Assessing the efficacy of different semen extenders on post-thaw quality of Bangladeshi buffalo sperm under a manual cryopreservation protocol

Cryopreservation has been used extensively for cattle in Bangladesh, however no study was conducted on the cryopreservation of buffalo sperm in Bangladesh. Thus, the current study aimed to evaluate the suitability of different semen extenders for improving the post-thaw quality of Bangladeshi buffalo sperm under a manual cryopreservation protocol. The manual cryopreservation protocol was compared and optimized with a commercial biofreezer protocol. Then, the efficacy of different diluters was evaluated using the optimized manual cryopreservation protocol. Meanwhile, the post-thaw sperm quality in terms of motility and morphology was evaluated by computer assisted sperm analyzer during the optimization process. During manual cryopreservation, the first cooling from 37 °C to 5 °C was done in an equilibration chamber and the second cooling from 5 °C to -120 °C in a Styrofoam box using liquid nitrogen vapor from different distances (0.5, 1.5, 1.6, 2 and 3 inches). Simultaneously, another batch of sperm was cryopreserved using a programmable freezer. The highest number of motile sperms (62.67±1.12; P<0.01) and progressive motility (38.97±1.10; P< 0.001) was observed at 1.6 inches above liquid nitrogen, which were similar to the results obtained from automated biofreezer protocol (65.94±4.65 and 45.54 ± 3.64, respectively). To evaluate the semen extenders' efficacy, one locally developed Tris-fructose-egg yolk-based diluter and two commercial diluters (Andromed, and Triladyl) were used in the freezing of buffalo sperm. The highest recovery and conception rates were observed in sperm diluted with tris-fructose-egg yolk-based (TFE) (82.4% and 80%, respectively). Therefore, it is suggested that this manual cryopreservation protocol and the TFE diluter could be a suitable and inexpensive alternative for Bangladeshi buffalo sperm considering post-thaw sperm quality and fertility.