Effects of different variables on the freezability, post-thaw longevity and fertility of buffalo spermatozoa in the tropics

A factorial experiment was conducted to determine the relative efficacy of 2 diluents (Tris and milk), 4 cooling rates (10/30 degrees C to 5 degrees C; 1 and 2 h each), 2 equilibration periods at 5 degrees C (0 and 2 h), 3 thawing rates (4 degrees C/5 min, 40 degrees C/1 min and 60 degrees C/15 sec), and their interactions in the cryopreservation of Murrah buffalo (n=3) semen on the basis of pre- and post-freezing motility, and post-thaw incubation (0, 30, and 60 min at 37 degrees C) and aging (6, 24 and 48 h at 5 degrees C) motility, and fertility to first AI (806). The pre-freeze sperm motility was significantly lower (P<0.05) following 1 and 2 h of direct cooling from 10 to 5 degrees C (65 and 70%) than the corresponding values of routine cooling from 30 to 5 degrees C (72 and 76%). Post-thaw forwardly motile spermatozoa at all intervals of incubation or aging including conception rates (65.4 and 68.1%; 90-d palpation confirmation) were significantly (P<0.01) higher following 2 h of prefreeze cooling both from 10 and 30 degrees C compared with 1 h of cooling from the respective temperatures (59.5 and 63.4%). Further, 2 h of equilibration at 5 degrees C compared with 0 h significantly improved the post-thaw recovery (48 vs 39%), incubation/aging survival (8 to 12%) and fertility rates (71 vs 56.5%) of frozen semen. Post-thaw recovery and incubation/aging survival of buffalo spermatozoa also increased significantly with each increment in thawing temperature from 4 to 40 to 60 degrees C. There were positive correlations between post-thaw motility and longevity of spermatozoa, and fertility. Overall, both Tris- and milk-based diluents were equally efficacious; slow cooling of straws from 30 to 5 degrees C for 2 h compared with faster cooling (1 h) or lower initial temperature (10 degrees C) and 2 h of equilibration at 5 degrees C appeared inevitable for successful cryopreservation of buffalo semen. A thaw rate of 60 degrees C/15 sec yielded better post-thaw recovery and longevity and could improve fertility of buffalo spermatozoa.