Effect of glutathione, ascorbic acid, and their combination on the quality and fertility of cryopreserved spermatozoa of Black Bengal buck

This study investigated the effect of the addition of reduced glutathione (GSH), l-ascorbic acid (AA), and their combination (GSH+AA) to the semen extender on the semen quality and fertility of post-thaw spermatozoa of Black Bengal bucks. Ejaculates (<i>n</i> = 30) collected from five Black Bengal bucks were evaluated, pooled (37 °C), and divided into four treatment groups: a control group (no antioxidants), a GSH group (control + 1 mM GSH), an AA group (control + 8 mM AA), and a combined group (control + 1 mM GSH and 8 mM AA). The semen samples were then filled into 0.25 mL French straws and cryopreserved following a standard cryopreservation method. Supplementation of GSH (1 mM) and AA (8 mM) significantly (<i>P</i> < 0.05) improved total motility, progressive motility, normal morphology (normal fraction, distal droplet, and bent tail), and kinematic parameters (average path velocity, straight-line velocity, curvilinear velocity, and beat cross frequency) of the spermatozoa. However, the maximum improvement was observed with the combined group. In terms of malondialdehyde (MDA) levels and hydrogen peroxide (H₂O₂) status, the values of MDA and H₂O₂ were reduced significantly (<i>P</i> < 0.05) in the GSH, AA, and GSH+AA groups compared to the control group. The fertilization capacity of the cryopreserved spermatozoa was significantly higher (<i>P</i> < 0.05) in the antioxidant-treated groups compared to the control group. In conclusion, supplementation of GSH and AA in freezing medium has a synergistic and beneficial effect on the quality and fertility of cryopreserved spermatozoa of Black Bengal buck.