摘要Gut microbiota not only biosynthesizes branched-chain amino acids(BCAA)but also catabolizes and utilizes them,while the effects of dietary BCAA supplementation on intestinal microbiota and meta-bolism remain largely elusive.Therefore,the present study aimed to investigate the impacts of dietary BCAA supplementation on productive performance,egg quality,gut microbiota and metabolism in laying hens.A total of 180 Fengda No.1 laying hens aged 41 weeks were randomly assigned to five groups,with each group consisting of six replicates of six hens,and the experiment lasted for 8 weeks.The control group(Ctrl AA)was fed a basal diet,while the other four groups were supplemented with 67％leucine(High Leu),isoleucine(High Ile),both leucine and isoleucine(High Leu+Ile),or a combi-nation of the three BCAA(High BCAA),respectively,based on the Ctrl AA.The results demonstrated that compared with Ctrl AA,both High Ile and High BCAA significantly decreased egg mass and laying rate(P＜0.05);High Ile also decreased egg weight(P＜0.001)while increasing the broken egg rate(P=0.020);only High BCAA significantly increased egg yolk color(P＜0.001).Compared with Ctrl AA,serum arginine was dramatically decreased by High BCAA(P=0.005),whereas serum ammonia level was significantly elevated by High Ile,High Leu+Ile,and High BCAA(P=0.002).Hepatic transcriptome analysis found that the expression of SLC7A2,CPS1,and ARG2 were downregulated in High BCAA group(P＜0.05),which was accompanied by inhibition of the urea cycle and upregulation of SCAP expression(P=0.024)compared with Ctrl AA.Compared with Ctrl AA,High BCAA significantly increased the relative abundance of ammonia-producing microbiota,including g__norank_f__Muribaculaceae,f__Muribaculaceae,g__Bifidobacterium,f__Oxalobacteraceae,g__Oxalobacter,and g__Eubacter-ium_hallii_group(P＜0.05),which were positively associated with increased serum ammonia levels.Untargeted metabolomics revealed High BCAA enhanced intestinal urea metabolism and glutamic acid production compared with Ctrl AA,leading to ammonia release and subsequent serum ammonia accumulation.In summary,High BCAA promoted ammonia-producing microbiota proliferation,resulting in increased ammonia overproduction.Excessive ammonia cannot be entirely detoxified through the hepatic urea cycle;instead,it stimulates SCAP to dissociate ammonia contributing to hyperammonemia and resulting in decreased productive performance.