Seasonal conditions shape mitochondrial genome organization and functional resilience in mature bovine sperm.

Sperm mitochondria are crucial for sperm quality and functional competence before and after ejaculation. However, the link between mitochondrial genome features and mitochondrial functional resilience in post-ejaculated mature sperm remains unclear. Seasonal variation offers a useful framework to investigate whether mitochondrial performance in ejaculated sperm reflects stable regulatory features established before ejaculation rather than de novo post-ejaculatory activity. Using a repeated-measures approach within bulls (n = 6), semen collected during spring (late March-early April) and summer (late July-early August) was cryopreserved and later incubated in vitro for 12 h at 37 °C or 41 °C. We evaluated sperm-borne mitochondrial DNA copy number and mitochondria-encoded transcript abundance, mitochondrial bioenergetic functions (membrane potential-dependent activity and ATP content), and sperm chromatin and acrosome integrity. Chromatin damage and mitochondrial membrane potential-dependent activity showed a significant overall effect of season, with condition-dependent differences. While ATP content and acrosome integrity showed significant overall effects of season and/or incubation condition, no consistent seasonal differences were detected within individual incubation conditions. Relative mtDNA copy number showed a significant overall effect of season, with differences only under 41 °C incubation. In contrast, mitochondrial transcript abundance showed gene-specific changes with incubation. No consistent increase in transcript abundance was observed at 41 °C compared with 37 °C. Taken together, these findings suggest that mitochondrial bioenergetic competence in mature sperm primarily reflects regulatory features established before ejaculation. In contrast, transcript abundance patterns did not uniformly parallel the functional readouts under the incubation conditions tested.