Distinct metabolic strategies drive variation in mass motility longevity between rams

CONTEXT: Motility longevity offers valuable insights into sperm function and metabolic resilience; however, the extent of inter-individual variation and its mechanistic underpinnings remain poorly defined. AIMS: This study aimed to characterise variation in mass motility duration in rams and determine whether differences in motility longevity are associated with distinct metabolic profiles. METHODS: Successive ejaculates (n = 314) were collected and assessed from 23 Merino rams. Seminal plasma from ejaculates exhibiting the shortest and longest motility duration was analysed immediately post-collection and at motility cessation using targeted mass spectrometry to characterise changes in metabolite abundance over time. KEY RESULTS: Mass motility duration varied markedly between rams, ranging from 11 min to over 20 h. Although mass motility duration, ejaculate volume, and sperm concentration were influenced by sequential ejaculate collection, mass motility duration was not strongly correlated with ejaculate volume or sperm concentration, indicating that variation in motility longevity is not simply explained by these traits. Rather, distinct metabolic strategies appear to drive variation among phenotypes. Short-lasting ejaculates showed rapid depletion of fructose and marked lactate accumulation, consistent with heavy reliance on glycolysis and limited mitochondrial engagement. In contrast, long-lasting ejaculates maintained stable fructose levels while exhibiting increased hexose phosphate abundance and substantial pyruvate utilisation, suggesting more controlled glycolytic flux and sustained mitochondrial oxidation. Accumulation of α-ketoglutarate and 2-hydroxyglutarate was exclusive to short-lasting ejaculates, indicating mitochondrial bottlenecks and redox imbalance. CONCLUSIONS: These findings highlight metabolic regulation, rather than substrate availability alone, as a key determinant of motility longevity in ram spermatozoa. IMPLICATIONS: Understanding how long-lasting ejaculates maintain coordinated glycolytic and mitochondrial activity provides a framework for developing metabolically informed strategies to prolong sperm survival during processing and storage.