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Objective:To investigate the activity of antioxidative enzymes in fresh and frozen thawed spermatozoa in relation to lipid peroxidation and semen quality in buffalo(Bubalus bubalis) bulls.Methods:Forty two semen ejaculates from seven buffalo bulls were collected by artificial vagina method and were used for the study.Sperm motility, livability, plasma membrane and acrosomal integrity, buffalo cervical mucous penetration test were assessed in fresh and frozen thawed semen.Intracellular antioxidative enzymatic activity such as super oxide dismutase (SOD), catalase(CAT), glutathione peroxidase(GSHPx) and reduced glutathione(GSH), reactive oxygen species(ROS) and lipid peroxidation(LPO) were estimated in fresh and frozen thawed semen.Results:A significant(P<0.01) reduction in activity of antioxidative enzymes(SOD by 47.7%,GSHPx by62.7% andGSH by58.6%) in frozen thawed spermatozoa as compared to fresh spermatozoa was found.Although the catalase activity was varied from0 to3.8IU/109sperm in fresh semen, but after freezing and thawing this activity was not detectable.These enzyme activities had a strong positive association with sperm motility, membrane integrity and distance traveled by vanguard spermatozoa in buffalo cervical mucus and negative correlation withLPO andROS. However, no significant correlation with acrosomal integrity was found.Conclusion:It was concluded that loss of activity of intracellular antioxidative enzymes was evident after freezing and thawing and there was a strong association between the antioxidative enzyme activities,ROS, lipid peroxidation and sperm function in buffalo semen.