Effect Of Feeding Probiotics On Fat Metabolism And Intra-Embryonic Injection Of Bile Acid On Intestinal Microflora And Growth Of Broilers

Antibiotic(ANTI)has the effects on improving growth,feed utilization and disease resistance in animals,but because of the negative effects of antibiotics,they are banned in my country.Probiotics(PB),a kind of bacteria,are beneficial to the host.Due to the "green,healthy and environment-friendly " traits,PBs become a potential substitute for poultry feed additives.Bile acids(BAs)are a class of substances synthesized in the liver by using cholesterol as a synthetic raw material.BAs are involved in the digestion and absorption of lipids in the small intestine,intestinal movement and energy metabolism and other physiological processes,and they have a regulatory effect on the structure and function of the intestinal flora.At present,probiotics and bile acids have been used as feed additives in livestock and poultry breeding,but due to their large variety and complex mechanism of action,there is still a lack of basic research materials for their application as feed additives.In addition,whether probiotics improve intestinal microbiota composition and epithelial development in poultry is related to BAs metabolism remains unknown.Therefore,this study preliminarily investigated the effects of probiotics treatment on BAs metabolism.This study includes three parts: the first part is to explore the effects of probiotics or antibiotics supplementation on lipid metabolism of broilers in the early stage of growth;the second part is to explore the effects of probiotics or antibiotics supplementation on BAs metabolism of broilers at the early stage of growth.In the third part,in ovo injection of ursodeoxycholic acid(UDCA)was used to explore the effects of BAs supplementation on the intestinal microbiota and growth performance during early growth of broilers.1、The effect of compound probiotics supplementation on fat metabolism in broilersIn order to investigate the effects of early probiotics supplementation on the growth performance of broilers,and compare with antibiotics treatment,120 healthy 1-day-old yellow-feather broilers were randomly divided into three groups,control group(control,CON),probiotics group(PB),and antibiotic group(ANTI).Each group had 8replicates(5 chickens/replicate).All groups were fed the same basal diets;the probiotic group was supplemented with compound probiotics(50mg/day per chicken)via drinking water from hatching to 28-day-old.The antibiotic group was supplemented with 3.3 mg colistin sulfate and 16.5 mg bacitracin/kg diet from hatching to day 28.Then all broilers were fed the same basal diet till 42 days old(d42).Broilers were slaughtered on day 28(d28)and 42,respectively.Samples of liver,intestinal tissue,and adipose tissue were collected for further analysis.The results showed that compared with CON group,the abdominal fat weight(P< 0.05)and the abdominal fat rate(P < 0.05)significantly reduced in PB group on d42;Antibiotics treatment significantly improved the abdominal fat weight on d28(P<0.05),but significantly reduced the abdominal fat rate on d42(P<0.05).HE staining showed that probiotic and antibiotic treatments slightly reduce the size of adipose cell(P>0.05).Compared to control group,Dgat2 m RNA expression in adipose tissue was significantly up-regulated in ANTI group(P<0.05),and the content of TG in adipose tissue slightly increased(0.05 <P<0.1).Probiotics has a tendency to increase the liver weight(0.05 <P <0.1).Both antibiotics and probiotics significantly decreased the relative weight of liver on d42 due to the increase of body weight(P <0.001).The hepatic morphology showed that compared with CON group lipid droplets significantly accumulated in ANTI group at early growth stage,and lipid droplets gradually reduced at d42;However,no obvious steatosis was observed in PB group.Probiotics supplementation significantly increased the levels of serum triglyceride(TG)and lowdensity lipoprotein cholesterol(LDL-C)on d28(P<0.05),and significantly increased the levels of serum TG on d42(P<0.05).Compared with the CON group,the levels of serum triglyceride(TG)and high-density lipoprotein cholesterol significantly increased(P<0.05)in ANTI group on d28(P<0.05).Probiotics and antibiotics supplementation did not change hepatic TG content.However,probiotics treatment significantly reduced the hepatic NEFA content on d42(P<0.05).Enzyme activity test results showed that PB treatment at 42 days of age can significantly increase the ability of liver fat hydrolysis(P<0.05).ANTI treatment up-regulated the fat hydrolysis capacity of the liver at 28 d,but significantly down-regulated HSL enzymatic activity at 42 d.Real-time PCR results showed that on d28,hepatic genes related to fatty acid synthesis(ACC and SCD)and lipolysis(LPL)significantly decreased in PB group(P<0.05).And antibiotics treatment significantly down-regulated hepatic genes expression of SCD(P<0.05),APOB(P<0.05)and SCD(P<0.05)on d42.In adipose tissue,genes related to lipolysis,such as lipoprotein lipase(LPL),peroxisome proliferator activated receptors(PPARs),adipose triglyceride lipase(ATGL)and perilipid droplet protein 3(PLIN3)significantly increased(P<0.05)compared to control.Antibiotics treatment significantly upregulated the gene expression of hormone-sensitive lipase(HSL)(P<0.05).Furthermore,PB treatment up-regulated genes expression of CD36 and APOAI in adipose tissue(P<0.05);and PPARγ,a gene related to adipocyte differentiation,was also significantly up-regulated by PB treatment(P<0.05).However,the expression of genes such as CD36,APOAI and PPARγ in adipose tissue was not altered in ANTI group(P>0.05).Western Blot results showed that on d28,PB treatment significantly increased the protein expression of CD36(P<0.05)in liver.And ANTI treatment significantly down-regulated the protein expression of HSL in adipose tissue(P<0.05).These results indicate that probiotics have a potential on inhibiting fat deposition,while probiotics administration increases hepatic fat synthesis and lipid deposition.2、Effects of probiotics supplementation on the bile acids metabolism in broilersThe experiment design is the same as the part one.The level of bile acids(BAs)and genes expression involved in BAs metabolism were analyzed among control,PB and ANTI groups.On d28 there was no obvious changes of bile acids metabolism among three groups.However,on day 42 probiotic and antibiotics treatments had significant effects on the bile acids metabolism.The level of hepatic BAs was increased significantly compared to control(P<0.05),whereas the level of bile acids in ilealepithlium tissue was decreased significantly(P<0.05).In addition,antibiotics treatment significantly reduced the bile acids concentrations in cecal contents(P<0.05).As the transcriptional level,several key genes,involved in classical and alternative pathways of bile acid synthesis,were significantly changed.Gene expression of FXR in the liver was significantly increased(P< 0.05),the expression of CYP27A1 gene was significantly up-regulated(P<0.05);while ANTI treatment significantly downregulated CYP7A1 m RNA expression(P<0.05)compared to control.In addition,two genes related to bile acid transport ABST and ABCC3 were up-related in ANTI group(P<0.05),and the bile acid nuclear receptor FXR was significantly activated(P<0.05).The above results indicate that feeding probiotics can activate the alternative synthesis pathway of bile acids at the late growth stage of broilers,thereby increasing the body’s production of bile acids;while antibiotic treatment increase intestinal reabsorption of bile acids and thus reduces the classical pathway of bile acids synthesis.3、The effects of BAs in ovo injection on growth performance and bile acids metabolism in broilersIn the experiment,150 Rose-308-eggs(mean weight,69.0 ± 5.0 g)were incubated following the standard conditions.After weighed and disinfected,eggs were randomly divided into 3 groups,control group injected with PBS(control group,CON);low-dose UDCA(LUDCA)and high-dose UDCA(HUDCA)injection groups.On the ninth day of hatching(E9),PBS and UDCA were injected into the yolk sac.After hatching 10 chickens from each group were slaughtered,blood and liver tissues were sampled.On day 23,colonic contents were collected to perform 16 s r RNA sequencing.The results showed that UDCA in ovo injection did not affect health,BAs metabolism or growth.However,the high-dose UDCA injection reduced serum glucose concentration compared to control(P<0.05).In addition,intestinal morphology showed that BAs injection improved the ileal morphological structure,low-dose UDCA treatment significantly increased the villus length(P<0.05).Compared with the control group,the growth weight and daily feed intake of broilers in low-dose UDCA group were increased(P<0.05).On day 23,UDCA injection changed bile acids metabolism.Compared with the control group,UDCA treatment decreased serum bile acid(P<0.05),whereas increased bile acids concentrations in the gallbladder(P<0.05)and liver(P<0.05).Moreover,the genes related to bile acids synthesis were significantly upregulated in the liver(P<0.05).16 s r RNA sequencing analysis showed that UDCA treatment changed the microbiota in colon.At the class level,the abundance of alphaproteobacteria(Alpha-proteobacteria)significantly reduced in the UDCA group(P<0.05);At the family and order levels,the abundance of Lachnospiraceae significantly increased in the UDCA group(P<0.05);the abundance of Ruminococcaceae and Marvinbryantia increased significantly in UDCA group(P<0.05).Results indicate that UDCA in ovo injection can promote bile acids metabolism,and show a potential benefit on growth in broilers.