of diameter differences between twin vesicles during the mobility phase on the mean day of completion of embryo reduction. Combined mean for differences of £1, 2, and 3 was significantly different from the combined mean for differences of 4 to 210 mm. Embryo reduction occurred later when the twins were similar in diam- eter and earlier when they were dissimilar. Adapted from a review (596). Reproductive Efficiency 553 The Deprivation Hypothesis DAY r\ 16 4—LOCATION or Euanvomc DISC 3 WALLS 2 WALLS 1 7 20 o..- o 25 @211. <9 8 CD BAC .. e 4o (3 FIGURE 12.24. The deprivation hypoth- esis for embryo reduction in unilaterally fixed twins. The thin two-walled portion (ectoderm and endoderm) of the yolk sac wall is depicted by a thin line and the thick three-walled portion (ectoderm, mesoderm, endoderm) by a heavy line. Uterine contractions continue after fixa- tion and subject the vesicles to a massag- ing action. As a result, singletons become orientated within the uterus, as shown, and twins become orientated relative to one another. The resulting spatial rela- tionship between the two vesicles deter- mines whether and when embryo reduc- tion will occur. Group A. The developing vascularized wall immediately surrounding the embry- onic disc in the vesicle on the right does not have contact With the uterine lumen. Embryonal-maternal exchange is reduced, and the deprived embryo undergoes early and rapid reduction. This relation- ship is most common when the vesicles mfl MASSAGE AND ROTATION AFTER FIXATION //\\ GO CEO GD no on or; 69 (3 GO GO CD CD EEO SB (3 (it) are dissimilar in diameter. Group B. The vascularized‘ wall of the vesicle on the right has partial Contact with endometrium. The vesicle therefore survives longer than for mares in Group A, but embryo reduction occurs before the ‘ emergence of the allantoic sac. . ‘ . Group C The contact area betWeen the vascularized yolk- sac wall and the endometrium is adequate for the Vesiclea on the right until the emergence" of the allantois. However, the emerging allan- tois is not adequately exposed to the uter- ‘ ine lumen and therefore is not able to assume its role as the major» membrane . for physiologic exchange by Day 80. Group D. Neither vesicle is deprived and both survive through the embryo stage; each has an adequate area of con tact between the endometrium and the vascularized portion of the wall of the con- ceptus. The relationships in Groups C and D are most common when the vesicles are of similar diameter. Adapted from (594). 554 Chapter 12 According to the deprivation hypothesis, the vesicle that has its vascularized wall covered by the adjacent vesicle is deprived of adequate embryonal—maternal exchange and, therefore, regresses. The uterine con- tractions that cause the extensive mobility of the embryonic vesicle continue for a few days after fixation (pg. 309). In mares with unilaterally fixed twins, the same forces that are postulated to rotate and orientate singletons (uterine contractions and tone) appear to cause twins to rotate and impinge upon one another until they are orientated relative to one another. The final vesicle—to-vesicle orientation in the majority of unilateral twins is with the thick wall (three-layered portion) of one vesicle impinging upon the thin wall (two- layered portion) of the other. The vesicle that loses contact between its thick vascu- larized wall and the uterine lumen is deprived and undergoes embryo reduction. The greater the proportion of thick wall that is blocked, the sooner and quicker embryo reduction occurs. This situation is most acute when the vesicles are dissimi- lar in size; the thick wall of the smaller vesicle impinges well into the thin wall of the other, and, because of its relatively small size, most or all of its thick wall is denied endometrial contact. The deprived smaller vesicle, therefore, undergoes early and rapid regression. The deprivation hypothesis is consistent with the following observations (594, 586): 1. Embryo reduction occurs only after the two vesicles become fixed in close apposition; 2. The most frequent and rapid occur— rence of reduction is during the first few days after fixation; 3. The process is efficient and unidirec- tional—the survivor is similar in size to a singleton, and the incidence of loss of both vesicles is similar to the incidence of loss of a singleton; 4. Unequal diameters favor a higher incidence and earlier occurrence of reduc- tion and a shorter interval from initiation to completion of reduction; 5. The survivor is sometimes disorien— tated, as indicated by attachment of the umbilical cord at the ventral hemisphere of the conceptus; and 6. The site of reduction is at the two- walled yolk—sac area of the survivor. In regard to Points 5 and 6, the mas- sage and rotation of twin vesicles appar- ently occurs during the time that a single- ton would become orientated relative to the mesometrial attachment. Therefore, the survivor of the reduction process is occasionally disorientated, even to the extreme of being upside down (586), espe- cially when the reductions occur late in the embryo stage (>Day 20). In all such reductions studied to date, the site of reduction was in the yolk-sac area of the survivor, and therefore the outer attach- ment of the umbilical cord of the survivor subsequently marked the site of reduc- tion. When the reductions occurred early (Day 25—correction of bilaterally fixed vesicles is increasingly difficult and the success rate increasingly reduced. In regard to Point 3, the high probabili- ty for unilateral fixation and early natural embryo reduction when the ovulations are on different days is fortunate; if a small vesicle is missed, there is a high probabili- ty that it will undergo natural reduction before Day 20 (pg. 551). There are peripheral considerations in selection of a twin—prevention program. Correction of twins during the late mobili- ty phase allows early removal of a prob- lem that otherwise may continue to plague both veterinarian and client. Furthermore, routine first pregnancy examinations during the mobility phase may disclose the following: 1) early return to estrus, based on the ultrasonic appear— ance of the endometrium and confirmed by the ultrasonic appearance of the corpus luteum, 2) small collections (e.g., 4 x 10 mm) of ultrasonically detected, intralumi- nal uterine fluid, and 3) loss of an embry- onic vesicle early in pregnancy. Any of these three findings will alert the operator to the probability of endometritis and cor— rective measures can be taken before reovulation. A first examination on Day 20 or later may fail to provide timely information for these three important diagnostic considerations. In conclusion, first examination for detection and correction of twins on Days 13 to 15 (equivalent to 14 to 16 days after mating) seems the optimal approach. Prefixation correction. Correction dur- ing the mobility phase involves compress- ing the uterine horn transrectally between finger and thumb caudal to the selected vesicle and moving the hand toward the tip of the horn (583). The vesicle may rup- ture in place during movement up the horn or upon being trapped at the tip of the horn. If the vesicles are in contact, the operator can wait (e.g., one hour or one day) until they separate due to the mobili— ty phenomenon, or an attempt can be made to separate them by gentle massage. Prefixation manual reduction is highly effective. In the initial developmental study (583), the procedure was successful in 8 of 8 mares. In extensive use on farms (1238, 240), success rates have exceeded 90% (e.g., 60/66). One veterinarian performed manual embryo reduction at 15 or 16 days or occasionally at 17 or 18 days after the last mating (597). The success rate for 54 mares was 93%, based on fall pregnancy examinations—that is, 93% of the mares in which twins were manually reduced had one conceptus and 7% had none. These data indicate that the probability that the remaining embryonic vesicle after prefixation reduction will develop normal- ly and enter the fetal stage is equivalent to the probability that a singleton of corre— sponding age will enter the fetal stage. The twinning tree. As an aid in design- ing twin-prevention programs, a twinning probability tree can be constructed based on known or expected double-ovulation rate and singleton pregnancy rate on a given farm (593). The twinning probability tree depicts the sequential events and their probabilities of occurrence, extend— ing from mating a group of 100 mares, including single and double ovulators, to the final outcome at Day 40 (Figure 12.26). As shown in the example, in herds with a 20% double-ovulation rate and 70% pregnancy rate per ovum, 4 of 10 expected twin sets can be expected to survive into the fetal stage—an undesirable outcome. Reproductive Efficiency 557 The Twinning Tree WWW’WWW 17\83%/\100% \0%/; W WWWWW’ 70% 30%/ WWW m 8"! 2/, 56”: DAY 16 Fixation DAYS 1 1- 15 W W W W W 149% 42961 9%2/ 600 FIGURE 12.26. A twinning probability tree that was developed from two assump- tions (20% double ovulation rate and 70% pregnancy rate for single ovulators) and three observed frequencies from a research herd (70% unilateral fixation rate and Day 40 twin-embryo survival rates of 17% for unilateral fixation and 100% for bilateral fixation). All other expected frequencies (percentages) were calculated by the rules of probability. The percentages represent the probability that a given event (beginning of a solid arrow) will have a given outcome (end of arrow). The bold numbers are number of mares (or percentage) of the original 100 that are expected to move from a given event 750%L 3/ DAY 0 Ovulation Principles of probability to a given outcome. To illustrate, the 20 double ovulators (arrow) at Day 0 pro: duced 10 twin sets on Days 11 to 15 (an outcome). The 10 twin sets involve 10% (a bold number) of the original 100 bred mares and 49% of the 20 double ovulators. For the given assumptions (20% double ovulations and 70% pregnancy rate for each ovum), 4 of the 100 mares are expected to have twins at the end of the embryo stage (Day 40)——one unilateral and three bilateral. The probability that a double ovulator will have surviving twins at Day 40 is 20% (1+3=4, 4/20); the proba- bility that a mare with twins during the mobility phase will have twins at Day 40 is 40% (1+3=4, 4/10). From (593). 558 Chapter 12 Postfixation correction. Correction after fixation is readily accomplished in bilateral twin sets by digital compres— sion of one of the embryonic locations or bulges. The success rate when done by Day 25 is equivalent to the success rate for prefixation correction (1238, 240). A disadvantage of postfixation correction is that unilaterally fixed vesicles are dif— ficult to correct. When done on the day of fixation or soon thereafter (Days 16 to 19), the unilateral vesicles can some- times be separated by persistent manip- ulation of the dorsal uterine wall at the site of the vesicles. When separation fails, an attempt can be made to rupture one vesicle in place, as described (240). The success rate for reduction of unilat- erally fixed twins on Days 16 to 19 was equivalent to the results of the above procedures. However, when attempted later, the success rate rapidly deterio- rated (e.g., 62% on Days 20 and 21). Manual reduction of unilaterally fixed twins is often tedious, time-consuming, and frustrating (240), especially begin— ning a day or two after fixation when the twin set has assumed a compressed irregular shape. In addition, if the ini- tial examination is done during Days 17 to 19, twin unilaterally fixed vesicles will occasionally be missed. An exten- sive study demonstrated the decreasing success rate with advancing pregnancy when manual rupture is done after Day 25 (1330). Surges of PGan have been shown to be released in proportion to the amount of manipulation required for rupture of a vesicle (1238). However, luteolysis did not occur, and it was con- cluded that rupture on Days 12 to 30 should not normally require administra— tion of supplemental progesterone or a PGFZoc synthesis inhibitor. 12.4F. Fetal Stage Twins Outcome of fetal twins. As described above, twins that are not manually or naturally corrected by the end of the embryo stage enter the fetal stage (>Day 40) intact. With fetal twin sets, abortion is more likely to occur than fetal reduction or birth. Limited critical monitoring information is available, however, on the outcome of twin sets that are still present and Viable (heart- beat detectable) at Day 40. In a study of farm transrectal palpation records (606), the presence of twins on Days 40 to 42 (n=16) resulted in abortion (loss of both fetuses) in 63%, birth of two foals in 31%, and fetal reduction in only 6% of the mares. In another study (1243), only 17 live foals were born from 130 mares carrying twins at approximately Day 42. In a study in Thoroughbreds in England (823), twin pregnancies were characterized by abortion (64.5%), birth of one of the pair (21.0%), or birth of both (14.5%). Only 25% of 124 fetuses were born alive, and only 14% survived to two weeks of age. In Thoroughbreds in Poland, diagnosed twins resulted in abortion (loss of both) in 73%, stillbirths in 11%, and birth of viable single or twin foals in 16% of the mares (404). Twinning was the single largest cause of abortion (externally observed fetuses) and accounted for 22 to 26% of fetal and neonatal material submitted to a diag- nostic laboratory in England (1769). An incidence of 35% stillbirths for twins born after 299 days, compared to a still- birth incidence of 2% for singletons, has been recorded (cited in 1769). Twinning thus results in a high rate of perinatal morality, as well as observed and unob- served fetal loss. Placental relationships. Placental fusion with blood exchange occurs in twin pregnancies, but a freemartin con— dition apparently does not result. This suggests that placental fusion may occur later in horses than it does in cattle (823). The loss of twin fetuses has been attributed to placental insufficiency. The combined surface area of the placentae of twins is only slightly larger than for singletons (823). A species difference that may con- tribute to high loss of twins in mares, when compared to cattle, may be related to uterine structure. In mares, the large uterine body forces a large proportion of nonproductive contact between the two placentae. An invagination of one pla- centa into the other may occur, and such areas of contact are aVillous. Whitwell (1769) has reviewed fetal and neonatal losses in horses and has described the various relationships between twin allantochorions. Management of fetal twins. Termin- ation of pregnancy (e.g., with PGFZoc) after the initiation of formation of endometrial cups (Day 36) may result in a long delay in the return to a cyclic condition (1953. 433). Many schemes have been reported for elimination of one fetus (>Day 40), including injection of a destructive agent into the placental sac or surgical removal of one fetus (796, 590). Transrectal crushing of a vesicle after Day 40 is difficult and seldom successful (1330, 300, 1243). Results of experimental injections of placental fluid into the uterine lumen suggest that loss of both conceptuses can be attributed to disruption caused by the free placental fluid from the ruptured vesicle (1240). Use of a prostaglandin synthetase Reproductive Efficiency 559 inhibitor in conjunction with the crush— ing procedure has been studied and reviewed (1240); administration of flunix- in meglumine was highly effective in inhibiting the release of PGFZoc during hysterotomy. Surgical removal of one conceptus at 41 to 65 days resulted in a single foal in 5 of 7 bilateral twins and in 0 of 7 unilateral twins (1567). An interesting procedure involving ultrasonically guided, fetal cardiac puncture also has been reported for cor- rection of twin fetuses (1312). One mem- ber of a twin set was treated at 66 to 168 days by inducing cardiac arrest with an intracardial injection of a potassium chloride (KCl) solution through the maternal abdominal wall; 8 of the 18 mares had a single live foal. It was con- cluded that this procedure may be a viable alternative in selected mares car- rying twins after 60 days. Fetal pericar- dial injection of KCl has been used suc- cessfully in women with multiple fetuses (cited in 460). Restricting the diet has been advo- cated for converting a twin pregnancy to a singleton (1085, 1675). Twin pregnan- cies were diagnosed at 3 to 7 weeks, and diet was then restricted (removal of concentrates and alfalfa), usually for 2 to 4 weeks (1085). About 60% of the mares gave birth to a single foal. These interesting findings need to be con- firmed with the inclusion of randomly selected untreated mares. In a clinical trial (1335), three mares with twins were given an exogenous progesterone regimen after midgesta- tion. In each mare, a single Viable foal and a mummified fetus were delivered at term. Thus, administration of proges- terone should be critically tested as a treatment for twin fetuses. 560 Chapter 12 HIGHLIGHTS: Reproductive Efficiency Pregnancy establishment 1. There has been an apparent 10% improvement in reproductive efficiency during . the past two decades Recent studies indicate that fertilization rate is high (>90%) in normal mares. ~ “ Recommended minimal farm goals are a 55% per‘estrus pregnancy rate over all mares and 80% for reproductively healthy individuals and an 80% liveefoal rate _r 7 for all mares per season. , \ * Reduced pregnancy rates are associated with the first postpartum estrus (e. g. ., 20% less), advanced age (e g., 50% less), and endometritis (e. g., 50% less). ” Much of the relationship between old age and poor reproductive efficiency is . attributable to oviductal and uterine pathology, but the role of other old- -age factors has not been adequately studied . - :~ Pregnancy loss 6. 10. 11. 12. Mating at the first postpartum estrus increased the embryo- and fetal- loss rate in . some studies but not in others. An extensive ultrasonic study did not find a Signif- icant increase in embryo loss but did find increased fetal loss ‘ Extensive uterine cysts are associated with increased embryo loss but apparently not fetal loss. However, the role of cysts has not been partitioned frOm the effects ‘ of other factors related to old age. It has not been demonstrated that chromosomal abnormalities are a cause of 1. fertilization or pregnancy failure in horses. 0 Recent studies suggest that rate of embryo loss in subfertile mares is very high on Days 1 to 5 (e. g., 50%) and progressively diminishes until Day 20; limited study failed to find a difference in loss rate between subfertile and normal mares OVer Days 20 to 50. The rate of loss diminishes after Day 60 ‘ ‘ The recovery rate was reduced and the incidence of morphologic defects was increased for embryos collected from subfertile donors on Days 7 to 9. Transfer of the embryos from subfertile mares resulted in reduced pregnancy rates and increased loss rates after pregnancy. ‘ Undersize of the embryo on Days 11 to 15 indicates high probability (a g., 60%) of eventual loss Undersized embryos on Days 11 to 15 usually grow at a normal rate. . . ., w \ - Expected farm pregnancy-loss rates are 6% for the last half of the embryo stage (Days 20 to 40) and 12% for the fetal stage. \ 13. 14. 15. 16. 17. Reproductive Efficiency 561 Most pregnancy losses under farm conditions are attributable to pathologic pro— cesses as indicated by macroscopic or microscopic lesions rather than to aberra— tions in physiologic mechanisms unaccompanied by pathologic change. Ultrasonically detectable intrauterine fluid collections are convenient indicators of endometritis and subfertility. Endometritis can activate the release of luteolytic quantities of PGFZoc from the endometrium, whereas pyometra or chronic severe endometritis can be associated with loss of PGF2a-producing cells and therefore luteal maintenance. Primary luteal insufficiency has not been shown to be a cause of embryo loss before Day 20, except in mares induced to ovulate when in the inactive phase of the anovu- latory season. A few cases of embryo loss (cessation of heartbeat) between Days 20 and 40 were preceded by a decrease in circulating progesterone concentrations. Pseudopregnancy is characterized by embryo loss followed by luteal maintenance and uterine turgidity. It is associated with a minority of losses On Days 10 to 20 (e.g., 25%) and with most losses on Days 20 to 40. 18. There are anecdotal accounts of progesterone inadequacy during the fetal stage and indications that severe stress may reduce progestins to dangerous levels. Twins 19‘. Almost all twin sets are attributable to double ovulations. 20. Unilateral fixation occurs in most (e.g., 70%) twin sets. 21. Dissimilarity in size (age) of twin embryos favors unilateral fixation and early and rapid embryo reduction. 22. Correction of twin embryos by the mare is done by embryo reduction and not by elimination of both. Expected postfixation embryo reduction rate for mobility- phase twins is 60%. Embryo reduction is negligible before fixation and in bilater— ally fixed embryos. Postfixation embryo reduction occurs in most (e.g., 85%) of unilateral twin sets; over half of the reductions occur before Day 20. 23. The time, incidence, and speed of embryo reduction apparently depends on the proportion of vascularized wall of one vesicle that is in contact with the opposite vesicle rather than with the endometrium (deprivation hypothesis). 24. In the past decade, manual correction of twin embryos by transrectal rupture has replaced attempted prevention of twin development by modifying the mating program when double follicles or ovulations were detected. 25. Twins that enter the fetal stage intact are more likely to undergo abortion (loss of both) than fetal reduction or birth of viable twins. 562 Chapter 12 MILESTONES: Reproductive Efficiency Transrectal palpation studies on pregnancy loss (review: 575). Extensive study of reproductive efficiency on horse farms (792). Development of biopsy techniques for assessing uterine health (review: 1661). Descriptions of the physical relationships between twin fetal membranes (823}. Reports that luteolytic doses of PGan are released by the uterus in response to endometritis, whereas PGFZa release may fail during pyometra (1136, 782) . 1981 Study of histopathology of the oviducts (723). 1983-85 Development (583) and testing (240) of manual reduction technique for twins during the mobility phase. 1984-87 Discovery that ultrasonically detectable intrauterine fluid collections are convenient indicators of endometritis and subfertility (614, 610, 4). 1984-89 Elucidation of the developmental characteristics of twin embryos and the probabilities for, and nature of, embryo reduction (602, 585, 586, 594). 1985-87 Demonstrations that endometritis is associated with low Day-11 pregnancy rates and a high incidence of ultrasonically observable embryo losses 90%) in normal mares (146) and retardation or loss of embryos in the oviducts or soon after entry into the uterus in subfertile mares (147). 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INDEX A Abdominal pouches, 9 Abortion after CG production, 451 before CG production, 450 definition of, 501 during CG production, 450 habitual, 544 treatments for, 450, 452 Absorptive arcades, 381, 391 Accessory corpora lutea, 324 Activin, 56 Adenohypophysis, 37 Adrenal steroids, see also specific hormones cortisol, 57 during puerperium, 479 general, 71 in relationship to expression of estrus, 96 in relationship to the estrous cycle, 244 role of, in parturition, 465 Adrenocorticotropin, 57 Adrenocortical nodules, 27 Age determining, of fetus, 392 effect on ovaries, 113 effect on pregnancy loss, 540 effect on pregnancy rate, 505 effect on reproductive seasonality, 111 Agonist, 54 Allantochorionic pouches histology of, 373 in terminal placenta, 462 origin of, 372 CG content of, 420 Allantoic fluid volume changes, 377 shifts, 411 Allantoic sac fluid volume, 377 origin of placental membranes, 367 transition from yolk sac to, 367 Allantois, see allantoic sac Amnion fetal, 377 fluid volume of, 37 7 histology of, 378 nodules of, 379 of discharged placenta, 462 origin of, 377 plaques and vascular system of, 37 9 Ampulla of oviduct, 25 Anabolic steroids, 287 Anatomy, see individual organs Androgenized teasers, 103 Androgens, including testosterone concentrations in blood during cycle, 243 concentrations during pregnancy, 427 effect on sexual behavior, 96 in adrenal, 71 in follicular fluid, 243 in placenta, 69 role during estrous cycle, 242 synthesis of, in thecal and granulosa cells, 64 and unseasonal estrus, 96 Androstenedione, 63, 64, 65, 242, 243, 251 Anestrus, see anovulatory season Anovulatory season, see also specific organs, specific hormones, reproductive seasonality, photoperiod, receding phase, inactive phase, resurging phase artificial termination of, 158 definition of, 106 follicular dynamics during, 136 sexual behavior during, 136 summary of endocrinology of, 156 Antagonist, 54 Antiluteolysin, 441 , Antipituitary preparations and potential for cycle control, 286 effect on corpus luteum, 265 effect on follicles, 258 Arcade space, see absorptive arcades Aromatization, 62, 63, 67 Arteries, see also specific organs role in uteroovarian relationships , 269 to reproductive organs, 34 vitelline, 363 628 Index Artificial control of ovulation of stallion, 77 GnRH for, 166, 281 repeatability of , 92 hCG for, 170, 279 stallion‘s choice of mares, 88 implementation of programs for , 287 unseasonal, 95 methods and purposes of, 278 Bilaminar omphalopleure, 365, 372, 390, 464 progestins and PGFZOL combinations for , 285 Biotechnology of gametes and embryos, 333 progestins for, 164, 282 artificial insemination, 333 prostaglandin for, 284 assisted fertilization, 335 Artificial insemination, 333 chronology of progress, 342 acceptance of, 333 embryo transfer, 337 frozen semen, 334 genetic engineering, 341 raw and cooled semen, 334 predetermining gender, 340 Assay of hormones preservation of embryos, 339 differentiating LH and CG, 423 Blastocoele, 350 general, 72 Blastocyst , see also conceptus kits, 72 definition of , 350 of CG, 50, 72 development of, at time of uterine entry , 350 of estrogens, 241, 427 formation of, 350 of PGF2a, 244 shedding of zona, 351 of progesterone, 237 Blastomeres, 348 Assisted fertilization, 335 Blood-typing, 301 collection and culture of oocytes, 336 Body condition, see nutrition gamete intrafallopian transfer, 336 Breeding, see mating and artificial insemination Atresia, see also follicles Breeding season, see seasonality, reproductive and androgens , 243 Broad ligament , 6 and selection mechanism, 186 Bromocriptine, 471 during anovulatory season, 137 Bursa, ovarian, 12 of oogonia in fetal ovary, 402, 403 C Attachment, placental Canals of Gartner , 27 absorptive arcades, 381, 391 Capsule of embryo, 352 early attachment, 380 changing investments, 352 early history, 380 definition and history of, 352 fetal-maternal, 380 function of, 354 microplacentomes, 380 origin of, 354 placental exchange, 389 structure of, 353 Autocrine, 41 Cavity Autumn follicles, see hemorrhagic follicles of corpus luteum, 198, 202 B of pelvis, 12 Behavioral signs, see also specific signs Cerebrospinal fluid, hormones in , 252 and hormones, 93 Cervical star . and pheromones, 93 during parturition, 459 causes of diminished, 91 origin of, 390 effect of exogenous steroids on, 97 Cervix effect of ovariectomy on, 96 changes in form and consistency of, 210, 211 incidence of , 84 function of , 29 _ intensity of , 89 gross anatomy of , 29 in herds, 84 histologic changes of , during cycle, 209 in jennies, 83 secretions of , 215 of nonreceptivity , 82 smears of, 103 of receptivity, see estrous behavior tone of , 210 Index 629 Chorion, 367 of postpartum period, 475 Chorionic girdle of site of sperm deposition and transport, 301 and formation of endometrial cups, 48, 370 of suspension of reproductive tract , 8 and production of CG, 375, 403 of time of embryo entry into uterus, 302 origin of, 367 of use of clenbuterol to delay parturition, 475 Chorionic gonadotropin Conceptus, see also blastocyst, embryo, fetus and the fetus, 423, 435 amnion, 377 assay of, 50, 72 blastocyst stage, 350 circulating concentrations, 419 cleavage or oviductal stage, 348 composition of, 49 embryo-uterine interactions, 305 content in endometrial cups, 48 fertilization, 347 cross activity of, 50 fetal mobility, 408 effect of conceptus removal on, 433 fetal-maternal attachment, 380 effect of GnRH on, 422 fetus, 392 effect on follicles, 434 growth rate of embryonic vesicle, 413 effect on small follicles, 449 steroid production by early, 66, 429, 446 experimental administration of, 435 survival mechanisms initiated by, 432 factors affecting concentrations of, 420 transition from yolk to allantoic sac, 367 half—life of, 51 yolk-sac stage, 364 history of, 47 Constrictor vulvae, 33 in allantochorionic pouches, 420 Contraception, 333 in urine, 51 Corpus albicans , 202 pulsatility of, 422 Corpus hemorrhagicum role in second luteal response, 443 development of central clot, 197 role in third luteal response, 444 during pregnancy, 326 secretion in uterine lumen, 435 gross characteristics of, 200 temporal association with endometrial cups, 419 palpable characteristics of , 197 Chromosomes ultrasonic morphology of, 197 characterization of X, 295 Corpus luteum, see also primary and secondary corpora defects in, 536 lutea, luteal responses to pregnancy number of, 292 deficiency leading to pregnancy loss, 525 Cilia, 26, 29, 207 during pregnancy, 324 Cleavage of fertilized oocyte, 348 effect of hysterectomy on, 267 Clenbuterol, to delay parturition, 475 effect of pituitary antiserum on, 265 Clitoral wink, 81 effect of uterine stimulation and pyometra on, 267 Clitoris, anatomy of, and function of , 31, 33 estrogen production by, during pregnancy, 446 Clomiphene citrate, 170 extension of life of, 265 Colchicine, 433 gross characteristics of, 200 Colostrum, 457, 471, 473, 475 histology of, 199 Comparative aspects lack of effect of exogenous oxytocin on, 275 of artificial control of estrous cycle , 278 palpable characteristics of , 197 of concentrations of LH, 266 pathway for regression of , 268 of effect of exogenous CG, 50 persistence of , see prolonged luteal activity of effects of ovarian steroids and oxytocin , 268, 275 regulation of, during pregnancy, 438 of embryo transfer, 339 role of gonadotropins in regulation of , 265 of local uteroovarian relationships, 268 role of uterus in regulation of , 266 of mating stimuli on ovulation, 81 steroid content of , 63 of ova trapping, 305 summary of regulation of , 272 of palpation of vesicular bulge, 320 ultrasonic morphology of, 197 of placental consumption, 461 ultrastructure of , 200 630 Index Cortex and medulla of ovary , 16 measures of, 502 Cortical steroids, see adrenal steroids, dexamethasone and pregnancy loss, 509 Corticosteroid-binding globulin (CBG), 448 and twinning, 546 Corticotropin releasing factor, 57 Embryo , see conceptus, embryo proper, etc. Cortisol, 57, 71 , 244 and fetus, terminology, 345 Covert estrus , 90 defects of, in pregnancy loss, 536 Critical period, see luteal response to pregnancy (first) oviductal stage, 348 Crown-rump length, 396 Embryo fixation, 309 Cuboni test , 331 day of occurrence, 309 Cumulus-oocyte complex, 17, 295 effect of reproductive status, 310 Cyclic AMP, 46, 59, 258 mechanism of, 309 Cysts role of, 311 fimbrial, 27 Embryo mobility, 305 fossal, 26 and pregnancy loss, 522, 542 in mesosalpinx and mesovarian, 26 and transuterine migration, 305 ovarian (follicular), 224 nature and mechanisms of, 306 tubal, 27 role in first luteal response, 439 uterine, 624 role of, 311 D time of occurrence of, 306 Dehydroepiandrosterone Embryo proper, 345 biochemical aspects of , 63 gross anatomy of, 368 during pregnancy, 68 reproductive organs of, 369 effects of dexamethasone on, 243 summary of characteristics of, 368 in follicular fluid and blood, 243 Embryo orientation, 312 Deprivation hypothesis, see embryo reduction Embryo reduction, 550 Dexamethasone before Day 16, 550 and delay of parturition, 468 days of occurrence of, 551 and immune response to hCG, 281 mechanism of, 552 as an abortifacient, 452 postfixation, 550 effect of, on dehydroepiandrosterone, 243 summary of deprivation hypothesis, 553 effect of, on estrous behavior, 96 Embryo transfer , 337 to induce parturition, 475 aspects of, 339 Diestrous ovulations, 179, 223 multiple embryo collections, 339 Diestrus preservation and transport of embryos, 339 effect of antipituitary preparations on, 265 uses of, 338 effect of season on length of, 173 Embryo—uterine interactions, 305, see also embryo follicular changes during, see follicles mobility, fixation, orientation ovulation during, see diestrous ovulations summary of, 319 Digestive organs, relationship to reproductive organs, 9 Embryonic bulge, 320 Dihydroprogesterone-200c, 64 Embryo death, see pregnancy loss concentrations during pregnancy, 426 Embryonic disc, 345, 367 Diurnal variation in hormones, 240 Endocrinology of parturition, 465 Double ovulation, see multiple ovulations adrenal cortical hormones, 465 E estrogens and progestins, 467 Early pregnancy factor, 332 oxytocin, 470 Ectoderm, 364, 378, 406 prostaglandin F20c, 469 Efficiency, reproductive, 499 prolactin, 471 reputation of horses, 499 prostaglandin E2, 470 progress in improving, 499 Endoderm, 364, 367, 378 terminology for, 500 Endometrial cups and formation from chorionic girdle, 48, 370 and formation of allantochorionic pouches, 372 and relationship to site of attachment, 372 and uterine glands, 376 and uterine milk, 37 3 CG content in blood, 51, 435 CG content in cup secretions, 48, 435 gross anatomy and morphogenesis of, 372 histology and histogenesis of, 373 origin of, 370 sloughing of, 372 Endometrial folds, 28 Endometritis diagnosis of, 519 role of, in pregnancy loss, 519 uterine defense and therapy, 524 Endometrium, see uterus Endoscopic examinations, 211 Epoophoron, 27 Epostane, 468 Equilenin, 67, 430 Equilin, 67, 430 Estradiol, see estrogens in pregnant mares, 430 in urine of nonpregnant mares, 65 seasonal effects on, during pregnancy, 431 Estrogen injection test for pregnancy diagnosis, 332 Estrogens, see also estrone, equilin, equilenin and the selection mechanism, 263 binding of, to uterus, 276 biosynthesis of, in follicles, 63 concentrations in blood and urine during cycle, 241 concentrations near parturition, 467 concentrations during pregnancy, 427 concentrations during puerperium, 479 concentrations in foal versus mare, 467 early history of, during pregnancy, 428 effect of exogenous, on corpus luteum, 274 effect of exogenous, on LH and FSH, 248 effect of exogenous, on parturition, 468 effect of hCG on secretion of, 281, 452 effect of month Within ovulatory season on, 241 effect on sexual behavior, 97 effect on uterus, 276 excretion of, in nonpregnant mare, 65 excretion of, in pregnant mare, 67 factors effecting, during pregnancy, 431 fetoplacental unit in production of, 448 functions of, in parturition, 468 Index 631 in association with first ovulation of the year, 148 in cerebrospinal fluid, 252 in follicular fluid, 65 in induction of abortion, 452 in luteal tissue, 63 in urine during anovulatory season, 145 in urine during cycle, 241 in urine of pregnant mares, 67 , 430 production of, by early conceptus, 66, 429 ring—B unsaturated, 61, 67 , 430 role of, in maintenance of corpus luteum, 274 role of, in pregnancy, 446 role of, in production of PGFZOL, 274 secondary peaks, 242 source of, in pregnancy, 68, 446 steroidogenesis, 63 summary of sources in early pregnancy, 447 Estrone, also see estrogens concentrations in blood and urine during cycle, 241 during pregnancy, 430 in urine of nonpregnant mare, 65 Estrous behavior, see also estrus, unseasonal estrus, detection of, teasing techniques, specific signs during the anovulatory season, 95, 102 during pregnancy, 321 effect of dexamethasone on, 96 in ovariectomized mares, 96 mating Without detection of, 286 Estrous cycle, see also specific organ, hormone, stage artificial control of, 278 effect of season on length of, 174 induction of ovulation during, 260 length of, 173 secretion of fluids during summary of hormonal and ovarian events, 288 summary of regulation of, 264 Estrus, see also estrous behavior, specific organs and hormones appearance of cervix and vulva during, 208 association of length of, and follicular growth, 190 during pregnancy, 321 during puerperium, 476 effect of antipituitary preparations during, 258 effect of season on length of, 17 4 follicular changes during, 176 length of, 173 Exocoelom definition and origin of, 367 in terminal placenta, 462 632 Index F Fallopian tube, see oviduct Fertilization assisted, 335 description of, 347 in vitro, 337 rate of, 50, 502 time of first cleavage, 348 Fetal activity, 413, see also fetal mobility Fetal adrenals, role in parturition, 465 Fetal attachment, see attachment Fetal gender diagnosis, 340 Fetal gonads, see also fetal ovary gross changes in, 397 interstitial cells of, 398 role in parturition, 468 role in steroidogenesis, 68 seminiferous tubules of, 400 size of, 397 Fetal membranes, germ cell origin of, 364 Fetal mobility allantoic fluid shifts, 411 and fetal activity, 410, 413 and fetal presentation, 410 and umbilical cord twists, 410 early studies, 409 overview of fetal kinetics, 408 recent studies, 410 Fetal ovary germinal area of, 403 oogenesis in, 400 Fetal pituitary gland, 406 Fetal tubular genitalia, 404 Fetus, see also the above characteristics of, for age determination, 392 crown-rump length, 396 external features of, 393 external reproductive organs, 392 summary of external characteristics, 394 Fimbria of infundibulum, 25 Fimbrial cysts, 27 First stage of labor, 459 Fixation, see embryo fixation Flehmen, 77 and parturition, 460 and vomeronasal organs, 95 definition of, 94 role of, in olfactory cues, 95 Foal heat, 476 Foaling rate, 508 Follicles, see also ovulatory follicle, atresia during anovulatory season, 141 dynamics of, during cycle, 176 dynamics of, during pregnancy, 322 effect of anti-pituitary preparations on, 258 effect of month on, 183 estrogen production by, during pregnancy, 446 experimental stimulation of, 260 experimental suppression of, 258 histology of, 19, 137 in hysterectomized mares, 448 preantral and intrafollicular controls, 257 preovulatory changes in, 189 regulation of, during cycle, 257 role of progestins in regulation of, 450 role of, in maintenance of pregnancy, 168 seasonal effects on, during pregnancy, 168, 431, 452 summary of regulation of, 184, 264 ultrasonic monitoring of, 179, 180, 182, 183 waves of, 178 Follicle stimulating hormone, see also microheterogeneity, pulsatility and the selection mechanism, 263 chemical characteristics of, 41 concentrations during cycle, 235 concentrations near parturition, 481 concentrations during pregnancy, 423 concentrations prior to puberty, 493 during anovulatory season, 140 during CG production, 449 during inactive phase of anovulatory season, 145 during resurgence of anovulatory season, 150, 155 effect of estrogen and progesterone on, 250, 259 effect of exogenous androgens on, 250 effect of exogenous follicular fluid on, 449 effect of GnRH on, 250, 252 effect of gonadotropin antiserum on, 258 effect of month within ovulatory season on, 236 effect of weaning on, 478 in newborn, 492 interaction of ovaries and season on control of, 254 reciprocal relationship of, with LH, 246 seasonal profile of, 122 summary of ovarian—seasonal control, 256 temporal association with inhibin, 248 temporal associations with estrogen and progesterone, 247 Follicular fluid and inhibin and related factors, 56, 257 effect of exogenous, on LH/FSH, 251, 449 loss of, in association with ovulation, 193 steroid content of, 63, 64 Follicular waves characteristics of, 182 definition of, 178 primary wave, 180 secondary wave, 178 Follistatin, 56 Fornix, 31 Fossal cysts, 26 Freemartin, 559 FSH, see follicle stimulating hormone G Gamete intrafallopian transfer, 336 Genital tubercle origin of, 392 for diagnosis of fetal gender, 392 Germ cell origin of fetal membranes, 364 Germ cells, 291 Germ layers, 364 Gestation length, see pregnancy length GnRH, see gonadotropin releasing hormone Golden slippers, 465 Gonadotropin releasing hormone administration of, 166 and down regulation, 53 and pituitary portal system, 52 chemical characteristics of, 53 concentration in pituitary effluent, 252 during resurgence of anovulatory season, 152 effect of estradiol on, 253 effect of, on melatonin, 121, 156, effect of, on ovulation, 262 effect on chorionic gonadotropin, 422 effect on gonadotropins, 252, 253 effect on LH during pregnancy, 423 for control of estrous cycle, 281 immunization against, 253 pulsatility of, 252 response to, during puerperium, 480 role of, in reproductive seasonality, 120, 123 to induce early onset of ovulatory season, 166 Gonadotropins of pituitary, see also luteinizing hormone and follicle stimulating hormone administration of, 124 antiserum of, 258 cross-activity of, 44 isoforms of, 43 nomenclature of, 41 pulsatility of , 45, 151 Index purification of, 41 regulation of, 246 structure and subunits of, 43 summary of ovarian-seasonal control of, 256 summary of role in cycle regulation of, 264 Gonads, see ovary and fetal gonads Granulosa cells and granulosum in steroidogenesis, 63 Green grass effect, 161, 163 H Hair coat and reproductive seasonality, 128 Half-life of chorionic gonadotropin, 51 of luteinizing hormone, 45 of progesterone, 65 of prostaglandins, 59 hCG, see human chorionic gonadotropin Heat, see estrus Hemorrhagic follicles, 224 Herding, 85 Hillock, 17 Hilus of ovary, 15 Hinny and CG, 420, 426 HIOMT, see Hydroxyindole-O-methyltransferase Hippomanes development of, 415 in discharged placenta, 464 Histology, see specific organs Homosexuality, 102 Horses versus ponies, see ponies versus horses Human chorionic gonadotropin effect of, on life span of corpus luteum, 265 effect of, on secretion of estrogen, 281, 452 for induction of abortion, 452 for induction of ovulation, 170, 262, 279 HY antigens, 341, 416 Hydroxyindole-O-methyltransferase, 57, 118, 145 Hydroxyprogesterone-17oc, 63 concentrations ‘during pregnancy, 426 Hydroxysteroid dehydrogenase-3I3 and parturition, 468 in follicles, 63 in embryonic vesicle, 66 in placenta, 68 Hymen, 30 Hypothalamus anatomy and function of, 36 role of, in reproductive seasonality, 120 633 634 Index Hysterectomy effect on follicles, 448 effect on FSH, 423 effect on primary corpus luteum, 267 effect on supplementary corpora lutea, 444, 450 I Identical twins, 546 artificial production of, 338 natural occurrence of, 546 Immunology, reproductive, 416 Inactive phase of anovulatory season, 141 follicles and tubular genitalia of, 141 endocrinology of, 145 Infertility, see also efficiency, reproductive seasonal, 129 Infundibulum of oviduct, 25 Inhibin see also follicular fluid assay of, 56 concentrations during cycle, 245 history of, 55 structure of, 56 temporal association with FSH, 248 Inner cell mass, 345, 351 Intercornual ligament, 27 Interferon, 441 Interovulatory intervals, 175 Interstitial cells of fetal gonads histology and morphogenesis of, 398 in postnatal filly, 490 Isoforms, see microheterogeneity Isthmus of oviduct, 25 J Jenny androgens, 243 chorionic girdle of, 416 chorionic gonadotropin of, 420 estradiol during pregnancy, 430 estrogens of, 241 gonadotropins of, 44 LH of, 235 progestins of, 238 seasonality of, 108 sexual behavior of, 83 species crosses with, 420, 426 K Karyotyping, 340, 537 Kurosawa method of pregnancy diagnosis, 332 L Lactational anestrus, 47 8 Lamina propria, 29 Latitude, effect on reproductive seasonality, 110 Lavage, uterine, 486 LH, see luteinizing hormone LHRH, see gonadotropin releasing hormone Ligament broad, 6 intercornual, 27 of bladder, 8 proper (round), of ovary, 12 round, of uterus, 6, 8 sacrosciatic, 11 suspensory, of the anus, 33 Live-foal percentage, see foaling rate Lordosis, see posturing Luteal response to pregnancy (first), 438 and days of progesterone divergence, 438 and pregnancy loss, 510, 522, 542 and the antiluteolysin, 441 interval between, and second response, 443 nature of, 439 role of embryo mobility in, 439 role of estrogens in, 442 time of occurrence of, 438 Luteal response to pregnancy (second) 443, 529 Luteal response to pregnancy (third), 444 Luteinizing hormone, see also microheterogeneity, pulsatility and the selection mechanism, 263 assay procedures for, 72 chemical characteristics of, 41 concentrations during pregnancy, 422 concentrations during puerperium, 480 concentrations during the cycle, 233 concentrations in fetal circulation, 423 concentrations prior to puberty, 493 disappearance rate of, 45 during anovulatory season, 140 during inactive phase of anovulatory season, 145 during resurgence of anovulatory season, 149, 155 effect of androgens on, 249 effect of estrogen and progesterone on, 248, 259 effect of follicular fluid on, 251, 260 effect of GnRH on, 250, 252 effect of GnRH on, during pregnancy, 423 effect of gonadotropin antiserum on, 258 effects of weaning on, 478 in association with first ovulation of the year, 254 in cerebrospinal fluid, 252 in newborn, 492 interaction of ovaries and season on control of, 254 reciprocal relationship of, with FSH, 246 role of, in ovulation, 264 seasonal profile of, 122 summary of ovarian-seasonal control of, 256 temporal association of, with estrogen and progesterone, 247 temporal association of, with other events, 264 Lymph vessels, 35 M Macrovilli, 381 Major histocompatibility complex, 416 Maternal recognition to pregnancy, see luteal response to pregnancy (first) Mating in pasture, 506 interference with, 88 interval between, 87 number of mares per stallion, 296 optimal time of, 296 postovulatory, 299 time versus sex of foal, 300 Median eminence, 37, 252 Medulla and cortex of ovary, 16 Meiosis arrested stage of, 295 during fertilization, 347 Melatonin, 121 during inactive phase, 145 effect of exogenous GnRH on, 121, 156 general, 57 in reproductive seasonality, 118 Mesoderm, 364, 367, 391 Mesometrial attachment, 7 Mesometrium, 6 Mesonephric ducts (Wolffian ducts), 370 Mesosalpinx, 6, 12 Mesovarium, 6, 12 Methallibure, 286 Microcotyledons, microcaruncles, microplacentomes, 381 and maternal—fetal exchange, 389 and placental attachment, 381 and their development from macrovilli, 381 avillous areas, 390 functional development of, 388 gross appearance of, 386 mature, 386 microcaruncles during puerperium, 485 of discharged placenta, 462 vascularity of, 386 Index Microheterogeneity effect of estradiol on, of LH, 249 of chorionic gonadotropin, 49 of FSH, 43, 237 of LH, 43, 237 Microvilli, see microcotyledons Morula, 302, 343, 348, 350 Mouth clapping or clamping, 83 Mucin test for pregnancy diagnosis, 332 Mullerian ducts, 27, 370 Multiple ovulations, 218 and multiple embryos, 339 as a cause of twins, 547 bilateral versus unilateral, 221 definition of, 218 diameter of, 221 effects of age on, 221 effects of breed on, 218 effects of reproductive status on, 219 effects of season on, 220 hormonal relationships to, 223 induction of, 166, 260 repeatability of, 219 synchronous versus asynchronous, 221 Myometrium activity during parturition, 461 contractions during cycle, 216 contractions during pregnancy, 313 N Nerves of reproductive tract, 37 Neural stalk, 37 Neurohypophysis, see posterior pituitary Nutrition effect on length of pregnancy, 329 effect on reproductive seasonality, 126, 161 0 Official birth date, 131 Olfactory cues, see pheromones Omphalopleure, see yolk sac, bilaminar, trilaminar Oocytes, see also ova collection and culture of, 336 cumulus—oocyte complex, 295 germinal vesicle, 292 intrafollicular, 292 meiotic arrest, 295 primary and secondary, 291 stage of ovulated, 295 Oogenesis, summary of, 292 Opioids, 54 Orientation,see embryo orientation 635 636 Index Ova, see also oocytes appearance of, 291 cleaved, production of humoral agents, 305 degeneration of unfertilized, 303 life span of, 296 time of entry of, into uterus, 302 transfer of, see biotechnology trapping of unfertilized, 303 Ovarian bursa, 12 Ovarian irregularities diestrous ovulations, 223 failure of ovulation, 224 hemorrhagic follicles, 224 multiple ovulations, 218 prolonged luteal activity, 227 Ovariectomy and estrous behavior, 96 during pregnancy, 437 effect on gonadotropins during puerperium, 480 effect on LH and FSH during cycle, 254 effect on LH and FSH throughout the year, 121 effect on parturition, 469 prior to puberty and effect on gonadotropins, 494 Ovary, see also corpus luteum, follicles, ovarian irregularities accessory structures and vestiges of, 26 attachment of, 14, 15 during pregnancy, 321 general function of, 17 gross anatomy of, 14 hilus of, 15, 17 histology during anovulatory season, 141 in prepuberal filly, 488 internal structure of, 16 ovulation fossa of, 12, 16, 489 proper (round) ligament of, 12 role of, in pregnancy maintenance, 437 summary of changes during pregnancy, 328 vasculature of, 34 Overt estrus, 90 Oviduct, see also ova accessory structures and vestiges of, 26 contractions of, 215 cysts of, 26 functional anatomy of, 26 gross anatomy of, 25 histologic changes during estrous cycle, 208 histology of, 26 masses in, 304 secretions of, 214 trapping of ova in, 303 vasculature of, 34 Oviductal embryos cleavage rate of, 348 in vitro culture of, 350 morphology of, 349 Ovulation, see also ovulatory follicle biochemical and biophysical mechanisms associated with, 191 clinical determination of, 195 during diestrus, see diestrous ovulations during pregnancy, see supplementary corpora lutea effect of exogenous GnRH on, see GnRH effect of hCG on, see hCG effect of pituitary extracts on, see pituitary extracts evacuation patterns, 193 failure of, 224 fate of discharged fluid during, 194 gonadotropin regulation of, 258 induction of multiple, 124, 154, 165 induction of, during anovulatory season, 158 induction of, during estrous cycle, 279 inequality in side of, 190 multiple occurrence of, see multiple ovulations nature of, 191 occurrence at night of, 192 prediction of, 192 seasonal distribution of, 106 sensation in association with, 193 site of, see ovulation fossa stage of oocyte at the time of, 295 synchronization of, 282 time of, in relation to estrus, 190 Ovulation fossa, 12, 192, 489 Ovulatory follicle, see also ovulation changes in, 189 characteristics of, 189 day, becomes the largest follicle, see selection mechanism effect of antipituitary preparations on, 258 growth rate of, 189 interval required for development of, 190 length of estrus and growth of, 190 maximum diameter of, 192 Ovulatory season corpus luteum during, 195 definition of, 106 endocrinology of, 233 estrous cycles during, 173 follicular dynamics during, 176 interovulatory intervals during, 175 ovarian irregularities during, 217 ovulation during, 190 preovulatory period during, 189 seasonal changes during, 174, 183 selection of dominant follicle during, 185 summary of folliculogenesis during, 184 tubular genitalia during, 207 Oxytocin concentrations during cycle, 275 effect of exogenous, on corpus luteum, 275 for induction of parturition, 473 general function of, 56 in corpus luteum, 57 role in production of PGFZOL, 275 role in parturition, 470 role in uterine contractions, 215 P Paracrine, 41, 57 Parentage, 301 Paroophoron, 27 Pars distalis, 37 Pars intermedia, 37 Pars tuberalis, 37 Parthenogenic cleavage, 303 Parturition, see also endocrinology of parturition characteristics of, 457 endocrinology of, 465 induction of, 473 myometrial activity during, 461 nocturnal nature of, 458 predicting imminence of, 457 rotation of fetus during, 459 stages of, 459 summary of control of, 472 Pelvic cavity, 11 Pelvic diaphragm, 12 Perimetrium, 7 Perineal body, 12 Perineum, 12 Peritoneum, Visceral and parietal layers, 7 Perivitteline space, 349 Persistent corpus luteum, see prolonged luteal phase PGFZOt, see prostaglandin F20L PGFM, 244, 275, 469 Phantoms, 103 Pheromones, 93 Photoperiod, also see seasonality, reproductive clinical alteration of, 113 description of natural, 110 Index 637 effect on hair coat, 128 effect on puberty, 494 experimental alteration of, 113 summary of effects of, 117, 162 summary of role in reproductive seasonality, 117 use of, for induction of ovulation, 158, 162 Photosensitive period, 116 Pineal gland antigonadal factors of, 118 effect of environmental nonlight factors on, 126 gross anatomy and histology of, 38 pathway from eye to, 118 removal of, 120 role of, in reproductive seasonality, 118 seasonal morphologic changes in, 118 superior cervical ganglionectomy of, 119 Pinealectomy, 120 Pituitary, anterior and portal system, 38, 52 embryonic origin of, 36 gross anatomy and function of, 36, 38 role of, in reproductive seasonality, 121 sampling effluent of, 52 Pituitary extract effect of, on ovulation during ovulatory season, 260 effect of, when given in anovulatory season, 165 for induction of multiple ovulations, 260 Placenta, terminal characteristics of, 462 retention of, 461 PMS, PMSG, see chorionic gonadotropin Polar bodies, 292, 347 Polyovular follicles, 547 Ponies versus horses cycle control programs in, 287 diestrous ovulations in, 223 general, 230 incidence of multiple ovulations in, 218 incidence of prolonged luteal activity in, 227 length of estrous cycle in, 173 reproductive seasonality in, 107 Pontine flexure, 368 Portal system, see pituitary Postpartum period, see puerperium Postpartum endocrinology, 479 LH/FSH, 480 steroids, 479 Posturing, 77 638 Index Pouches abdominal, 9 allantochorionic, 373 Rathke’s, 406 Pregnancy diagnosis, 330 Pregnancy length factors affecting, 329 prolonged, 330 shortened, 330 Pregnancy loss after Day 40, 515 and etiology of late fetal loss, 545 and fate of dead embryos, 544 and habitual abortion, 544 and luteal progesterone, 525 critical periods for, 516 definition of, 501 due to chromosomal defects, 536 due to embryo defects, 536 due to gamete aging, 537 during postpartum period, 541 effect of age on, 540 effect of stress on, 538 immunologic aspects of, 538 in yearlings, 539 in oviducts, 511, 518 on Days 6 to 10, 512 on Days 11 to 40, 514 overall loss rate, 510 relationship to conceptus undersize, 542 role of uterine inflammation in, 519 study of, 509 summary of causes of embryo loss, 535 summary of embryo loss following luteolysis, 523 summary of time of occurrence, 517 time of occurrence, 511 Pregnancy protein—one, 441 Pregnancy rate, 503 definition of, 500 during pasture mating, 506 effect of age on, 505 effect of breed on, 504 effect of puerperium on, 505 effect of reproductive status on, 504 effect of season and other factors on, 507 for end of season, 508 Pregnanes concentrations during pregnancy, 426 general, 69 Premature birth, 330 Prepartum milk test, 458 Primary corpus luteum during pregnancy, 324 effect of CG on, 443 effect of early embryo on, 430 Primary oocytes, 291 Primordial follicles, 293 Primordial germ cells, 291, 400 Proceptivity, 76 Progesterone and progestins, see also assay, luteal responses to pregnancy and the selection mechanism, 263 binding in blood of, 66 concentrations during cycle, 237 concentrations near parturition, 467 concentrations during pregnancy, 424 concentrations during puerperium, 479 concentrations following administration of, 66 concentrations in corpora lutea, 237 concentrations in foal versus mare, 467 concentrations postovulatory, 238 deficiency in, leading to pregnancy loss, 525, 539 effect of exogenous, on parturition, 468 effect of month on, 240 effect of species crosses on, 426 effect of stress on, 539 effect of, on estrous behavior, 98 effect of, on follicles, 450 effect of, on uterine tone, 315 effect of injections of, on LH, 248 effect on uterus, 276, 315 for artificial control of estrous cycle, 163 for artificial control of postpartum ovulation, 487 for termination of anovulatory season, 163 formation of, in placenta, 69 functions of, in parturition, 467 half-life of, 65 in cerebrospinal fluid, 252 in newborn, 467 in milk, 240 in uterine flushes, 240 in uterine vein during pregnancy, 70 luteal sources during pregnancy, 436 metabolism of, to pregnanes by placenta, 69 rapid assays of, 72 role in production of PGFZOL, 274 role of, during pregnancy, 437, 525 role of, in release of uterine luteolysin, 274 steroidogenesis, 63 summary of sources during pregnancy, 445 therapy to prevent pregnancy loss, 525, 526 Prolactin during pregnancy, 432 general, 47 role of, in reproductive seasonality, 124 Prolonged luteal activity, 227 pseudopregnancy, 228 study of, by ultrasound, 229 terminology, 227 Proper ligament of ovary, 14 Prostaglandin E2 during parturition, 470 exogenous, to soften cervix, 470 Prostaglandin F20L and endotoxins, 522 and first luteal response to pregnancy, 440 binding of, to corpus luteum, 273 clearance of, 27 3 concentrations during cycle, 244 concentrations during parturition, 469 concentrations of metabolite of, 244 effect of, on corpus luteum during cycle, 267 first assay report for, 244 for artificial control of estrous cycle, 282, 284 for control of postpartum ovulation, 487 for induction of parturition, 474 for postpartum myometrial stimulant, 486 for termination of pseudopregnancy, 271 general effects of, 59 induction of abortion with, 433, 450 mechanisms in production of, 274 minimal luteolytic dose of, 267, 271 resistance to luteolytic action of, 27 3 role of, in uterine contractions role of, in luteolysis, 273 role of, in parturition, 469 site of action in luteolysis, 273 summary of production of, 277 synthesis of, 59, 274 tissue culture studies, 441 transient effect of, on several hormones, 272 Prostaglandins, general, 58 Protein transforming growth factor, 56 Pseudopregnancy and effects of steroids on uterine tone, 314 and prolonged luteal activity, 228 definition of, 228 effect of prostaglandin F20c on, 271 subsequent to embryo loss, 528 Index Puberty age of, 490 colts versus fillies, 494 effect of health on, 492 effect of nutrition on, 491 639 effect of ovariectomy on LH and FSH prior to, 494 in feral fillies, 492 LH and FSH prior to, 492 nature of first ovulatory season, 492 ovarian anatomy prior to, 488 summary of gonadotropins, 496 Puerperium, see also postpartum uterine involution artificial control of, 487 effect of suckling on, 478 effects of body condition on, 478 effects of weaning on, 479 first estrus of, 476 first ovulation of, 476 importance of, 475 seasonal effects on, 476 use of artificial lights during, 160 Pulsatility, see also specific hormones and synchrony of LH and FSH, 235, 246 general, 45 of FSH, 46, 237, 481 of GnRH, 53, 252 of LH, 46, 234, 481 of progesterone, 240 Pyometra, 522 Q Quiet ovulation, 91 R Rathke’s pouch, 406 Rauber’s layer, 351, 367 Receding phase of anovulatory season definition of, 135 endocrinology of, 140 follicular dynamics of, 140 Receptors , for LH in corpus luteum, 266 for PGF20c in corpus luteum, 273 of steroids in uterus, 276 Recrudescence, see resurgence Rectogenital pouch, 9, 432 Relaxin concentrations during pregnancy, 431 general, 57 source of, 432 Reproductive efficiency, see efficiency, reproductive Reproductive hormones, see also specific hormones 640 Index Reproductive seasonality, see seasonality, reproductive Resurgence of anovulatory season, 146 definition of, 134 endocrinology of, 148 follicular dynamics of, 146 late resurging phase, 155 pulsatility of gonadotropins during, 151 role of GnRH during, 152 transitional period, 135 Resurgence of corpus luteum, 443, 529 Retained placenta, see placenta, terminal Round ligament of ovary, 14 of uterus, 6, 8 S Season effect on estradiol during pregnancy, 431 effect on follicles during pregnancy, 168, 452 effect on length of pregnancy, 329 effect on postpartum ovulations, 476 effect on supplementary corpora lutea, 452 Seasonality, reproductive and LH and FSH profiles, 122 association of hair coat changes with, 128 effect on distribution of estrous periods, 108 effect on distribution of ovulations, 106 effect of age on, 111 effect of light on, 113 experimental alteration of, 113 in jennies, 108 in zebras, 109 interaction with ovaries, 254 mechanisms of control of, 112 near the equator, 111 neural pathway for, 117 problems associated with study of, 106 relationships to photoperiods, latitudes, breeds, 110 role of environmental factors in, 126 role of hypothalamus in, 120 role of intrinsic factors in, 127 role of pineal in, 118 role of pituitary in, 121 role of prolactin in, 124 seasonal infertility and, 129 summary of role of daylength on, 125 terminology used for, 106 Second stage of labor, 460 Secondary corpora lutea, see also diestrous ovulations during pregnancy, 324 effect of CG on, see luteal response, third Secretions of reproductive tract, see uterine secretions Selection mechanism (follicular), 185 hormonal control of, 262 methods of study, 186 summary of, 188 time of, 186 Semen, see sperm, mating, artificial insemination Senescence, 111 Serotonin, 57 Sexual behavior, see estrous behavior Siderophages, 485 Silent estrus, 91 Sinus terminalis, 365, 390, 464 Somatopleure, 367 Sperm capacitation of, 296, 337 deposition and transport of, 301 entry of, into oocyte, 347 life span in mare of, 296 reservoir for, 301 sex separation of, 340 Splanchnopleure, 367 Split estrus, 90 Squatting, see posturing Stabling to induce early onset of ovulatory season, 161 Steroids, see also specific hormones adrenal, 71 alternate pathways for, in follicles, 63 biochemistry, 60 during inactive phase of anovulatory season, 141 during resurgence of anovulatory season, 148 early studies of, 60 in nonpregnant mares, 62 in pregnant mares, 66 production by embryonic vesicle, 66 research obstacles, 59 steroidogenesis, 62 Stratum compactum, 29 Stratum spongiosum, 29 Subestrus, 90 Suckling, effect on ovaries and gonadotropins, 478 Superior cervical ganglionectomy, 119 Supplementary corpora lutea, 324, 325, see also luteal response to pregnancy (third) Suspensory ligaments of the anus, 33 of the reproductive tract, 6 Syngamy, 347 T Teasing techniques alternatives to, 103 group methods of, 102 individual methods of, 99 use of androgenized teasers in, 103 use of marking devices in, 103 Testosterone, see androgens Third stage of labor, 461 Third ventricle, 52, 252 Transitional period, see resurgence Transuterine migration, see embryo mobility Transverse fold of vagina, 30 Trilaminar omphalopleure, 365 Trophoblast, definition of, 351 Trophoblastic vesicles, 352 Trophoblastic protein-one, 441 Tubal cysts, 27 Tubal membrane, 12 Tubular genitalia, definition of, 1 Twinning tree for twin probabilities, 556 Twins, 546, see also embryo reduction during fetal stage, 558 early growth, 548 embryo reduction of, 550 incidence and origin, 546 management of twin embryos, 554 management of twin fetuses, 559 mobility and fixation of, 548 probability of (twinning tree), 556 Two-cell theory, 63 U Umbilical cord of discharged placenta, 464 Unseasonal estrous behavior, 95, 108 Urachus, 415 Urinary bladder, ligaments of, 8 Urination, as a behavioral Sign, 81 Uterine artery, 34 Uterine capacity, 414 Uterine contractions and embryo-uterine interactions, 313 control of, 313 during cycle, 216 summary of, during cycle, 217 techniques for measuring, 216 Uterine echotexture during anovulatory season, 148 during estrous cycle, 212 Index 641 Uterine glands and absorptive arcades, 391 and endometrial cups, 376 during puerperium, 485 general anatomy of, 29 histologic changes of, during cycle, 208 Uterine histology during anovulatory season, 143 during early pregnancy, 317 during estrous cycle, 208 during puerperium, 485 Uterine involution, 481 histology of, 484 hormonal aspects of, 486 importance of, 481 lavage and, 486 lochial contents and uterine secretions, 484 myometrial stimulants, 486 size changes, 482 summary of rapidity of, 482 tone and contractility during, 483 Uterine luteolysin, see prostaglandin F20c Uterine milk and absorptive arcades, 391 and endometrial cups, 373 Uterine secretions during early pregnancy, 317 during estrous cycle, 214 Uterine tone association of, with diameter, 213, 316 cause of, 315 changes during estrous cycle, 209 during pregnancy, 314 Uterine tube, see oviduct Uterotubal junction, 25 Uterus, see also uterine echotexture, —tone, —histology, — contractions, —secretions, —involution association with digestive organs, 9 changes of, visible by endoscope, 212 contractions 05215, 313 diameter of, during cycle, 213 during anovulatory season, 141 general histology of, 28 gross anatomy of, 9, 27 handling artifacts of, 27 hormonal regulation of, 276 intercornual ligament of, 27 round ligament of, 6, 8 vasculature of, 34 642 Index V Vagina electrical resistance of, 193 general histology of, 31 gross anatomy of, 30 histologic changes in, during cycle, 209 secretions of, 215 smears of, 215 vasculature of, 34 Visible changes during cycle, 211 Vaginal artery, 34 Vaginal fornix, 31 Vaginal process, 8 Vaginal smears, 103 Vasculature, see artery, vein Vasopressin, 57 Veins to reproductive organs, 34 vitelline, 363 role in uteroovarian relationships, 269 Vesicogenital pouch, 9 Vessels, see artery, vein, lymph Vestibular bulbs, 33 Vestibular glands, 32 Vestibule, 31 Vomeronasal organ, 95 Vulva gross anatomy of, 31 histology of, 32 labia of, 33 vasculature of, 34 . Visible changes in, during cycle, 211 Vulval wink, 81 Vulvar cleft, 32 W Winking of clitoris or vulva, 81 Wolffian ducts, 370 Waxing, 457 Y Yolk sac bilaminar omphalopleure of, 365 endodermal encirclement of, 364 histology of, 365 in discharged placenta, 464 mesodermal invasion of, 365 morphogenesis of, 367 shape changes in, 366 sinus terminalis of, 365 transition to allantoic sac, 367 trilaminar omphalopleure of, 365 Z Zebras seasonality of, 109 gonadotropins of, 45 Zona pellucida, 349 shedding of, 351 Zygote, 347