15 THERMORESISTANCE SPERM TEST FOR EPIDIDYMAL STALLION SPERM FROZEN WITH TWO CRYOPROTECTANTS

Abstract

The Peruvian Paso Horse has become a flagship product of Peru, for which it is very important to work on promoting and preserving their genetic material through semen. The objective of this study was to evaluate the thermoresistance of epididymal sperm of stallion frozen with 2 cryoprotectants (dimethylformamide and glycerol). The testes with the epididymides (n = 16) of 8 adult stallions in good health condition were obtained in the slaughter house and transport at isothermal conditions (30°C). Spermatozoa from the epididymis were recovered and incubated with lactated ringer's solution (Hartman solution) for 30 min, centrifuged at 2000 RPM for 15 min, and resuspended with an extender based on sucrose (7%) and egg yolk (3%) with different levels of cryoprotectants: dimethylformamide-DMF (T4%), glycerol-GLI (4%), and a combination of dimethylformamide-DMF (2%) + glycerol-GLI (2%) with an equilibrium time of 20 min at 5°C. The final dilution was 160 million motile spermatozoa placed on 0.5-mL straws. These straws were kept over liquid nitrogen fumes for 15 min over a tripod stand. After cryopreservation, the straws were stored in liquid nitrogen tanks (–196°C). The thawing process of the straws was initiated at 75°C for 7 s, immediately followed by an evaluation of sperm motility at 4 times 0, 30, 60, and 90 min in which sperm was incubated on water bath at 37°C (thermoresistance test). The data was evaluated (ANOVA) with the SAS 9.12 program (SAS Institute Inc., Cary, NC, USA) and, for the media, Tukey's test was carried out. The results showed an effect of the treatment on the motility of the frozen-thawed semen; dimethylformamide, the cryoprotectant, had the best performance.

Table 1.  Motility of frozen-thawed stallion epididymal sperm (thermoresistance sperm test)¹