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OviPlus® significantly preserves ram sperm quality better than INRA96® during liquid storage at 4 °C over a 96-hour period. Novelty: ClaimNovelty.NOVEL_FINDING. Consensus alignment: ConsensusAlignment.NEUTRAL.

February 19, 2026Open Access

Evaluation of Milk-Based and Egg-Based Semen Extenders for Extended Storage of Ram Semen at 4 °C

Key Points

The study aims to compare the effectiveness of two commercial extenders, INRA96® and OviPlus®, on ram sperm quality during liquid storage.
Collected semen from mature rams and diluted it into INRA96® and OviPlus® extenders.
Measured sperm quality parameters at multiple time points over 96 hours.
Assessed membrane integrity, acrosomal remodeling, ROS levels, and motility using image-based flow cytometry and Computer Assisted Semen Analysis.
OviPlus® showed less plasma membrane destabilization and acrosomal damage compared to INRA96® at several timepoints.
Both extenders exhibited increased ROS levels over time, but OviPlus® maintained significantly lower levels at specific intervals.
Zinc signature profiling indicated that OviPlus® preserved more viable, uncapacitated sperm than INRA96® over 60 hours.

Abstract

Abstract 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-hour period. Semen samples were collected from mature rams and diluted into each extender, then assessed at 0, 12, 24, 36, 48, 60, and 96 hours 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 hours, 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.

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