72 COMPARISON OF CRYOPRESERVATION METHODS: SLOW COOLING VERSUS RAPID COOLING BASED ON SPERM VIABILITY OF SPERMATOZOA OBTAINED FROM THE CAUDA EPIDIDYMIS OF ALPACA (VICUGNA PACOS)

Abstract

In alpacas, the male has a low reproductive performance due to the small size of the testes, extended period of ejaculation, and low quality of semen. This work had an objective to evaluate 2 methods of cryopreservation on sperm viability of spermatozoa obtained from the cauda epididymis of male alpacas. Testes from 19 male alpacas (>3 years old) were obtained from the slaughterhouse of Huancavelica and transported to the laboratory in isothermal conditions within 3 h of slaughter. The spermatozoa were obtained by slicing the head of the epididymis, diluting in tris-yolk-glycerol at environment temperature, and then refrigerating for 2.5 h at 5°C. The freezing process was carried out by 2 methods, slow cooling and rapid cooling, the results for percentage of progressive motility, vital staining (eosin nigrosin staining), and hypoosmotic swelling test for each method were evaluated. Cryopreservation of spermatozoa by slow cooling was using 0.25-mL straws immediately after the addition of the extenders and sealed with polyvinyl alcohol. The freezing procedure consisted of placing a metallic rack in a polystyrene foam box of 25 × 20 × 15 cm (length × width × height) and pouring LN (–196°C) within the box, keeping the level of LN below the surface of the metallic rack by 6 cm. The straws were placed onto the metallic rack exposing them to the vapors of the LN and closing the box hermetically by 10 min to freeze and then store by immersion in LN. The cryopreservation of spermatozoa by rapid cooling was carried out in pellets of 0.25 mL immediately finishing the addition of the extenders a final concentration of 60 × 10⁶ sperm mL^–1. The freezing process consisted of placing the suspension of spermatozoa in holes made on the surface of a block of solid carbon dioxide (dry ice, –79°C) with a micropipette placing aliquots of 0.25 cc quickly and successively, trying to not let the time between the first pellet formed and the last exceed a minute, and then stored by immersion in LN. Semen was thawed at 37°C in hot bath for 1 to 2 min for pellets and 30 s for straws. Percentage of progressive motility, vital staining, and hypoosmotic swelling test were analysed statistically using ANOVA at a significance level of P < 0.05 using the statistical program SAS® 8.0 (Statistic Analysis System, SAS Institute Inc., Cary, NC, USA). There were statistical differences between the 2 methods slow cooling and rapid cooling for percentage of progressive motility (21.7%^a v. 36.2%^b), vital staining (30.4%^a v. 39.7%^b), and hypoosmotic swelling test (21.6^a v. 19.0^a) for the epididymis spermatozoa. We conclude according to the viability parameters for frozen-thawed spermatozoa that the method of rapid cooling (pellets) is a good alternative for cryopreserved spermatozoa from male alpaca epididymis.