Minimization of apoptosis-like changes in cryopreserved buffalo bull sperm by supplementing extender with Bcl-2 protein

Aim: This study was aimed at evaluating the anti-apoptotic effects of Bcl-2 protein in cryopreserved buffalo bull sperm. Materials and Methods: A total 10 ejaculates from two buffalo bulls (5 each) were collected using artificial vagina method, and semen was evaluated using a standard protocol. Semen was extended by Tris egg yolk extender supplemented with Bcl-2 protein at 5, 10, and 15 mu M. Semen was cryopreserved at ultra-low temperature using traditional vapor freezing method. Pre-freeze and post-thaw semen samples were evaluated for percent motility, viability, hypo-osmotic swelling test (HOST) reactive sperms; status of mitochondrial membrane activity and status of sperm phospholipase A1 and phospholipase A2 activity. Results: There were no significant effects of Bcl-2 protein supplementation on pre-freeze sperm quality. Percent motility and active mitochondria in post-thaw Bcl-2 supplemented and control groups were also similar. However, viable sperms were significantly (p<0.05) higher (74.29+/-4.23%) in Bcl-2 supplemented group (5 mu M) as compared to control (51.6+/-5.77%). The proportion of HOST reactive sperms was also higher (63.1+/-6.73%) in Bcl-2 supplemented (5 mu M) group as compared to control (50.7+/-6.98%). The sperm with low PLA activity (non-apoptotic) was significantly (p< 0.05) higher in all the supplemented doses of Bcl-2 protein, i.e., at 5 mu M (73.42+/-5.79%), 10 mu M(75.51+/-6.22%), and 15 mu M (74.78+/-5.89%) as compared to control (60.23+/-4.45%). We found that Bcl-2 protein supplementation at 5 mu M dose improved the post-thaw semen quality indicated by higher viability, HOST reactive sperms, and sperm with low PLA activity (non-apoptotic sperms). Conclusion: Bcl-2 protein supplementation exerts its protective effect on spermatozoa against apoptosis-like changes developed during cryopreservation.