Functional and Proteomic Changes in Ram Sperm During 48-Hour Liquid Storage at 5 °C Across the Breeding Season

This study evaluated functional and proteomic changes in ram sperm during 48 h of liquid storage at 5 °C, comparing samples collected at two breeding-season stages (July and November). Semen from six Assaf rams was diluted in INRA 96® and stored at 15 °C for 6 h and at 5 °C for 24 and 48 h. Sperm functionality (motility and kinetics, viability, apoptotic-like changes, and mitochondrial reactive oxygen species content) and proteomic profiles were assessed at each storage protocol. Sperm collected in July showed a significantly faster decline in motility and kinetics, viability, and mitochondrial reactive oxygen species levels, along with increased apoptotic-like changes during storage (p < 0.05), resulting in lower values for several functional parameters after 48 h compared with November samples. Proteomic analyses revealed more pronounced changes in protein abundance during storage in July samples, including enrichment of metabolic pathways and reduction in proteins associated with membrane stability and ion regulation. In contrast, November samples showed fewer changes in protein abundance, mainly involving mitochondrial and regulatory proteins. These results indicate that ram sperm resistance to liquid storage varies across breeding-season stages and is associated with distinct proteomic profiles. The findings provide descriptive molecular insights that may support future studies aimed at improving semen preservation strategies in ovine reproduction.