Dietary antimicrobial peptides are associated with improved colonic immunity and barrier function and altered microbiota–metabolite profiles in fattening Holstein bulls

High-concentrate diets can impair gastrointestinal mucosal integrity, induce hindgut acidosis, and promote intestinal inflammation in ruminants. Antimicrobial peptides (AMP) are considered promising alternatives to antibiotic feed additives because of their antimicrobial and immunomodulatory functions. This study aimed to characterize the protective effects of dietary AMP supplementation on colonic barrier integrity under high-concentrate feeding conditions and to explore the regulatory relationships among colonic microbiota, metabolites, and immune responses in finishing Holstein bulls. Eighteen Holstein bulls (300 ± 10 days, 346.03 ± 19.94 kg) were randomly assigned to a control group (CON, basal diet) or an AMP group (basal diet supplemented with 8 g·d − 1 ·head − 1 AMP) for 270 days. Colonic tissues and contents were collected to assess morphology, barrier and immune function, inflammatory responses, short-chain fatty acids (SCFAs), microbiota, metabolites, and transcriptomic profiles using histological and immunostaining assays, enzyme linked immunosorbent assay, gas chromatography–mass spectrometry, 16 S rRNA gene sequencing, LC–ESI–MS/MS-based widely targeted metabolomics, RNA sequencing, and qPCR. AMP supplementation reshaped the colonic microbiota, increasing Firmicutes-affiliated genera, including Negativibacillus , Christensenellaceae_R-7_group , and Lachnospiraceae_UCG-010 . It also increased microbial metabolites, particularly propionate, butyrate, 3-aminobenzamide, hispidol, and N-acetyl-5-hydroxytryptamine. These changes were accompanied by enhanced barrier function, as indicated by upregulated expression of tight junction-related genes ( ZO-1 , claudin-1 , and claudin-4 ) and mucin genes ( MUC1 and MUC2 ). AMP supplementation also reduced proinflammatory cytokine gene expression ( TNF-α , IL-1β , and IL-6 ), increased IL-10 and PIGR expression, and elevated secretory immunoglobulin (sIg)A and sIgM concentrations, indicating improved mucosal immunity. Dietary supplementation with AMP was associated with improved colonic health in finishing Holstein bulls, accompanied by reduced intestinal damage, attenuated inflammation, and changes in colonic barrier function and gut microbiota–metabolite profiles.