Abstract / Summary
This exploratory study applied untargeted LC–MS/MS metabolomic profiling to investigate metabolic differences between Holstein bulls with high-quality (HQ) and low-quality (LQ) semen in both spermatozoa and seminal plasma. Bulls were classified based on long-term routine semen quality records and ejaculate rejection rates, providing a stable and biologically meaningful grouping. A total of 249 unique metabolites were annotated across both biological matrices. Differential analysis identified 46 LC–MS features corresponding to 44 unique metabolites in spermatozoa and 11 LC–MS features corresponding to 9 unique metabolites in seminal plasma that met the predefined multi-criteria selection thresholds (FDR < 0.05, |log2FC| > 1, VIP > 1, and AUC ≥ 0.80 or ≤0.20). The HQ group was characterized by higher levels of metabolites involved in amino acid and energy metabolism, including glutamine, glutamate, aspartate, succinate, ornithine, citrulline, and arginine. Conversely, the LQ group showed increased abundance of metabolites potentially related to altered redox homeostasis and compounds potentially originating from dietary or microbial sources. As this is an exploratory study, mechanistic interpretations regarding oxidative stress, microbiome-derived metabolites, and actual fertility should be considered strictly hypothesis-generating. The annotated metabolites represent candidate indicators associated with semen quality parameters rather than validated biomarkers. Their relationship with actual field fertility and their underlying biological mechanisms require confirmation in larger, independent populations with direct functional assays and field fertility records.