Genome-resolved functional characterisation of the broiler caecal microbiome following commercial gut health interventions

Background The broiler caecal microbiome harbours a diverse repertoire of functional genes that contribute to microbial adaptation and ecosystem functioning. However, the distribution of these features among microbial community members and their response to commercial gut health interventions (GHIs) remain poorly characterised. Utilising a genome-resolved approach on 307 metagenome-assembled genomes (MAGs) from broiler caecal samples, this exploratory study investigated how GHI combinations, including coccidiosis management agents, probiotics, prebiotics, reduced crude protein diet, and essential oils, shape gut microbial functional features, encompassing virulence-associated (VAG), antimicrobial resistance (ARG), stress response (SAG), and biogeochemical function genes. Results A total of 208 putative functional genes were annotated, with VAGs (51.92%) and ARGs (43.75%) being most prevalent. Phylum Firmicutes A carried the most diverse resistance classes, and glycopeptide resistance was the most widely distributed, identified in 65.08% of ARG-carrying MAGs. These findings reflect ecological adaptation to the gut environment rather than selective antimicrobial pressure. Notably, no ionophore resistance determinants were detected. Among GHI treatments, the Bacillus -based probiotics A-essential oils-coccidiosis vaccination combination was associated with significantly reduced resistome and virulome alpha diversity ( p <0.05) and lower macrolide ( mef (En2) ) and lincosamide ( lnuA , lnu(AN2) ) resistance gene abundance. Among individual interventions, probiotics A was uniquely associated with reduced SAG and VAG abundance without significant influence on ARG abundance, demonstrating a functionally neutral-to-beneficial profile. Metabolic reconstruction revealed no predicted capacity for methane or nitrous oxide production, although methane consumption potential was identified in 19 MAGs. Pathways associated with ammonium and hydrogen sulphide production were detected through nitrogen fixation and sulphite reduction, respectively. Probiotics A, probiotics B, finisher-stage ionophore administration, and prebiotic supplementation did not alter core biogeochemical-cycling functions. Conclusions This study provides a metagenomic baseline describing how commercial GHI programmes impact the functional gene repertoire of the broiler caecal microbiome. Overall, our research demonstrates that strategic GHI selection and combination design can meaningfully modulate the functional gene landscape of the broiler gut, with probiotic-containing combinations emerging as the most consistently favourable approach. These findings contribute to the evidence base for rational GHI selection, advancing One Health initiatives and the long-term sustainability of global poultry production.