Exploratory metabolomic analysis of boar semen using GC-MS and NMR. Insights toward fertility biomarker identification.

Microbial contamination of boar semen remains a major challenge in artificial insemination systems, where high bacterial loads can compromise sperm quality, reduce fertility, and accelerate extender degradation. Although commercial extenders contain antimicrobial compounds, increasing evidence of antimicrobial resistance raises concerns about their long‑term efficacy. This study characterises the microbial composition, resilience, and antibiotic susceptibility of bacteria present in boar semen, alongside a comprehensive analysis of the seminal metabolome.

Untreated boar semen consistently exhibited a high microbial load, producing dense lawns on nutrient agar and reaching 4.17 × 108CFU/mL on nutrient agar and 1.29 × 108 CFU/mL on MacConkey agar. Growth under aerobic and anaerobic conditions demonstrated that the dominant bacteria were Gram‑negative facultative anaerobes, frequently displaying green or yellow pigmentation consistent with Pseudomonas spp.

Semen-derived bacteria repeatedly survived conditions that eliminated soil-derived microbes, including exposure to the JSR extender, which suppressed soil bacteria but allowed persistent semen-associated growth. Antibiotic susceptibility assays revealed consistent resistance to ampicillin (10ug), while susceptibility to tetracycline (25ug), colistin sulphate (25ug), sulphatriad (200ug), cotrimoxazole (25ug), and streptomycin (10ug) was retained, an important finding given that many extenders rely on ampicillin as their primary antimicrobial agent. A sealed extended semen sample later developed spontaneous contamination, confirming that resistant bacteria were intrinsic to the semen rather than introduced during handling.

Metabolomic profiling revealed a highly conserved metabolomic profile dominated by core metabolites such as glucose (489mM), citrate (720mM), and intermediates of energy, amino acid, and lipid metabolism. These metabolites underpin fundamental processes, including energy provision, osmotic balance, membrane integrity, and redox regulation. Fraction-dependent differences were minimal, though low-abundance compounds pointed to additional processes such as nitrogen metabolism, oxidative stress responses, and micro

bial or exogenous influences.

Despite these insights, the absence of poor-quality semen, spectral masking by abundant glucose, and relatively small sample sizes limited the detection of subtle metabolic shifts and associations with sperm function. Future research integrating compromised semen samples, targeted metabolite analysis, larger cohorts, and comparative cross-species studies will be essential to uncover sensitive biomarkers of fertility. Extending these investigations to humans may validate candidate biomarkers identified in livestock and reveal novel metabolites relevant to reproductive health, with applications for personalised fertility care and assisted reproduction.