Effects of Melatonin on Cryopreserved Semen Parameters and Apoptosis of Thai Swamp Buffalo Bull (Bubalus bubalis) in Different Thawing Conditions

Semen cryopreservation process leads to over production of reactive oxygen species (ROS) compromising the semen quality. Melatonin is a neuroendocrine hormone which acts as an antioxidant to protect the cells from oxidative stress. Hence, the present study was conducted to investigate the effects of melatonin supplemented to the semen extender on the cryopreserved semen quality parameters and apoptosis of swamp buffalo semen after thawing with different methods. Semen, collected from four bulls by artificial vagina, was divided into two equal aliquots, diluted with (0 mM and 1 mM) melatonin supplemented tris-egg yolk-citrate-glycerol extender, and finally cryopreserved in liquid nitrogen. Two different thawing methods were performed: 30°C for 60 seconds and 37°C for 30 seconds. The computer assisted semen analysis results showed that the percentage of total motility and progressive motility in the melatonin treated samples were significantly higher (p ˂ 0.01) than those in the untreated groups at both thawing methods. All the sperm kinetic parameters, excluding the wobble, were higher in the samples treated with melatonin and thawed at 37°C for 30 seconds. The flow cytometry results showed that there was lower apoptotic sperm in all thawing conditions in the samples treated with melatonin. The percentage of apoptotic spermatozoa was found significantly lower (p < 0.01) in the treated samples using 37°C for 30 seconds of thawing procedure than that in the samples when thawed at 30°C for 60 seconds. Hence, the overall results of the present study confirmed that 1 mM melatonin supplementation to the semen extender can improve the motility and viability of spermatozoa and reduce the number of apoptotic sperm in the frozen-thawed semen of swamp buffalo.