key: cord-0706200-8kvchxu0 authors: Tan, Xian; Li, Jia; Li, Yali; Li, Jianzhong; Wang, Qingping; Fang, Lin; Ding, Xueqin; Huang, Pengfei; Yang, Huansheng; Yin, Yulong title: Effect of chicken egg yolk immunoglobulins on serum biochemical profiles and intestinal bacterial populations in early‐weaned piglets date: 2019-05-30 journal: J Anim Physiol Anim Nutr (Berl) DOI: 10.1111/jpn.13129 sha: fd259551a9bfb092f0507736753f7f72f34a9249 doc_id: 706200 cord_uid: 8kvchxu0 This study was conducted to test the hypothesis that dietary supplementation with anti‐E. coli, chicken egg yolk immunoglobulins (IgY), may affect early weaned piglet (EWP) intestinal functions and enteric micro‐organisms. One hundred and forty‐eight ([Landrace × Yorkshire] × Duroc) piglets, weaned at age day 21, were randomly assigned to receive one of three diets for 14 days. Treatment group one (control group) was fed the base diet. Treatment group two (antibiotics group) was fed the base diet which was supplemented with 100 ppm colistin sulphate and 15 ppm enramycin; treatment group three (IgY group) was fed the base diet which was supplemented with 500 mg/kg anti‐E. coli IgY. The study evaluated the effects on EWPs of IgY on growth, serum biochemical, inflammatory profiles and also digestion content intestinal bacterial populations. Results showed no significant difference in diarrhoea rates between IgY‐fed EWPs and antibiotic‐treated EWPs. Serum biochemical analysis showed that EWPs fed an IgY‐containing diet had both lower (p < 0.05) cholesterol and low‐density lipoprotein compared to antibiotic‐treated EWPs. Escherichia coli populations measured in IgY‐fed EWP ileal contents, compared to the control group, were significantly reduced (p < 0.05). Enterococcus, Lactobacillus, Clostridium and Bifidobacterium populations were unaffected by the IgY treatment. Larger (p < 0.05) Enterococcus populations and lower (p < 0.05) expression levels of heat‐stable enterotoxin b (STb) were observed in IgY‐fed EWP caecal digesta compared to the control group. Enteric Lactobacillus significantly decreased (p < 0.05) in EWPs fed antibiotics while it was unaffected by IgY treatment. Dietary supplementation with anti‐E. coli IgY has the potential to suppress enteric E. coli growth, but not Lactobacillus, Clostridium and Bifidobacterium. This promotes and maintains a healthy EWP intestinal environment. These findings suggest that IgY may be used as an alternative to antibiotics in EWP diets. Weaning triggers significant psychosocial, and physical, stress in piglets including maternal and littermate separation, and abrupt diet change (Campbell, Crenshaw, & Polo, 2013; Qiao, Li, Wang, & Wang, 2015; Xiong et al., 2015) . Abrupt weaning can contribute to intestinal and immune system dysfunctions and lead to diarrhoea (Kuang et al., 2015; Pluske, Hampson, & Williams, 1997) . In order to address problems caused by weaning, antibiotics have been widely used (Cromwell, 2002; Yin et al., 2009) . Misuse of antibiotics in feed has resulted in serious complications due to drug residues in animal products and increased bacterial resistance (Yen, Lai, Lin, & Chiang, 2015) . Dietary antibiotics change enteric microflora that are important maintaining intestinal health and function (Guarner & Malagelada, 2003) . IgY derived from egg yolks by immunizing hens. It is actively transported from hen serum into the embryo via the egg yolk and provides passive immunity to embryos and offspring (Muller, Schubert, Zajac, Dyck, & Oelkrug, 2015; Sui, Cao, & Lin, 2011) . IgY is resistant against specific pathogens based on the antigen the hens are immunized against. It has been shown to be effective against a variety of intestinal pathogens particularly diarrhoea pathogens such as bovine and human rotaviruses, bovine coronavirus, enterotoxigenic Escherichia coli (ETEC) and Salmonella (Diraviyam et al., 2014; Muller et al., 2015; Sui et al., 2011; Thu et al., 2017; Xu et al., 2011) . IgY has attracted considerable interest as an alternative to antibiotics for the control of infectious diseases in the alimentary tract (Li, Wang, Zhen, Li, & Xu, 2015) . The present study was conducted to test the hypothesis that supplementing early weaned piglet (EWP) diets with anti-E. coli IgY may affect their enteric Escherichia coli, without affecting other micro-organisms, and also beneficially intestinal function. One hundred and forty-eight (148) ([Landrace × Yorkshire] × Duro c) piglets were weaned day 21. Their initial body weight (BW) was 7.37 ± 0.26 kg. They were used in a 14-day feeding trial. EWPs were assigned to one of three possible treatments (3 replicate/treatment; 13-17 piglets/replicate). They were the control group (base diet), the antibiotics group (base diet + 100 ppm colistin sulphate + 15 ppm enramycin) and the IgY group (base diet + 500 ppm specific IgY). The base diet formulation (Table 1) Initial and final body weight and feed consumption were recorded throughout the trial. Average daily gain (ADG), average daily feed intake (ADFI) and feed/gain (F:G) ratio were calculated. Each pig was clinically monitored throughout the experiment. Diarrhoea score was recorded Blood was sampled via 10-ml vacutainer tubes that contained EDTA as an anticoagulant. They were centrifuged at 3,000 × g for 10 min at 4°C (Yin et al., 2009) Total RNA was isolated from jejunal mucosal samples using a TRIzol reagent (100 mg tissue per 1 ml TRIzol; Invitrogen Life Technologies) following manufacturer instructions. RNA integrity was checked using 1% agarose gel electrophoresis stained with 10 µg/ml ethidium bromide. The quantity and quality of RNA were determined using a NanoDrop ND-2000 spectrophotometer system (Thermo Fisher Scientific). All RNA samples were reverse transcribed into cDNA using a Superscript First-Strand Synthesis System (Invitrogen Life Technologies) with a PrimeScript RT-PCR kit (TaKaRa) using OligodT Primer. cDNA samples were then tested for IL-1β, IL-6, IFN-r, TNF-α, ZO-1, Claudin-1 and Occludin-1 expressions via real-time RT-PCR performed as described by Yang, Wang, Xiong, and Yin (2016) . Results were normalized to β-actin expression. Relative quantification was calculated using the 2 −ΔΔCT method. The sequences for the sense and antisense primers used to quantify mRNA were designed using Oligo 6.0 (Molecular Biology Insights) and appear in Table 2 . Table 3 . Results were expressed as mean ± SEM. Statistical differences were determined using one-way ANOVA with spss 22.0 software (SPSS). Duncan differences were determined to compare differences among the groups. Values were considered significantly different at p < 0.05, while 0.05 < p < 0.10 was used to indicate a tendency towards significance. Early weaned piglet growth during the 14-day experimental period appears in Table 4 . ADG, ADFI and F:G were similar for all dietary treatments. EWPS fed antibiotic-containing diets had lower (p < 0.05) diarrhoea rates than controls. There were no diarrhoea rate differences between antibiotic-treated and IgY-fed EWPs. Serum biochemical analysis showed that EWPs fed an IgY-containing diet had lower (p < 0.05) CHOL and LDL than did antibiotic-treated EWPs. There were no differences in serum TP, ALT, AST, BUN, GLU, TG, HDL, DAO, C 4 , IgM or NH 3 among the three treatment groups (Table 5 ). IL-1β gene expression decreased (p < 0.1) in the groups receiving antibiotics compared to the control group and the IgY group. There were no significant differences in the mRNA expression of pro-inflammatory cytokines (IL-6, IFN-r and TNF-α). Tight junction proteins (ZO-1, Claudin-1 and Occludin-1) were observed in all treatment groups (Table 6 ). In-feed antibiotics significantly decreased (p < 0.05) E. coli and Lactobacillus populations as well as heat-stable enterotoxin b (STb) expression in ileal contents compared to the control group ( Enterotoxigenic E. coli is a major cause of diarrhoea and death in neonatal and EWPs (Wu et al., 2012) . E. coli can adhere to the intestinal epithelial cells and elaborate enterotoxins (LT, STa or STb). This induces diarrhoea and intestinal inflammation (Heo et al., 2013; Wang et al., 2017) . In the experiments described here, dietary supplementation of antibiotics or E. coli K88-specific IgY had no effect on ADG or ADFI compared to the control group. Heo et al. (2015) reported that egg antibodies did not significantly affect growth performance in 21-day-old EWPs in the first phase (14-day period and unchallenged) of the investigation. It has been reported that pro-inflammatory cytokines, such as TNF-α, IFN-r, IL-6 and IL-1β, play a crucial role in the modulating inflammatory response (Al-Sadi, Boivin, & Ma, 2009 ) and also participate in intestinal barrier integrity regulation (Hu, Xiao, Luan, & Song, 2013; Wang et al., 2016) . The present study analysed gene expressions of pro-inflammatory cytokines and tight junction proteins in the EWP intestines. No significant differences were observed. This demonstrates that adding antibiotics or IgY to diets results in no differences in intestinal inflammatory responses or intestinal barrier integrity. Digestive system microflora play important roles in maintaining intestinal health and function (Dowarah, Verma, & Agarwal, 2017) . A previous study on intestinal microbiota of weaned piglets has shown that after weaning, E. coli concentrations increased while the number of Lactobacillus decreased (Konstantinov et al., 2006) . As we know, E. coli is one of the major sources of intestinal pathogens, and a few strains can induce serious illness, including diarrhoea (Hu et al., 2014) . pig's gut. Antibiotics seriously affect the activity and composition of the gut microflora. It is reported that most cases of antibioticassociated diarrhoea (AAD) may be due to direct toxics effects of antibiotics on the intestine, altered digestive function secondary to reduced concentrations of gut bacteria or overgrowth of pathogenic micro-organisms (Beaugerie & Petit, 2004) . Additionally, it has been reported that an increment of Lactobacillus results in competitively exclude potentially pathogenic species from colonizing the intestine (Collier et al., 2003) . In our study, Clostridium and Bifidobacterium are not affected by IgY supplementation. In healthy intestinal tracts, Lactobacillus dominates (Dowarah et al., 2017) . Lactobacillus is considered to produce lactate from sugars as the only or major end product with some minor products such as acetate, formate or ethanol (Tsukahara & Ushida, 2002) . Previous studies demonstrated Lactobacillus potential to increase beneficial bacteria and inhibit pathogenic bacteria (Hossain, Begum, & Kim, 2015; Qi et al., 2011) . Lactobacillus produced lactic acid, hydrogen peroxide and lactoferrin which may exhibit antagonistic activity against E. coli (Li, Ni, et al., 2015) . IgY supplementation significantly decreased cholesterol and low-density lipoprotein concentrations and confirms this positive effect of IgY. Jeon, Kang, Kim, Hwangbo, and Park (2016) reported findings consistent with this report that IgY significantly decreases total cholesterol compared to the control group. The decreased cholesterol concentration could be attributed to assimilation (or uptake) by Lactobacillus (Buck & Gilliland, 1994) or to coprecipitate of cholesterol with deconjugated bile salts (Jin, Ho, Abdullah, & Jalaludin, 1998) . Chen, Wang, Yan, and Huang (2013) reported probiotics reduced serum cholesterol and inhibit hydroxyl-methyl-glutaryl coenzyme-A, which is involved in cholesterol synthesis. Thus, the decreased cholesterol concentration could be attributed to the reduced synthesis of cholesterol. Low-density lipoprotein (LDL) is also referred to as "bad" cholesterol, because it constitutes a major risk factor for cardiovascular disease (Toth et al., 2013) . In order to prevent or treat enteric infections, IgY must resist degradation and reach the small intestine without activity loss (Hong et al., 2004) . Several strategies to protect IgY from hydrolysis have been developed including liposomes (Chang, Lee, Chen, & Tu, 2002) , polymeric microspheres (Torche et al., 2006) and multiple emulsifications (Cho et al., 2005) . Further investigations are indispensable to determine how robust of IgY application can be. Optimizing IgY dose effectiveness via a suitable formulation to withstand the gastric environment is warranted, and we hope explore any synergistic effects of combining IgY with other therapeutic strategies, such as probiotics or plant extracts in order to improve performance. In this work, dietary supplementation with IgY has the potential to suppress the growth of bacterial pathogens, thus promoting and maintaining a healthy EWP intestinal environments. These findings suggest that IgY may be used as an alternative to the use of antibiotics in diets for weaned EWPs. Research of the Chinese Academy of Sciences (QYZDY-SSW-SMC008), Natural Science Foundation of Hunan Province (2017JJ1020) and Young Elite Scientists Sponsorship Program by CAST (YESS20160086). 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