P-113: Quality Improvement of Buffalo Frozen -Thawed Spermatozoa by Supplementation of Cysteine and Glutamine in Cryopreservation Extender

Background: Sperm cryopreservation has been associated with over production of reactive oxygen species (ROS). Buffalo spermatozoa are susceptible to ROS inducing damages due to insufficient level of cytoplsmic antioxidant along with high amount of poly unsaturated fatty acids composition in membrane structure. Therefore, appropriate antioxidant in buffalo cryopreservation extender would reduce this oxidative stress, leading to achieve optimal fertility with frozen-thawed buffalo spermatozoa. Cysteine and glutamine are the main component of glutathione which act as a major antioxidant in sperm cell. Therefore this study aimed to assess functional and structural characteristics of buffalo spermatozoa in order to detect the optimum concentrations of cysteine and glutamine for buffalo semen cryopreservation Materials and Methods: Twenty ejaculates of four buffalo bulls were diluted in tris-egg yolk extender and divided in to seven equal groups consisting of cysteine (5, 7.5 and 10 mM), glutamine (10. 15 and 20 mM) and no additive. Diluted sample equilibrated, filled in 0.5 straws and frozen in liquid nitrogen. The post thawed sperm motion characteristics, membrane integrity were assisted with computer assisted sperm analyzer (SCA, Spain) and hypo-osmotic swelling test. Also flowcytometric evaluation of intracellular ROS and mitochondrial membrane potential were analyzed with Dihydroethidium and JC-1 dye respectively Results: Addition of cysteine at 5 and 7.5 mM and glutamine at 15 mM in cryopreservation extender significantly increased post thawed motility and plasma membrane integrity with significant reduction of intracellular ROS compared to control groups (p<0.05). Also 7.5 mM cysteine elevated both progressive motility and mitochondrial membrane potential as compared with the control group (p<0.05). Supplementation of both amino acids did not present any significant effect on sperm motion characteristics following cryopreservation Conclusion: It is concluded that addition of 7.5 mM cysteine and 15 mM glutamine could be suitable for cryopreservation of buffalo spermatozoa.