Safety And Effectiveness Evaluation Of Bile Acids On Laying Hens And Its Preventative Mechanism Of Fatty Liver

In this study,four experiments were conducted to systematically evaluate the safety and effectiveness of bile acids on laying hens,and to clarify its mechanism of preventing FLHS in laying hens,in the hope of providing a scientific theoretical basis for the application of bile acids in laying hens.Experiment 1:Three bile acids doses(0,400 and 800 mg/100 mL PBS)were used to emulsify the water-oil mixture,and the emulsifying effect was observed.An in vitro digestion model was established to simulate the intestinal digestion environment.Ten concentration gradients of bile acids(0-45 mg/25 mL PBS)and lipase were added to the water-oil mixture(soybean oil and corn oil).The acid value was used to measure the effect of fat hydrolysis.The release rate of fat-soluble vitamins A,D and E was measured at the same time.The results showed that the upper oil of 800 mg bile acids group showed milky white foam shape,indicating that the emulsification effect was obvious.With the increase of bile acids concentration,the acid value of soybean oil and corn oil digestive juice increased significantly,and tended to be stable when it reached 25-30 mg bile acids group.At the same time,the acid value of soybean oil and corn oil did not change significantly in the lipase free group.Moreover,the release rate of the three fat-soluble vitamins increased with the increase of bile acids content in soybean oil and corn oil.In the dose range of this study,the highest release rate of vitamin A in the two oils was far higher than the other two vitamins.Experiment 2:A total of 240 healthy 58-week-old HyLine Grey laying hens with similar body weight were randomly divided into 5 treatments with 6 replicates in each treatment and 8 hens in each replicate.In the experiment,the corn-soybean meal based diet was added with 0,60 mg/kg,300 mg/kg(5-fold),and 600 mg/kg(10-fold)bile acids respectively.The experimental period was 56 days.The results showed that there were no adverse changes in egg production rate,average egg weight,daily egg production,average daily feed intake and feed-to-egg ratio in each bile acid group.There were no significant adverse changes in egg quality in the 10-fold bile acid group(P>0.05).There were no significant changes in heart,liver,spleen,lung,kidney,pancreas and oviduct and ovary indices in the 10-fold bile acid group(P>0.05).There was no significant effect of each addition level of bile acids on the blood routine indicators of the hens(P>0.05).No significant changes in the serum biochemical indices occurred in the 10-fold bile acid group(P>0.05).Based on the observation of H&E stained sections of each organ,it was known that there were no significant pathological changes in the heart,liver,spleen,kidney,ovary,oviduct and small intestine segments of the laying hens in the 10-fold bile acid group.Experiment 3:A total of 450 healthy 45-week-old HyLine Grey laying hens with similar body weight were selected.Birds were randomly divided into 5 treatments with 6 replicates in each treatment.And 15 birds in each replicate.For each treatment,0,30,60,90 and 120 mg/kg bile acids were added to the corn-soybean meal basal diet.The experimental period was 168 days.The results showed that supplementing 60 mg/kg bile acids to the diet of laying hens increased egg production rate and feed conversion rate,but had no effect on daily feed intake and average egg weight.According to the quadratic regression equation,the optimal amount of bile acids in the diet of laying hens was 50-60 mg/kg.At the same time,90 and 120 mg/kg bile acids reduced egg yolk color,and other levels of supplementation had no effect on egg quality.Supplementing 60 mg/kg bile acids to the diet reduced the abdominal fat rate of laying hens and increased the total number of follicles.The supplementation of bile acids increased the utilization rate of crude fat,and the 30～90 mg/kg group also increased the utilization rate of total energy.The supplementation of 60-120 mg/kg bile acids also increased the content of vitamin A in the liver of laying hens,but did not affect the content of vitamin D and E.The dietary supplementation of 30-120 mg/kg bile acids reduced the content of serum total cholesterol(TC)and low-density lipoprotein cholesterol(LDL-C).Also,supplementing 60 mg/kg bile acids to the diet of laying hens increased the content of vitamin A in serum,but the content of vitamin D and E in serum has no obvious change.Supplementation of 90 and 120 mg/kg bile acids to the diet of laying hens increased the content of serum immunoglobulin A,but had no effect on the content of immunoglobulin G and M.In terms of serum antioxidant capacity,the serum malondialdehyde content in the 60 and 90 mg/kg bile acids groups was reduced.Glutathione peroxidase activity increased in the 60-120 mg/kg bile acids group,but total superoxide dismutase activity did not change.Supplementing 60-120 mg/kg bile acids to the diet of laying hens improved the lipase activity in duodenal mucosa.And 60-120 mg/kg bile acids groups improved the lipoprotein esterase activity of duodenum and jejunum mucosa.The content of hormone-sensitive lipase in duodenal mucosa of laying hens in 90 and 120 mg/kg bile acid groups was significantly reduced.When 90 mg/kg bile acids was supplemented,it optimized the cecal microbiota structure,increased the abundance of Turicibacter,and promote the intestinal health of laying hens.Experiment 4:A total of 60 healthy 51-week-old HyLine Grey laying hens with similar body weight were selected.Birds were randomly divided into 4 treatments with 15 replicates in each treatment,and fed in a single cage.The basal diet of the experimental diet was cornsoybean meal type.The high-fat diet replaced 8%corn starch with 8%soybean oil on the basal diet.The treatments included normal energy diet(NC),high-fat diet(HFD),high-fat diet+60 mg/kg bile acids(MBA),and high-fat diet+120 mg/kg bile acids(HBA).The experimental period was 70 days.The results showed that the average daily food intake of HFD group was lower than that of NC group.The average daily food intake of MBA and HBA groups was not different from that of HFD group.The daily gain of HFD laying hens was higher than that of NC group,and the daily gain of HBA laying hens was alleviated.The abdominal fat rate of laying hens in HFD group was higher than that in NC group.However,the abdominal fat rate of laying hens in MBA and HBA groups decreased,which reduced the abdominal fat rate of laying hens to the level of NC group.Moreover,due to the increase of dietary fat level,the activities of glutamic oxaloacetic transaminase(AST),glutamic alanine transaminase(ALT)and alkaline phosphatase(ALP)in the serum of laying hens increased.The supplementation of bile acids reduced the activities of ALT and AST in the serum,but increased the activities of ALP.It can be seen from the H&E staining sections of the liver that there were a lot of vacuoles(fat drops)in the HFD liver,and the liver cells are arranged in disorder,with obvious fatty liver phenomenon.The liver in MBA,HBA and NC groups was normal.Correspondingly,the NAFLD score of HFD group increased significantly,while that of MBA and HBA groups decreased.Compared with HFD group,the levels of TC,TG and LDL-C in serum of laying hens in MBA and HBA groups decreased,and the HDL-C/LDL-C values were higher.Also,the supplement of bile acids significantly increased the content of various bile acids in the liver,duodenum,jejunum,ileum and caecum of laying hens.The serum total antioxidant capacity of laying hens in MBA and HBA groups was higher than that in HFD group.The results of immunofluorescence staining showed that the expression of FAS-ligand in MBA and HBA groups decreased compared with HFD group.But in HBA group,FXR expression was up-regulated.For TGR5,it only shows an upward trend in HFD.The expression of PPARa increased significantly in HBA group.In addition,bile acids reduced the abundance of cecal microbiota in laying hens,increased the proportion of Firmicutes and Bacteroides.Meanwhile bile acids enriched beneficial bacteria in cecum,such as Lactobacillus and Turicibacter.The supplementation of bile acids reshaped the function of cecal microbiota,namely that it enhanced the ability of fatty acid degradation and weakened the ability of energy metabolism.Moreover,the change of cecal microbiota was closely related to the change of fatty liver severity in laying hens.Based on the results of four experiments,this study concludes that bile acids can safely and effectively improve the production performance of laying hens,and can prevent fatty liver disease in laying hens by regulating liver and serum lipid metabolism,optimizing the structure of cecal microbiota.