Effects of cooling and warming conditions on post-thawed motility and fertility of cryopreserved buffalo spermatozoa

The principal objective of this study was to derive an improved procedure for cryopreservation of swamp buffalo (Bubalus bubalis) spermatozoa. Experiments were conducted to determine effects of cooling rate, intermediate plunge temperature and warming rate on motility and acrosome integrity of spermatozoa. Spermatozoa were obtained from three bulls (three ejaculates/bull) and were subjected to nine cooling conditions before being frozen in liquid nitrogen: cooling at 10, 20, or 30°C/min each to −40, −80, or −120°C before being plunged into liquid nitrogen. The spermatozoa frozen under a given condition were then thawed either at 1000 or 200°C/min. Cooling rate, intermediate temperature and warming rate significantly affected survival of spermatozoa obtained from the three bulls. Cooling spermatozoa from 4 to −120°C either at 20 or 30°C/min yielded better progressive motility compared to other cooling conditions (50 versus 30%). Rapid warming was superior to slow warming. In an additional study, motility and fertility of spermatozoa frozen after being cooled to −120°C at 20°C and 30°C/min and those frozen by a standard protocol used routinely for semen processing were assessed. Progressive motility of cryopreserved spermatozoa cooled at 20°C and 30°C/min was 40%, while that of spermatozoa cryopreserved using a standard protocol was 25%. A total of 178 buffalo cows were inseminated with cryopreserved spermatozoa obtained from one bull, and their pregnancy status was assessed 60 days later by rectal palpation. Out of the 60, 26 (43%) and 23 of 58 (40%) cows inseminated with sperm cooled at 20 and 30°C/min, respectively, became pregnant, whereas 17 of 60 (28%) cows inseminated with sperm frozen by a standard protocol became pregnant. This study demonstrates that an effective cryopreservation procedure for buffalo spermatozoa can be derived by systematic examination of various cryobiological factors.