The Effects Of Supplement With Ruminal Fluid,Probiotics On The Gastrointestinal Microbiota And Immunity Of Lambs Aged 28 Days

Newborn lambs as model animals,were fed with healthy adult sheep ruminal fluid(experiment one)and probiotics(Lactobacillus plantarum,Bacillus subtilis and combined probiotic,expriment two)at 1-day-old,once daily over a period of 14 days,were euthanized to obtain the gastrointestinal tissue and digesta on d 28,applied of high-throughput sequencing technology,molecular techniques and conventional immunoassay,for determination of the gastrointestinal microflora and related parameters of theintestinal mucosal immunization,explored the effects on gastrointestinal microbiota and immune of the lambs by ruminal fluid or probiotics.Results of the first experiment: 1,Supplement with ruminal fluid reduced Firmicutes(P<0.05)and Actinobacteria(P<0.05)of ruminal digesta,increased Bacteroidetes(P<0.01)and Lactobacillus(P<0.05).Increased Bifidobacterium(P<0.05)of ileal digesta.Increased Lactobacillus,while reduced Bacteroidetes and Escherichia of jejunal and ileal mucosa(P>0.05).Increased Lactobacillus(P<0.01)of colonic digesta.2,In the duodenum,the mRNA expression of TLR2(P<0.05),TLR4(P<0.05)and CD14(P<0.01)increased compared with control,and the significant increase of expression of TLR2(P<0.01),TLR4(P<0.05)and NF-κB(P<0.01)in ileum tissue was related to orally administered ruminal fluid.3,IgG increased in ileum signigicantly(P<0.05),and SIgA significantly increased in the jejunum(P<0.05)and ileum(P<0.05)of the ruminal fluid group.4,The concentration of IL-6(P<0.05)in doudenal and TNF-α(P<0.05)in jejunal intestinal mucosa was increased supplemented with ruminal fluid,while IL-1b concentration in jejunal and ileal intestinal mucosa was reduced compared with control lambs(P<0.01).5,Supplement with ruminal fluid had no significant influence on immunoglobulins and cytokines in plasma(P>0.05),IgG and Fc in intestinal digesta(P>0.05),and had no significant effect on length villous,cryth depth,and the number of iIEL and MC(P>0.05).Results of the second experiment: 1,Compared with control group,the combined group reduced Firmicutes(P<0.05),Bacillus subtilis and combines reduced Actinobacteria(P<0.05)of ruminal digesta.Bacillus subtilis reduced Bacteroides(P<0.01),increased Lactobacillus(P<0.05)and reduced Escherichia(P<0.05)of duodenal digesta.In jejunal digesta,supplement with Lactobacillus plantarum increased Bifidobacterium(P<0.01),supplement with Bacillus subtilis increased Clostridium(P<0.05).In jejunal mucosa,supplement with probiotics reduced Escherichia(P<0.05)and Bacteroides(P<0.01),Bacillus subtilis and combined increased Clostridium(P<0.05).In ileal digesta,Bacillus subtilis increased Coprococcus(P<0.01),while reduced Escherichia(P<0.01),supplement with combined probiotic increased Bacteroides(P<0.01),Verrucomicrobia(P<0.01),Prevotella(P<0.01),Lactobacillus(P<0.05)and Ruminobacter(P<0.05),reduced Turicibacter(P<0.05).In ileal mucosa,supplement with Bacillus subtilis increased Actinobacteria(P<0.05)and Chloroflexi(P<0.05),and combined probiotic increased Bacteroides(P<0.05).In colonic digesta,supplement with Lactobacillus plantarum increased Clostridium(P<0.01),Bacillus subtilis increased Prevotella(P<0.01).2,In duodenum,compared with the control group,gene expression of TLR2 in the experimental groups were 1.27-(P>0.05),0.44-(P<0.05)and 1.27-flod(P>0.05),TLR4 were 3.41-(P<0.01),0.76-(P>0.05)and 1.34-flod(P>0.05),CD14 were 4.84-(P<0.01),0.76-(P>0.05)and 1.34-fold(P>0.05),NF-κB were 0.54-(P<0.05),0.38-(P<0.01)and 0.40-flod(P<0.01).In jejunum,compared with the control group,gene expression of TLR2 in the experimental groups were 1.89-(P>0.05),2.87-(P<0.05)and1.38-flod(P>0.05),TLR4 were 1.48-(P>0.05),1.56-(P>0.05)and 4.3-flod(P<0.01),CD14 were 1.35-(P>0.05),2.09-(P<0.05)and 3.95-fold(P<0.01),NF-κB were 3.88-(P<0.01),2.23-(P>0.05)and 4.49-flod(P<0.01).In ileum,compared with the control group,gene expression of TLR2 in the experimental groups were 1.04-(P>0.05),2.72-(P<0.01)and 2.34-flod(P<0.05),TLR4 were 2.20-(P>0.05),1.98(P>0.05)and 2.38-flod(P>0.05),CD14 were 0.75-(P>0.05),1.98-(P<0.05)and 2.01-fold(P<0.05),NF-κB were 1.23-(P>0.05),1.59-(P<0.05)and 1.56-flod(P<0.05).3,In duodenum,supplement with Bacillus subtilis and combined probiotic significantly reduced SIgA(P<0.05),respectively.In jejunum,supplement with Lactobacillus plantarum increased SIgA(P<0.01),combined probiotic reduced IgA(P<0.05)and increased IgG(P<0.05),there were no significant difference in the content of on IgA,SIgA and IgG(P>0.05).4,Supplement with Lactobacillus increased IL-6(P<0.01),while TNF-α was reduced(P<0.05)in duodenum,supplement with Bacillus subtilis increased IL-6(P<0.01)and TNF-α(P<0.01)in jejunum,increased TNF-α(P<0.01)in ileum.5,Supplement with probiotics didn’t play a role in the content of immunogloblulins in plasma(P>0.05),IgG and Fc in intestinal digesta(P>0.05),increased length villous and cryth depth(P>0.05),and Lactobacillus increased the number of iIEL in ileum(P<0.01),and MC in duodenum and jejunum(P<0.05).The results showed that there are rich kinds and large amount of bacteria in the gastrointestinal tract of 28-day lamb,with the dominant phylum of Firmicutes and Bacteroidetes.Supplement with ruminal fluid or probiotics was advantageous to establishment of the gastrointestinal microflora,and could enhance the innate immune response of the intestinal mucosa,while not cause excessive immune of lamb.