Initial stage glycerolization prevents the incidence of backward sperm motility during cryopreservation and increases buffalo semen freezability

Abstract

Present study aimed to overcome the problem ofbackward motility ofbuffalo spermatozoa during cryopreservation, by examining the effect of osmolarity of extender and the stage of glycerolization. Ejaculates (40) collected during summer from 6 buffalo bulls were divided into 4 aliquots each. Two aliquots were diluted with extender EI (osmolarity 298 mOsm), either already glycerolated (El-O) or glycerolated after cooling the half extended semen to SOC (El-5). The remaining 2 aliquots were extended with E2 (osmolarity 285 mOsm), glycerolated similarly at 2 stages, viz. E2-0 and E2-5. Pre- and post-freezing motility ofspermatozoa was recorded as per cent forward and backward moving spermatozoa. Osmolarity of extenders did not influence the sperm motility, but the stage of glycerolization significantly influenced the incidence of backward sperm motility, irrespective of osmolarity of the extender, with a significant decline in the proportion of backward moving spermatozoa in EI-0 and E2-0. During winter, one extender El (298mOsm) with 2 glycerolization stages was studied in 21 ejaculates, leading to similar results i.e. no incidence of backward motility following initial stage glycerolization. Overall, pre-freezing and post-thaw forward motility ofsemen was better during winter. Accordingly, the semen freezing protocol was modified to undertake glycerolization at the initial stage ofdi lutian. An analysis of 1 year semen freezing records revealed that the original freezing protocol, glycerolization after cooling extended semen to SoC, resulted in a high rejection rate (36.3%) of ejaculates. Data for subsequent year, when freezing protocol was suitably modified with glycerolisation at initial stage, revealed greatly reduced rejection rates. Overall for the year, modified protocol resulted in successful freezing ofapproximately 20% more ejaculates. It is therefore, concluded that buffalo semen freezing protocol should include glycerolization at the initial stage of dilution for better forward motility of spermatozoa.