The effect of varying equilibration times on post-thaw viability of Bactrian camel spermatozoa abstract
Semen cryopreservation efficiently extends genetic materials within a population, especially for those on the brink of extinction. Moreover, the success of breeding programs, such as artificial insemination, largely depends on the quality of fresh or cryopreserved semen, as seen in the camelid species. The equilibration time (ET) is one of the contributing factors defined as the pre-freezing period during which glycerol rapidly penetrates the spermatozoon to establish equilibrium between its intracellular and extracellular compartments. Although long exposure to glycerol appears detrimental to spermatozoa, an optimal period is required for the plasma membrane to adapt to lower temperatures and for water to exist in the cell, reducing the damage caused by ice crystals during freezing-thawing. Therefore, this study was conducted to determine an optimal ET for its semen freezing ability. Semen samples from four healthy adult males were collected, processed, and pooled. They were then subjected to a completely randomized design with two equilibration times (ET; 1 or 2 h at 5 ºC). After freezing and thawing, sperm viability was assessed. Sperm viability was evaluated by the eosin nigrosin staining method (eosin Y 0.67 g, nigrosin 10 g, NaCl 0.9 g solved in 100 mL double-distilled water). Briefly, 10 µL of diluted semen was mixed with 20 µL of stain on a prewarmed slide and smeared. Each slide was prepared in duplicate. The percentage of unstained heads of spermatozoa (live) and stained/partially stained heads of spermatozoa (dead) was determined by counting 200 spermatozoa under a phase-contrast microscope at 400× magnification. According to the results, ET showed that 1 h equilibration resulted in significantly higher sperm viability (51.37 vs 48.12%) compared to 2 h of ET (P < 0.05). In conclusion, the highest improvement in viability of post-thawed sperm was obtained with an hour equilibration time. Therefore, this procedure may become an effective approach for cryopreservation of
Bactrian camel sperm.
