Evaluation of milk-based and egg-based semen extenders for extended storage of ram semen at 4°C

Ram semen cryopreservation and liquid storage are essential tools for reproductive management in sheep, but remain limited by the sensitivity of sperm to cold shock and oxidative damage. Commercial extenders aim to preserve sperm function, yet differences in biochemical composition may influence their efficacy across key indicators of fertility. This study evaluated the effects of two commercial extenders, INRA96 and OviPlus, on ram sperm quality during liquid storage at 4°C over a 96-h period. Semen samples were collected from mature rams and diluted into each extender, then assessed at 0, 12, 24, 36, 48, 60, and 96 h post-collection for membrane integrity, acrosomal remodeling, reactive oxygen species (ROS), and zinc signatures using image-based flow cytometry (IBFC), as well as motility parameters measured with Computer Assisted Semen Analysis (CASA). While both extenders supported initial sperm viability, OviPlus had significantly less plasma membrane destabilization (timepoints 0, 12, 36, 48; P ≤ 0.03) and acrosomal damage (timepoints 12, 24, 36, 48, 60 and 96; P ≤ 0.002) across timepoints when compared to INRA96, as indicated by lower PI and PNA labeling. ROS levels increased over time in both groups but remained significantly lower in OviPlus (timepoints 0 and 36; P ≤ 0.01) preserved samples, suggesting a more protective oxidative environment. Zinc signature profiling revealed time-dependent shifts in capacitation status, with INRA96-treated sperm undergoing accelerated zinc efflux and increased transition into capacitated (Signature 3) states by 60 h, whereas OviPlus maintained a higher proportion of viable, uncapacitated sperm over the same interval. These findings demonstrate that extender formulation significantly influences ram sperm stability during liquid storage. OviPlus offered increased preservation of structural and functional sperm parameters, supporting its potential utility for extending the fertility window in ram artificial insemination protocols. Future research is needed to explore fertilization competency of stored ram sperm and extender-specific differences.