Metabolomic and metagenomic insights into WFBG-mediated regulation of gut microbiota and metabolism in broilers

The steady state of gut microbiota is a key factor in regulating the growth of broilers. The regulatory role of wet-fermented brewer’s grain (WFBG) in broiler gut development and microbiota is still elusive. In this study, non-targeted metabolomics and 16S rRNA sequencing analysis were used to investigate the effects of WFBG supplementation on serum metabolites and gut microbiota in 42-day-old broilers. Serum metabolomic analysis identified 546 differentially expressed metabolites (DEMs), with GO and KEGG enrichment analyses showing that specific DEMs were enriched in intestinal development-related pathways, including phenylalanine, tyrosine, tryptophan biosynthesis, and alpha-linolenic acid metabolism. 16S rRNA sequencing analysis showed significant intergroup differences in the relative abundances of Ligilactobacillus , Olsenella , Erysipelatoclostridium , and Blautia at the genus level in broiler gut microbiota between the control and WFBG groups. Integrative analysis of 16S rRNA sequencing and non-targeted metabolomics demonstrated that bacterial genera, including Streptococcus and Proteus , were positively correlated with N6,N6-dimethyllysine and quercetin but negatively associated with 18 DEMs, such as 4-methylbenzenesulfonic acid and deoxycholic acid derivatives. Furthermore, we identified potential biomarkers associated with intestinal development induced by 20% WFBG supplementation. Our findings suggest that the maximum recommended inclusion level of WFBG in broiler feed should not exceed 20%. This study provides novel insights into the molecular mechanisms underlying fiber utilization and intestinal maturation in broilers. IMPORTANCE This study investigated the regulatory mechanism of wet-fermented brewer's grain (WFBG) on gut development and microbiota in commercial broilers. Through integrated 16S rRNA sequencing and non-targeted metabolomic analysis, the study not only identified differential gut microbiota, serum metabolites, as well as their correlations, but also discovered potential biomarkers associated with intestinal development induced by 20% WFBG and clarified the maximum recommended inclusion level of WFBG (≤20%). This not only filled the gap in the molecular mechanism underlying WFBG-mediated regulation of fiber utilization and intestinal maturation in broilers but also provided a theoretical basis and practical guidance for the resource utilization of agricultural by-products, precision feeding of broilers, and intestinal health monitoring.