Effect of reduced glutathione supplementation on cryopreservation induced sperm cryoinjuries in Murrah bull semen

•Cryopreservation leads to cryoinjuries in the sperm due to various stresses during cooling and freezing.•Supplementation of an antioxidant, reduced glutathione in Murrah bull semen protect sperm from cryoinjuries.•Study has been undertaken in Murrah buffalo bull, which is an elite class of buffalo breed in India. The experiment was conducted to study cryopreservation induced sperm cryoinjuries and the protective effect of reduced Glutathione supplementation in Murrah bull semen. A total of 20 semen ejaculates were split into two parts after initial examination and were extended in glycerolated egg yolk TRIS diluter (Control group) and glycerolated egg yolk TRIS diluter + 0.5 mM reduced Glutathione (Treatment Group). The diluted semen samples were loaded into 0.25 ml French mini straw and sealing of straws were done. Thereafter, semen straws were kept for equilibration for 4 h at 5 °C and semen was frozen using a standard cryopreservation protocol in automatic biological freezer. Post-thaw analysis was performed after 24 h of semen storage in liquid nitrogen. Fresh and post-thaw sperm assessments included sperm motility, viability (SYBR-14/PI assay), mitochondrial function (JC-1/PI assay) and plasma membrane integrity (HOST). Cryopreservation of semen in liquid nitrogen induced a marked reduction in post-thaw sperm motility, viability, mitochondrial function and plasma membrane integrity and increased moribund type of sperm (SYBR-14/PI assay) in control group as compared to reduced glutathione treated group. There were significant (P