Effects Of Cordyceps Militaris On The Internal Morphology And Barrier Function Of Finishing Pigs

Cordyceps militaris(C.militaris)contains many active ingredients such as cordyceps polysaccharide,D-mannitol,and cordycepin.It not only has the functions of enhancing immunity,anti-inflammatory,and regulating the intestinal microbiota,but also has the advantages of fewer adverse effects and no residue.C.militaris has been proven to improve performance and immunity of pigs.With the development and maturity of the artificial cultivation technology of C.militaris,its production efficiency is improved and the cost is reduced.This makes it has potential application prospects in animal husbandry.Moreover,the intestinal mucosal barrier affects the body’s physiology and immune regulation,and is of great significance for regulating the body’s health.Therefore,this study estimated the effects of C.militaris on the intestinal morphology,physical barrier,chemical barrier,immunological barrier,and biological barrier function of the finishing pigs by adding C.militaris to the basal diet,as well as the mechanism of regulating the immunity.In this study,a total of 160 Duroc ×(Landrace × Yorkshire)pigs(36.82 ± 0.20 kg,half females and half castrated males)were randomly divided into two groups: control group and CM group,each group consisted of 80 pigs and they were housed in five pens,with sixteen pigs per pen.The two groups were fed basal diet and basal diet supplemented with 300 mg/kg C.militaris respectively.When the body weight reached the slaughter weight,two pigs(one male and one female)close to the average weight were selected to slaughtered.In our study,the effects of C.militaris on duodenal morphology,goblet cells,and intraepithelial lymphocytes(IELs)were investigated by staining the duodenal sections.The levels of secretory immunoglobulin A(SIg A)and cytokines in the duodenal mucosa of finishing pigs were detected by enzyme linked immunosorbent assay(ELISA).Quantitative real-time polymerase chain reaction(RT-q PCR)was used to detect the expression levels of intestinal tight junction proteins(TJP),mucins,and TLR4/NF-κB signaling pathway related genes in pigs.Western blot was used to determine the proteins expression levels of TJP and TLR4/NF-κB signaling pathway related proteins.The Hiseq high-throughput sequencing was used to detect the composition of intestinal microbiota in the duodenal and colonic digesta,and the concentration of short-chain fatty acids in the colonic digesta was determined by gas chromatograph.Get the following main results:1.CM group reduced the duodenal crypt depth and increased the ratio of villus height to crypt depth(P < 0.01),which indicated that C.militaris improved the duodenal morphology of pigs.In terms of genes,CM group increased the m RNA expression of Zona occluden-1(ZO-1),Occludin and Claudin-1(P <0.05).In terms of proteins,CM group elevated the expression of ZO-1 and Claudin-1(P <0.05).These findings indicated that C.militaris improved the intestinal physical barrier function of the duodenum in pigs.Furthermore,the number of goblet cells in CM group was more than that in control group(P <0.01),and the gene expression level of mucin-2 was also higher than that in control group(P <0.05).So,C.militaris enhanced the chemical barrier function of the duodenum in pigs.2.The number of IELs in CM group was more than that in control group(P < 0.05).ELISA results showed that the CM group also increased the levels of SIg A and pro-inflammatory cytokines of IL-10 and IL-4 in the mucosa(P < 0.05),while CM group reduced the levels of anti-inflammatory cytokines of IFN-γ(P < 0.05),TNF-α(P <0.01),and IL-12(P < 0.05).RT-q PCR results showed that the CM group downregulated m RNA expression levels of My D88(P < 0.05),TRAF6(P < 0.05),and NF-κB(P <0.01).Western blot results showed that the CM group also downregulated the protein expression levels of TLR4,My D88,TRAF6,and p I-κB-α(P < 0.05),and significantly downregulated the protein level of p-NF-κB p65(P < 0.01).These results showed that C.militaris improved the immunological barrier function of the duodenum in pigs.C.militaris may regulate the immune reaction by inhibiting the TLR4/My D88/NF-κB signaling pathway.3.In the duodenum,the abundance of Firmicutes(P < 0.01)and Clostridiaceae(P < 0.05)of CM group were lower,and the abundance of Proteobacteria was higher(P < 0.05).In the colon,the abundance of Tenericutes and Ruminococcaceae of CM group were higher(P < 0.05),and the abundance of Christensenellac(P < 0.05)and Prevotellaceae(P < 0.01)were lower.Moreover,CM group increased the concentration of acetate(P < 0.05)and butyrate(P < 0.01)in colon,and lower the p H of the colon.Collectively,these findings showed that C.militaris improved the intestinal biological barrier function in pigs.In summary,the diet supplemented with C.militaris could improve the intestinal morphology and barrier function of finishing pigs.C.militaris regulated the immune reaction by inhibiting the TLR4/My D88/NF-κB signaling pathway.This study identified a new role of C.militaris in regulating the intestinal barrier function,which may construct a new framework for nutritional interventions to improve the intestinal health in pigs.