Integrated Metagenomic and Metabolomic Profiling of Boar Semen During Ambient-Temperature Storage

The reproductive efficiency of breeding boars substantially influences swine industry productivity. Sperm viability during ambient-temperature storage is critically affected by environmental factors, including microbial activity. This study aimed to elucidate the dynamics and interactions between the seminal microbiome and metabolome during boar semen storage at 17 °C. Using integrated 16S rRNA sequencing and untargeted metabolomics, we analyzed semen samples from six healthy boars (31–33 months old) collected at day 0 (control), 2, 4, and 6 of storage. Our results demonstrate that storage leads to a marked decline in microbial diversity, progressive enrichment of the opportunistic genus Proteus, depletion of key antioxidant and cofactor metabolites such as vitamin B6, and extensive metabolic reprogramming—including alterations in short-chain fatty acid, purine, and lipid oxidation pathways. Multi-omics correlation analysis further revealed strong associations between microbial succession and metabolic shifts, highlighting their combined role in driving sperm functional decline. These findings provide a mechanistic basis for improving semen preservation strategies through microbiome and metabolite-targeted interventions.