Temperature-processing effect on equine semen cryopreservation

Abstract

Advances in semen cryopreservation, alongside the implementation of new laboratory techniques to better protect sperm cells viability, have allowed increased quality and fertility of frozen semen in mammals. However, this process can impair the acrosomal integrity, viability, and motility of spermatozoa to some degree. Deleterious effects of cryopreservation are largely due to shock temperature during the freezing process that causes osmotic and oxidative stress. The aim of the study was to assess whether an early semen cooling could influence main parameters of equine frozen-thawed semen. Eight healthy and fertile Sicilian Oriental Purebred stallions underwent semen collection by using a Missouri artificial vagina. Semen samples were processed according to two different freezing protocols. Semen was extended 1:1 ratio (semen:extender) in INRA-96 extender prewarmed at 37°C; thereafter in protocol A diluted samples were placed into conical tubes with 2ml Maxifreeze cushion and centrifuged at room temperature for 10 min at 600g. After removal of supernatant and cushion, the sperm pellet was resuspended with INRA-freeze to a final concentration of <200 × 10 6 spermatozoa/ml. The obtained semen was then drawn at room temperature into 0.5-ml straws (Minitube, Germany), sealed thermally, and cooled at 5°C over one hour. Protocol B differed from protocol A as diluted samples were cooled at 5°C for one hour before being centrifuged at 5°C and all other steps of semen processing were performed at 5°C. Straws obtained with both protocols were placed 4 cm above liquid nitrogen in the vapor phase in a foam box for 10 min before being plunged into the liquid phase. The straws were then stored in goblets and kept immersed in liquid nitrogen until computer-assisted semen analysis (CASA). Student's T-test was performed to highlight differences in total, progressive, and immotile sperm, p ≤ 0.05 was considered statistically significant. After thawing, equine semen frozen according to protocol A showed higher total (p=0.03) and progressive (p=0.01) motility, in comparison with protocol B. In addition, immotile sperm were significantly lower in protocol A than B (p=0.03). Stallions show high individual variation in semen freezability, and equine spermatozoa are known to be very sensitive to shock temperature, hence several studies focused on exploration of temperature drop rates and freezing protocols. In this study, a higher quality of frozen-thawed semen was observed with room temperature centrifugation and packaging, rather than after one-hour semen cooling at 5°C before refrigerated centrifugation.