Comparison of Thawing Techniques for Optimizing Post-Thaw Sperm Viability in the Somali Wild Ass (Equus africanus somaliensis)
The Somali wild ass (Equus africanus) is a critically endangered species that would benefit from biological resource banking. Development of sperm cryopreservation protocols often focus on freezing, but not thawing rates. The objective of this study was to compare post-thaw motility and viability of sperm frozen in either an egg yolk-based or milk-based extender, and thawed in a water bath at a control temp (37°C) or a higher temp (50°C). Cryopreserved semen from two adult animals was frozen at a concentration of 25–50 x 106 sperm/ml in 0.5 ml straws in equine extender EQ (20% egg yolk, 4% glycerol) or INRA96 (5% egg yolk, 6% glycerol) and was thawed at 37°C for 55 seconds or 50°C for 20 seconds. Sperm motility (% of total and progressive motile) and sperm viability (% of viable sperm) using live/dead stain were assessed. Significant post-thaw differences between INRA and EQ diluents were observed for total motility (EQ 0 h = 45.5±5.6%, 1 h = 27.1±5.6%, 2 h = 12.3±2.7%; and INRA 0 h = 28.5±3.7%, 1 h = 5.3±1.1%, 2 h = 0.9±0.3%; p<0.001) and viability (EQ 0 h = 58.2±5.64%, 1 h = 57.0±5.87%, 2 h = 49.1±5.72, 4 h = 53.9±5.24%; and INRA 0 h = 48.4±3.85%, 1 h = 51.9±3.92%, 2 h = 45.2±3.63%, 4 h = 43.1±3.37%; p<0.001), but not for thawing temperature at time 0, 1, 2 and 4 h (p>0.05). This study indicates that sperm tolerate fast thawing rates (∼700°C/min) at 50°C vs. 37°C (∼230°C/min) as long as the straw internal temperature does not exceed 37°C, potentially allowing greater flexibility for field procedures.