Sperm kinetics of equine semen cooled at 5°C in solid state

Abstract

Refrigeration of equine semen is widely used for transportation and storage for time periods of up to 48 hours. In addition, it is a technique that supports several reproduction biotechnologies. The semen and extender medium, in the liquid state, is exposed to a low temperature of 5 or 15°C, reducing sperm metabolism and increasing its longevity. However, viability usually decreases after 24 hours of cooling, t least in part because of precipitation of spermatozoa to the bottom of the tube containing the semen which will cause pH fluctuation. An approach to solve this problem would be the use of a more viscous or even solid extender medium, decreasing the sperm sedimentation process. The objective of the present study was to evaluate the if addition of gelatin (2%) can solidify the medium and maintain sperm in cooled-stored extended stallion semen in the solid phase. Twenty stallions were used, and the ejaculates were split into two aliquots: Control (CG): semen diluted with BotuSemenGold®; and solid group (SG): semen diluted with BotuSemen Gold® extender plus 2% gelatin. The samples were refrigerated and evaluated at 24, 48 and 72 hours of storage. Thekinetic parameters total motility (MT), progressive motility (PM) and fast spermatozoa (RAP) were evaluated by computer-assisted semen analysis (CASA) by the HTR-IVOS-10 system (Hamilton Thorne Research – Animal Version 12.0 L, USA). Analysis of parametric data was done by analysis of variance (ANOVA) with repeated measures, using the GraphPad Prism® v9.4.1 software (GraphPad Inc., San Diego, CA, USA). Differences were considered significant when p<0.05. The addition of 2% gelatin to the cooling extender was effective in altering the physical state from liquid to solid, being completely reversed after warming to 37°C for up to 10 minutes. The addition of gelatin did not increase the evaluated parameters: MT, MP and RAP at any time assessed: 24, 48 and 72 hours (P<0.05). In summary, the addition of gelatin at a concentration of 2% to the equine cooling medium did not improve sperm kinetics in cooled equine semen. However, it was not harmful to the evaluated sperm parameters. Therefore, additional studies are needed to evaluate the potential advantages of semen cooled-storage in a solid medium.