| Intestinal inflammation severely affects the growth performance of chickens.It was further found that the imbalance of immune cells in the intestine is an important factor in intestinal inflammation.Especially the increase of T helper 17(Th17)cells and the decrease of regulatory T cells(Treg cells),and the imbalance of the two cells could lead to various inflammatory bowel diseases.Studies have shown that intestinal bacteria play an important regulatory role in the Th17/Treg cell balance.Fecal microbiota transplantation(FMT)is an effective method to reconstitute the intestinal flora,but little has been reported on the mechanism by which FMT affects the growth and development of chickens by regulating the balance of Th17/Treg cells in the jejunum.Therefore,in this study,we aimed to investigate the imbalance of Th17/Treg cells in the jejunum as a key factor affecting the growth performance of chickens based on the screening of chicken populations with extreme differences in their growth performance.To confirm the mechanism that fecal transplantation could promote the balance of Th17/Treg cells in the jejunum and improve the growth performance of chickens,16 S r RNA sequencing and other molecular techniques were performed.The main findings are as follows:1.The selection of chickens with extreme differences in growth performanceIn order to obtain chickens with extreme differences in growth performance,three different breeds of chickens Turpan cockfighting × White Leghorn chickens,white feathered chickens and yellow feathered chickens,were screened respectively.Among each group of chickens in good health,20 males with the higher and lower body weight were selected as high and low body weight chickens,respectively.A comparative study of their growth performance revealed that the body weight(P < 0.0001),leg muscle weight(P < 0.01),breast muscle weight(P < 0.01),and individual cell cross-sectional area of leg and breast muscles(P < 0.0001)were significantly higher in the high body weight chickens than in the low body weight chickens among the three breeds,suggesting significant differences in growth performance between the high and low body weight chickens.2.The comparison of expression levels of Th17/Treg cell-associated transcription factors and cytokines in the jejunum of chickens with extreme differences in growth performanceIn order to reveal that Th17/Treg cell imbalance in the jejunum is a key factor affecting chicken growth performance,the expression levels of Th17/Treg cellassociated transcription factors and cytokines in the jejunum of chickens with extreme differences in growth performance were studied.At the m RNA level,the expression levels of retinoid-related orphan receptor gamma t(rorγt),signal transducer and activator of transcription 3(Stat3)forkhead Box P3(foxp3)and other cytokines associated with Th17 and Treg cells respectively in chickens with extreme differences in growth performance were compared.The relative m RNA expressions of Stat3,interleukin 6(IL-6),IL-17 A,IL-17 F and IL-21(P < 0.05)were all significantly higher in the low body weight chickens than those of high body weight chickens,while the relative m RNA expressions of Treg cell-associated transcription factor foxp3,transforming growth factor β(TGF-β),and IL-10(P < 0.05)were significantly lower in the low body weight chickens than those of high body weight chickens.At the protein level,the expressions of rorγt(P < 0.001)and foxp3(P < 0.01)associated with Treg cells were significantly lower in low-body weight chickens than in high-body weight chickens.The above results suggest that the increase of Th17 cells in the jejunum and the decrease of Treg cells decreased the growth performance of chickens.3.Influence of fecal microbiota transplantation on the growing performance of chickensIn order to confirm the influence of fecal microbiota transplantation on the growing performance of chickens,the fecal suspension from the three-month-old chickens with high body weight was transplanted to a day-old chicks for 30 days.The body weight(P < 0.0001),leg muscle weight(P < 0.001),breast muscle weight(P < 0.001),jejunum length(P < 0.05),jejunum villus length(P < 0.0001),and cross-sectional area of individual cells of leg and breast muscles(P < 0.0001)were significantly higher in the FMT group compared with the control group.The above findings suggest that fecal microbiota transplantation could significantly enhance the growth of chickens.4.Effect of fecal microbiota bacteria transplantation on Th17/Treg cells in chicken jejunumIn order to confirm that fecal microbiota transplantation could improve Th17/Treg cell balance in the jejunum to promote chicken growth performance,the effect of fecal microbiota transplantation on the expression levels of Th17/Treg cell-associated cytokines were investigated.At the m RNA level,the relative expressions of Th17 cellassociated rorγt(P < 0.01),Stat3(P < 0.05),IL-6(P < 0.05),IL-17A(P < 0.05)and IL-21(P < 0.05)were significantly lower in the FMT group compared with the control group,while the relative expressions of Treg cell-associated foxp3(P < 0.05),TGF-β(P < 0.05)and IL-10(P < 0.05)were significantly higher in the FMT group compared with the control group.At the protein level,the expression of foxp3(P <0.001)was significantly higher,while the expression of rorγt(P < 0.001)was significantly lower in the FMT group compared with the control group.The above results indicated that fecal microbiota transplantation significantly reduced Th17 cells and significantly increased Treg cells in chicken jejunum,which in turn improved the Th17/Treg cell balance.5.Effect of fecal microbiota transplantation on the jejunum bacteria of chickensIn order to investigate whether fecal microbiota transplantation improves the growth performance of chickens by remodeling the intestinal bacteria,16 S r RNA gene high-throughput sequencing was performed on the jejunal contents of chickens in the FMT and control groups.At the genus level,the results showed that the relative abundances of Lactobacillus,Gardnerella and Actinomyces in the jejunum of the FMT group were significantly higher compared with the control group,while some opportunistic pathogenic bacteria such as Enterococcus and Streptococcus were significantly less abundant in the FMT group compared with the control group.These results suggest that fecal microbiota transplantation increased the abundance of probiotic bacteria such as Lactobacillus in the jejunum,while decreased the abundance of opportunistic pathogenic bacteria.6.Effect of fecal microbiota transplantation on metabolites in chicken jejunumTo investigate whether the reconstituted jejunum bacteria affect the growth performance of chickens through the production of specific metabolites,Comparison of jejunal metabolite composition between the FMT and control groups by liquid chromatography-tandem mass spectrometry.The results showed that the differential metabolites in the jejunum of the FMT group and the control group were mainly amino acids and lipids,among which the differences in tryptophan-related metabolites were particularly obvious.KEGG functional analysis of the differential metabolites showed that the enriched metabolic pathways were mainly benzoxazine biosynthesis,zeatin biosynthesis,biotin metabolism,tryptophan metabolism,etc.Among these,tryptophan metabolic pathway was the most significantly enriched,and its three related tryptophan metabolites such as serotonin,indole and 5-methoxytryptamine were also significantly enriched in the FMT group.The above findings suggest that fecal bacteria transplantation increased the level of tryptophan metabolism.7.Correlation analysis of differential jejunal bacteria and differential metabolitesIn order to investigate whether differential metabolites are produced by increased intestinal probiotics,Spearman correlation analysis was used to investigate the relationship between Lactobacillus and tryptophan metabolites.The results showed that Lactobacillus was significantly(P < 0.05)and positively correlated with the tryptophan metabolites serotonin,indole and 5-methoxytryptamine,while Enterococcus and Streptococcus,the opportunistic pathogenic bacteria were negatively(P < 0.05)correlated with tryptophan metabolites.The above results indicated that Lactobacillus in the jejunum promoted tryptophan metabolism.8.Correlation analysis of differential bacteria and tryptophan metabolites with growth performance and Th17/Treg cell-associated transcription factors and cytokines in the jejunumTo investigate whether fecal transplantation of reconstituted intestinal flora and increased tryptophan metabolites are associated with growth performance and Th17/Treg cells,the relationship between differential bacteria,tryptophan metabolites and growth performance,Th17/Treg cell-associated cytokines and transcription factors were explored by Spearman correlation analysis.The results showed that Lactobacillus and tryptophan metabolites serotonin,indole and 5-methoxytryptamine were significantly and positively correlated with body weight(P < 0.05),leg muscle weight,chest muscle weight,and Treg cell-related transcription factors and cytokines such as foxp3,TGF-β,IL-10,while Enterococcus and Streptococcus were significantly and negatively correlated(P < 0.05)with growth performance indicators and were significantly(P < 0.05)and positively correlated with Th17 cell-associated transcription factors and cytokines such as rorγt,Stat3,IL-6 and IL-17.These results suggest that increased Lactobacillus and tryptophan metabolites in the jejunum are closely associated with growth performance and Th17/Treg cell balance in chickens.9.How tryptophan metabolites affect Th17/Treg cell homeostasisThe tryptophan metabolic pathway and aryl hydrocarbon receptor(AhR)pathway were validated using q PCR and immunohistochemistry in the jejunum of chickens.The cytochrome P450 family 1 subfamily A polypeptide 2(CYP1A2)(P < 0.01)and IL-22(P < 0.05)were significantly more expressed in FMT group compared with the control group.At the protein level,the relative expression of AhR(P < 0.01)was significantly higher in the FMT group compared with the control group.Taking all these findings together,Th17/Treg cell imbalance in the jejunum is a key factor affecting chicken growth performance.Fecal microbial transplantation could increase the relative abundance of Lactobacillus in the jejunum,promote the production of tryptophan metabolites,activate the AhR signaling pathway,promote the expression of CYP1A2 and IL-22,balance Th17/Treg cells,improve intestinal inflammation,and promote chicken growth and development. |
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