key: cord-0326857-nwrv0152
authors: Campolina, Joana P.; Coelho, Sandra G.; Belli, Anna Luiza; Machado, Fernanda S.; Pereira, Luiz G. R.; Tomich, Thierry R.; Carvalho, Wanessa A.; Silva, Rodrigo O. S.; Voorsluys, Alessandra L.; Jacob, David V.; Campos, Mariana M.
title: Effects of a blend of essential oils in milk replacer on performance, rumen fermentation, blood parameters and health scores of dairy heifers
date: 2020-03-17
journal: bioRxiv
DOI: 10.1101/2020.03.17.995209
sha: 735792aa71c44a8599d70455772a9eb7dd33d701
doc_id: 326857
cord_uid: nwrv0152

The objective of this study was to evaluate how the inclusion of a blend of essential oils in milk replacer (MR) affects different outcomes of dairy heifers. The outcomes evaluated: feed intake, performance, body development, blood cells and metabolites, insulin-like growth factor-1 (IGF-1), rumen fermentation, fecal scores and respiratory scores. All outcomes were evaluated during pre-weaning (4 – 60 d of age), and carryover effects on post-weaning (61 – 90 d of age) periods. The experimental units utilized were 29 newborn Holstein × Gyr crossbred dairy heifers, with genetic composition of 5/8 or more Holstein and 3/8 or less Gyr and body weight (BW) at birth of 32.2 ± 5.2 kg. Experimental units were randomly assigned to either a control group (CON, n = 15) or a treatment group, consisting of supplementation of a blend of essential oils (BEO, n = 14) with1 g/d/calf (Apex Calf, Adisseo, China). During the pre-weaning phase, all heifers were fed 5 L of MR/d reconstituted at 15% (dry matter basis), divided into two equal meals. Water and starter were provided ad libitum. During the post-weaning, animals received a maximum 3 kg of starter/d, and ad libitum corn silage, divided into two meals. The outcomes feed intake, fecal and respiratory scores were evaluated daily. BW was measured every three days, while body development was recorded weekly. Blood samples were collected on 0, 30 and 60 d of age for total blood cell count, weekly to determinate ß-hydroxybutyrate, urea and glucose, and biweekly for IGF-1. Ruminal parameters (pH, volatile fatty acids, ammonia-N and acetate:proprionate proportion - C2:C3) were measured each 14 days. A randomized complete block design with an interaction between treatment and week was the experimental method of choice to test the hypothesis of effect of BEO on all outcomes. An ANOVA procedure was used for continuous outcomes and a non-parametric test was used for the ordered categorical outcomes both adopting a C.I. = 95%. Results indicated that there was not enough evidence to accept the alternative hypothesis of effect of BEO in MR on: feed intake, performance, body development and blood metabolites during both pre-weaning and post-weaning periods. However, results indicated that the inclusion of BEO in MR significantly affects the proportion of C2:C3 during pre and post-weaning (P ≤ 0.05). Similarly, the effect is significant for basophil (P ≤ 0.001) and platelet (P ≤ 0.05) counts in pre-weaning. The interaction between week and treatment was also significant for lymphocytes (P ≤ 0.001), revealing a long-term treatment immunological effect. Lastly, the effect on fecal scores was also significant (P ≤ 0.05) during pre-weaning, with lower values for BEO. BEO contributed for ruminal manipulation on pre-weaning and carry over effect on post-weaning; immunity improvement and a decrease morbidity of neonatal diarrhea in pre-weaning phase.

141 box. They were then mixed with a 10 mL of milk, homogenized and incorporated in 142 0.49 L of MR (0.5 g/calf at morning meal and 0.5 g/calf at afternoon meal) to ensure 143 total ingestion of the product. After ingesting 0.5 L MR with 0.5 g of the blend of 144 essential oils, the rest of the meal was given.

145 

The analysis of each outcome was performed independently of all others using 286 linear mixed models (package: nlme). Each independent outcome was modeled as a 287 function of the following fixed effects: treatment, experimental week, the interaction 288 between treatment and week. Birth weight and serum Brix refractometer were tested as 289 a covariate but did not improve statistical significance. Therefore they were eliminated 290 from the model. The genetic composition of the animal was included as a blocking 291 effect. The effect of heifer within treatment was included in the models to account for 292 individual variability.

The continuous outcomes such as intakes, performance, ruminal and blood 294 parameters were analyzed with ANOVA. A 95% Confidence Interval was adopted to 295 accept or deny the null hypothesis and P-values were produced with a Fisher test. In 296 order to meet the required assumptions of this model, all outcomes were tested for 13 297 normality, and variable transformation was applied to milk replacer intakes to meet that 298 assumption.

The categorical outcomes fecal and respiratory scores were analyzed using a non-300 parametric aligned rank transformation test, implemented in the R package ARTool. A 301 95% Confidence Interval was also adopted for the non-parametric tests.

303 Results and discussion 304 Intake and heifer performance 305 Most studies evaluating essential oils or a supplement with BEO to dairy calves, 306 feed the additive in the starter, to benefit rumen development and accelerate growth.

307 However, the intake of starter in the firsts weeks of age is small (23). Due to the calf's 308 limited capability of ingesting large solid feed amounts in the first days of life, the 309 supplement intake could be compromised during pre-weaning period and possibly mask 310 any effects. Therefore, in this trial, it was decided to offer BEO in the liquid diet.

The tested BEO has a strong aroma. Nevertheless, due to the way it was offered, 312 no rejection of the mixture BEO and MR was observed. The supplemented heifers 313 consumed liquid diet equally to the control treatment, with no refusal and good 314 acceptance (Table 2 ). Some essential oils have different acceptability by animals. Table 2 . Pre and post-weaning milk replacer (MR) intake, starter intake, total dry 322 matter intake (DM), total crude protein intake (CP), total gross energy and water 323 intake of heifers (n = 29) of control (CON) and supplemented with blend essential 324 oils (BEO) in milk replacer during pre-weaning. (Table 3) 355 during pre and post-weaning. As was also observed for intake and ADG, a week effect 16 356 (P ≤ 0.05) was detected in all variables due to normal animal growth. Table 4 ). Since there were no differences between treatments during pre-375 weaning, the carryover effect may not be assumed to be the answer for this difference.

376 Although no differences on intake were observed, heifers' ingestion behavior might 377 justify the difference in post-weaning pH. However, this parameter was not evaluated. 378 Nevertheless, previous studies with essential oils supplementation also found low pH 379 values for pre-weaning calves, but no essential oils treatment effect (16,25,27). 458 Since essential oils can increase of insulin sensitivity, not finding glucose differences 459 between treatments does not mean that there were no changes in glucose pathway.

460 Therefore, further investigations over theses aspects is needed.

All blood cells count were within age and specie normality. According to Benesi 

The average age for diarrhea occurrence was 12.2 ± 3.6 d for BEO and 13.6 ± 3.8 516 d for CON with no statistical difference. Diarrhea incidence on pre-weaning in BEO 517 treatment was 85% against 93% for CON treatment with no statistical difference. Fecal 518 score was different between treatments (P ≤ 0.05), with lower values for BEO, and 519 changed through time (P ≤ 0.05, Table 7 ). Days with diarrhea and days with severe 520 diarrhea were not different within treatments (Table 7) . Three animals of each group 521 were treated for diarrhea with anti-inflammatories, and the treatment duration was of 522 1.6 ± 0.57 d of treatment for BEO and 3.0 ± 1 d for CON. It is noteworthy that this 523 treatment was done outside the total cell count.

524 525 Table 7 . Pre and post-weaning mean values of fecal score, health score, days with 526 health score above 4, days with fever, days with diarrhea, days with severe 527 diarrhea of control heifers (CON) and heifers supplemented with blend of essential 528 oils (BEO) in milk replacer during pre-weaning. 

Powder integral milk, wheat isolated protein, acidifying additive, whey, coconut oil

calcitic limestone, sodium chloride, corn gluten 152 meal, defatted corn germ, wheat bran, soybean meal, rice hulls, kaolin, molasses, 153 flocculated corn, ground corn, corn grain, alfalfa hay, monensin, citrus pulp, dried 154 sugarcane yeast, whole toasted soybean, sodium selenite, copper sulfate, manganese 155 sulfate, cobalt sulfate, iron sulfate, zinc sulfate

Heifers were weaned at 60 d of age. During the postweaning period

161 d of age, all heifers received starter and corn silage (Table 1). The amount of corn silage 162 provided was enough to result in at least in 10 % orts, and the starter intake was fixed in and received local anesthesia (5.0 mL/horn, Lidovet, Bravet, Engenho Novo, Brazil) 165 and 2 d of non-steroid anti-inflammatory (0.025 mL/kg, Maxicam 2%

Feed intake (MR, starter, water and corn silage) was measured daily. Samples of

PA) through a 173 1-mm screen before analysis. Starter, corn silage and MR were analyzed to determine 174 DM (Method 934.01), CP (Method 988.05), ether extract

Wither 183 height (distance from the base of the front feet to the withers), rump height (distance 184 from the base of the rear feet to the rump), rump width (distance between ileus) and 185 heart girth (circumference of the chest) were measured on birth date and, thereafter, at 14, 28, 42, 60, 74 and 90 d of age and pH was assessed using a 193 portable potentiometer (Phmetro T-1000

They 212 were immediately transported on ice to the laboratory and were centrifuged at 3000 x g 213 for 10 min at room temperature (22 -25 °C). Two aliquots of each metabolite and 214 hormone sample were individually allocated into microtubes and frozen at -20 ºC for 215 further analysis. BHB and urea serum concentrations were determined by use

Plasma glucose was measured in a microplate Spectrophotometer EON

VT) using the enzymatic colorimetric method

IGF-1 plasma concentrations were analyzed using 222 chemiluminescence essay

Blood samples were collected for complete blood count during preweaning at 0, 225 30 and 60 d of age, by jugular vein puncture into EDTA tubes (Labor Import

Brazil) was used to perform: red 228 blood cell count (RBC), packed cell volume (PCV), hemoglobin (Hb), mean 229 corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), 230 platelet and total white blood cell count. Manual white cell blood differential counting magnification for: total leukocyte count, basophils, eosinophils, neutrophils, 233 band neutrophils, segmented neutrophils, lymphocytes, monocytes. Morphological 234 changes were also performed, such as toxic neutrophils, reactive lymphocytes and 235 activated monocytes (22)

Health measurements (fecal and respiratory scores) were performed daily, in the 240 morning, before other animal management. Fecal scores were graded according to

as follows: 0 -normal (firm but not hard); 1 -soft (does not hold form, 242 piles but spreads slightly); 2 -runny (spreads readily to about 6 mm depth); and 3 -243 watery (liquid consistency, splatters). A heifer was considered to have diarrhea if fecal

for that reason, a low pH is frequently found in pre-weaning calves

Therefore, acidosis has not been reported at this phase, since calves can stand lower 394 rumen pH values when compared to adult ruminants

Changes in these profiles in rumen fluid 402 would also alter the acetate:propionate (C2:C3) proportions. Since butyrate and 403 propionate are important for ruminal papillae development, and especially propionate is 404 used in the gluconeogenesis route (5), a smaller C2:C3 ratio is wanted

This could reduce the chances of essential oils entering the rumen and alter 413 local microbiota as well as VFA. Nevertheless, since the MR amount was small and 414 given at beginning of the feeding, it is possible that the esophageal groove was still 415 open, permitting essential oils content extravasation to the rumen

Corroborating these findings, Santos et al. (25) did not present differences for VFA 422 in supplemented calves, but also observed age impact on the VFA profile. Since VFA 423 are associated with rumen development

As for nitrogen ammonia, there was no treatment effect during pre-or

contrast, Santos et al. (25) reported greater nitrogen ammonia

For all ruminal parameters it was found week effect during preweaning

That could be related to the increase in starter intake, rumen development and

Similar patterns of BHB, glucose (14,25), urea (16), 437 total plasma protein, and IGF-1 (29) were found for essential oil supplemented calves

Table 5), since they are 439 directly correlated with fatty acid metabolism and ruminal ammonia concentration, 440 respectively (30)

4 0 -normal (firm but not hard); 1 -soft (does not hold form, piles but spreads slightly)

532 2 -runny (spreads readily to about 6 mm depth); and 3 -watery (liquid consistency, 533 splatters). A heifer was considered to have diarrhea if fecal score was 2 or 3

Respiratory score evaluations considering the sums of: rectal temperature score = 0 -respiratory score or number of affected animals. However, it was observed a week 552 and an interaction week x treatment effect on pre-weaning, with difference between 553 treatments scores and lower values for BEO

Week 2 was the period in which animals had higher incidence of diarrhea. Thus, it can 555 be assumed that respiratory signs were related to previous enteric disease since a higher period, with an average of treatment days of 1.3 ± 1.4, and 3 for CON with 560 an average of treatment days of 2

Post-weaning respiratory scores revealed higher 564 mean values

Gram positive bacteria 568 are the most sensitive to the essential oils microbial activity (7,12), but it is possible that 569 gram negative bacteria and some types of parasites are also susceptible (9) to different 570 essential oils. Thus, essential oils could reduce the incidence and severity of diarrhea did not inhibit bacterial growth

573 at this concentration BEO did not have any antibacterial in vitro effect. However, 574 besides no direct influence found over the bacterial evaluation, BEO calves presented 575 differences on basophil (Table 6) and lymphocyte cell populations

Feeding BEO to pre-weaned heifers on MR did not affect intake, performance 583 parameters, blood metabolites or IGF-1 concentration. However, it changed C2:C3 on decrease morbidity of neonatal diseases. Therefore, essential oils are a 588 health additive option to modern production sytems

Professor Ângela Quintão, Professor Fabiola Paes Leme and Vera Carsoso Ferreira

Aiken for helping on this paper. We also thank Coordenação de Aperfeiçoamento de

Fundação de Amparo à Pesquisa 596 do Estado de Minas Gerais

Embrapa Dairy Cattle 599 (Minas Gerais, Brazil) and Adisseo Company for financial support of this research

606 Investigation: Joana P. Campolina, Anna Luiza Belli

Anna Luiza Belli and Mariana 608 M. Campos

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