Cryopreservation of the Endangered Mahseer ( Tor khudree ) Spermatozoa: Effect of Dimethyl Sulfoxide,Freezing,Activating Media,and Cryostorage on Post-Thaw Spermatozoa Motility and Fertility

Abstract

The present study was conducted with the goal of evaluating the viability of spermatozoa of the Deccan mahseer (Tor khudree, Cyprinidae) cryopreserved using different strategies. Immotile spermatozoa pooled from 2–4 males were diluted with modified fish Ringer's solution (pH: 7.48) and protected with dimethyl sulfoxide (Me₂SO) at 5%, 10%, and 15% and subjected to different equilibration periods. Diluted samples (1:10) were drawn into 500 µL plastic straws and frozen at a distance of 5 cm from the level of liquid nitrogen (LN₂) and preserved for 385 days in LN₂. Me₂SO at 10% resulted in higher post-thaw spermatozoa motility rate (46.7%) than 5% or 15% (up to 33.3%) after 385 days of cryopreservation. Of the different equilibration periods, 20–40 min generally produced higher motility (%) rates than 0, 10, 50, 60, 70, 80, or 90 min. The highest post-thaw motility of spermatozoa was obtained when they were frozen at 2 cm (−120.3°C) above the level of LN₂ and the optimum freezing rate was found to be 14.5 ± 0.1°C /min. Freezing spermatozoa at 1 cm (−160.4°C) or 3 cm (−103.0°C) or 4 cm (−77.9°C) from the level of LN₂ resulted in a significant decrease in post-thaw motility. Among the different activating media tested in the presence of different extenders and cryoprotectants, NaCl (0.3%) was found to be the best, followed by glucose (1%), and tap water. In the presence of different concentrations of Me₂SO, NaCl (0.3%), glucose (1%), and NaCl plus urea (0.3% + 0.4%) generally produced comparable post-thaw motility percentage and duration at all the concentrations of the cryoprotectant. When the spermatozoa were diluted in the Ringer's solution having pH 7.5 and 7.9, cryoprotected with Me₂SO at 10% (equilibration period: 30 min) and frozen at 2 cm from LN_2, the motility rates were 43% and 40%, respectively, after 810 days of cryostorage. High fertilization rate (98%) was obtained for spermatozoa cryopreserved up to 780 days and was close to that of fresh spermatozoa (98.5%). However, the hatching rate and fry survival were marginally lower in the treatment than that of control. After 1 year of rearing in ponds, the growth and survival of fish obtained from cryopreserved and fresh spermatozoa were comparable. The fish produced from cryopreserved spermatozoa were as normal as normally produced fish. The importance of cryopreservation of mahseer spermatozoa vis-à-vis establishment of a frozen gene bank is discussed.