The Promoting Effect Of Lactobacillus Salivarius And Lactobacillus Agile On The Repair Of Intestinal Mucosal Injury In Laying Hens

Lactobacillus,as a probiotic,has attracted wide attention in recent years due to its prominent role in promoting growth,regulating intestinal flora,improving gastrointestinal function.Many strains of Lactobacillus were widely used in poultry industry,however,the types of Lactobacillus which can effectively enhance the intestinal mucosal barrier function in laying hens were unclear and their mechanism needs to be further clarified.In this study,Hyline brown layers at the laying peak period were selected.Three strains of lactobacillus screened previously were fed in various combinations.Then the intestinal mucosal barrier function was analyzed.At the same time,further analyze the effect of the optimal combination of lactobacillus to promote the repair of intestinal mucosal damage in laying hens,to provide a theoretical basis for the development of new feed additives for laying hens.1.Regulation effect of Lactobacillus on intestinal mucosal barrier function of laying hens.Based on the preliminary screening of lactobacilli,Lactobacillus salivarius(L.sa.),Lactobacillus agile(L.ag.)and Lactobacillus avium(L.av.)were fed with various combinations.Hyline brown layers were randomly divided into 7 groups,30 in each group:control group(C group),L.sa.group,L.ag.group,L.av.group,L.sa.+L.ag.group,L.sa.+L.av.group,L.ag.+L.av,group.Each day,take 1010 CFU individual corresponding lactobacillus and mix with feed by equal increment method for the different groups.Another group is equal amount of common feed(the blank control group).They were fed continuously for 30 days.The results of the intestinal flora analysis showed that various lactobacillus treatments could increase the beneficial bacteria and reduce the harmful bacteria in the intestine.The analysis of tight junction protein expression in the intestinal mucosa showed that the combination of L.sa.+L.ag.could significantly increase the expression of tight junction protein genes Claudin-1,Claudin-2 and Occludin(P<0.05).The analysis of the expression of cell nuclear proliferation antigen(PCNA)showed that the combination of L.sa.+ L.ag.significantly increased the proliferation activity of the intestinal mucosa of laying hens(P<0.05).The results of qRT-PCR analysis showed that the combination of L.sa.+ L.ag.can up-regulate the relative expression of proliferation-related genes CCND1 and CDK2(P<0.05).The analysis of genes related to the activity and differentiation of intestinal stem cells showed that the combination of L.sa.+L.ag.could significantly up-regulate the relative expression of Lgr5,Lysozyme,MUC2 and SI(P<0.05).These results suggested that the combination of L.sa.+L.ag.could effectively regulate the structure of intestinal flora,change the activity and direction of differentiation of intestinal stem cells,and enhance the intestinal mucosal barrier function.2.The promoting effect of Lactobacillus on the repair of intestinal mucosal damage in chickens.Firstly,different doses of LPS(0,8,40,200 ng/μL)were used to treat the intestinal "organoid" of chicks in vitro.The results showed that the damage of organoids was gradually aggravated with the increase of LPS dose at 24 h.At 48 h,the damaged organoids began to recover,especially in the 8 ng/μL and 40 ng/μL groups.The above results showed that low-dose LPS could promote the intestinal stem cells to start the repair process.In order to further study the regulating effect of Lactobacillus on intestinal mucosal damage and repair of laying hens,this study selected L.sa.,L.ag.and L.av.to feed chickens in various combinations.In this study,newly hatched Hyline white laying hens were randomly divided into 8 groups,5 in each group:C group,LPS group,L.sa.+L.ag.group,L.sa.+L.ag.+LPS group,L.sa.+L.av.group,L.sa.+Lav.+LPS group,L.ag.+L.av.group,L.ag.+Lav.+LPS group.All the chickens in the Lactobacillus group and the Lactobacillus+ LPS group were fed the basal diet supplemented with Lactobacillus from 7 days old(1010 CFU Lactobacillus mixed with the feed by the equal amount increasing method every day),and the rest groups were fed the same amount of basal diet.They were fed continuously for 30 days.When the chicks were 37 days old,the LPS group and the Lactobacillus+LPS group were intraperitoneally injected with 0.1 mL LPS(10 mg/kg BW),and the C group was intraperitoneally injected with the same amount of sterile saline.The analysis of intestinal mucosal barrier function damage showed that the content of FITC-dextran in the serum could significantly increase,the relative expression of tight junction protein Ckaudin-1,Claudin-2 and Occludin in duodenal could significantly decrease after LPS injection,indicating that LPS treatment can cause intestinal mucosal barrier damage.On the contrary,compared with LPS group,L.sa.+L.ag.+LPS group could reduce the FITC-dextran content in the serum by 48.75%(P=0.0013),increase the expression of Claudin-1 by 57.91%(P=0.0001),alleviate the intestinal mucosal hemorrhage and inflammatory cell infiltration induced by LPS,increase the number of goblet cells by 111.64%(P<0.05),alleviate the up-regulation of pro-inflammatory factors TNF-α,IL-6 and the decrease of proliferation-related genes CDK1 and CDK2 induced by LPS.The above results indicate that the combination of L.sa.+Lag.can promote the repair of intestinal mucosal damage induced by LPS in chickens.In summary,this study found that the combination of L.sa.+L.ag.in layers during the peak laying period can influence the intestinal flora,change the proliferation activity and differentiation direction of intestinal stem cells,and enhance the intestinal mucosa barrier function.At the same time,the combination of L.sa.+L.ag.can promote the repair of intestinal mucosal damage induced by LPS.