The effect of seminal plasma on the mass motility of epididymal ram spermatozoa

CONTEXT: Mass motility assessment is widely used to evaluate semen quality in the sheep breeding industry, yet the biological factors influencing motility duration remain poorly characterised. At ejaculation, spermatozoa are combined with seminal plasma (SP), which initially supports motility and function but may compromise long-term metabolic endurance. AIMS: This study aimed to investigate the effects of SP on mass motility duration and pH regulation of epididymal ram spermatozoa. METHODS: Epididymal sperm were incubated with whole SP, saline, or left undiluted to assess motility duration. Subsequently, sperm were incubated with varying SP concentrations and molecular weight-separated fractions (protein-free 3 kDa) and assessed for motility and pH over 24 h. Metabolomic and lipidomic profiling via targeted mass spectrometry characterised the biochemical composition of each SP fraction. KEY RESULTS: SP exposure significantly reduced mass motility duration (195 ± 11 min) compared with undiluted sperm (1797 ± 185 min; P = 0.0123) and saline dilution (2667 ± 134 min; P < 0.0001). All SP-treated groups exhibited a progressive pH decline, consistent with increased glycolytic flux, whereas SP-naïve sperm showed stable or increasing pH, indicating a more regulated metabolic state and greater metabolic flexibility. The protein-free SP fraction was most detrimental to motility, which was likely to be the result of the depletion of key proteins, lipids, and metabolites associated with energy production. CONCLUSIONS: Prolonged exposure to SP impairs ram sperm motility longevity by promoting a glycolysis-biased metabolic state and reducing metabolic endurance. IMPLICATIONS: Understanding how seminal plasma composition influences sperm metabolic regulation may inform strategies to better preserve sperm motility during semen handling and storage.