Improvement in cryosurvival of buffalo bull (Bubalus bubalis) sperm by altering freezing rate within critical temperature range

Objective: To optimize the cryopreservation of buffalo bull semen by altering freezing rates within critical temperature range (4 °C to -60 °C).Methods: A total of 20 ejaculates each from 5 Murrah buffalo bulls were cryopreserved using programmable biofreezer in 2 phases. In the 1st phase, 9 freezing rates were applied at -2, -5, -10, -20, -30, -40, -50, -60 or -4 °C/min (control) from 4 °C to -15 °C ; at -40 °C/min from -15 °C to -60 °C. In the 2nd phase, a fixed freezing rate was applied at -30 °C /min from 4 °C to -15 °C. Six freezing rates were applied at -10, -20, -30, -40 (control), -50 or -60 °C/min from -15 °C to -60 °C. The freezing from -60 °C to -140 °C were fixed at -50 °C/min in both the phases. Post thaw semen quality was assessed in terms of motility, viability, membrane integrity (hypo-osmotic swelling test), sperm abnormalities, and active mitochondria. Data were arc sine transformed and analyzed through one-way analysis of variance using SPSS software.Results: In the 1st phase, percent individual motility, progressive motility and viability were similar among various protocols. Percent hypo-osmotic swelling reactive sperm was higher with freezing at -30 °C/min. In the 2nd phase, percent individual motility, viability and hypo-osmotic swelling reactive sperm was higher with freezing at -50 °C /min. Sperm head abnormalities were lower at -30 °C /min in the 1st phase, but were similar among the protocols of the 2nd phase. Percent active mitochondria were higher at -30 °C /min in the 1st phase and at -50 °C/min in the 2nd phase.Conclusions: The optimum post thaw semen quality of buffalo bull could be obtained by applying freezing rate at -30 °C/min (4 °C to -15 °C) and at -50 °C/min (-15 °C to -140 °C ), followed by plunging of straws in into liquid nitrogen for storage