Development of a Sperm Cryopreservation Protocol for Critically Endangered Mohashol, Tor tor (Hamilton)

This study dealt with the development of a sperm cryopreservation protocol of Tor tor. Sperm was collected from hormone-induced males and the concentration and pH of sperm were estimated as 4.3 ± 0.1 × 10¹⁰ cells/mL and 8.6 ± 0.2, respectively. Activation of sperm motility was evaluated in different osmolalities of NaCl solution where motility of sperm decreased with increasing osmolality of extenders, and was completely inhibited at 319 mOsmol/kg. Similarly, the swimming duration of activated sperm was affected as the osmolality of the extender increased. The duration of initial motility of sperm was recorded as 8.4 ± 0.4 minutes at 48 mOsmol/kg, while the highest motility was recorded as 68.0 ± 7.2 minutes at 128 mOsmol/kg. To evaluate the toxicity of cryoprotectants, sperm was incubated with dimethyl sulfoxide (DMSO) and methanol at 5%, 10%, and 15% concentrations, respectively, for 5-45 minutes. Alsever's solution with 5% and 10% DMSO produced better motility during 5-10 minutes of incubation and 15% DMSO seemed toxic to sperm. For the cryopreservation of sperm, Alsever's solution, egg yolk citrate, and urea egg yolk were used as extenders with DMSO and methanol. Alsever's solution with 10% DMSO provided the highest equilibration (90.0% ± 3.5%) and post-thaw (80.0% ± 3.5%) motility followed by that of 87.0% ± 2.0% and 79.0% ± 1.9% with egg yolk citrate plus DMSO, and 82.0% ± 2.6% and 78.0% ± 2.0% with urea egg yolk plus DMSO, respectively. The sperm cryopreservation protocol developed through this study can be applied for long-term preservation of genetic materials of the critically endangered T. tor, and eventually, it will be an effective tool for protecting them from extinction.