Does the equilibration time prior to freezing affect post-thaw motion characteristics of stallion spermatozoa?

Abstract

In stallions, around 20% of ejaculates suffer damage during the freezing process, leading to significant declines in sperm quality. Consequently, numerous studies are dedicated to refining techniques to enhance fertility rates using frozen semen. One factor to consider is the equilibration time, whereby sperm cells are in contact with cooling/ freezing media components at a temperature of 4°C, providing a proper osmotic balance between the intra- and extra-cellular milieu. Our research aimed to assess the impact of preserving semen at 4°C for varying periods (2, 4, or 6 hours) following dilution before freezing, and its subsequent effect on sperm motility after thawing. To achieve this, we collected 16 ejaculates from five stallions. The spermatozoa were separated from the seminal plasma via centrifugation in INRA^Ⓡ 96 medium. After removal of the supernatant, the pellet was mixed with INRA FREEZE^Ⓡ extender at a concentration of 100 million sperm per milliliter. Subsequently, the semen was frozen in 0.5 ml straws after being kept at 4°C (Equilibration) for either 2, 4, or 6 hours. Post-thaw motility and progressive motility were assessed using computer-assisted sperm analysis (CASA). Our results indicate that an equilibration period of 6 hours led to higher percentages of motile spermatozoa after thawing (average of 46.3% P ≤ 0.05), compared to equilibration periods of 4 hours (average of 40%) and 2 hours (average of 33%). Additionally, samples frozen 2 hours after cooling exhibited lower percentages of progressively motile spermatozoa (average of 15.1% P≤ 0.05) compared to samples frozen 4 hours (average 18.8%) and 6 hours (average 20.6 %) after cooling, which showed no significant difference. Based on our initial findings, we suggest that semen should be maintained at 4°C for 6 hours before being frozen to yield higher sperm motility.