Effect of antioxidants on quality and DNA methylation of frozen Anglo-Nubian goat semen

This study investigated the effects of different concentrations of antioxidants (glutathione, cysteine, and vitamin E) on the cryopreservation of Anglo-Nubian goat sperm. The antioxidants were administered in a tris-based extender designed to achieve iso-osmolality with seminal fluid and contained combinations of soybean lecithin (3 %) and glycerol (5 %). Sperm with motility higher than 70 % was pooled and used for cryopreservation. The sperm was diluted with a tris-based extender and different concentrations of each antioxidant were supplemented at different concentrations (1, 3, and 5 mM for glutathione, 6, 9, and 12 mM for cysteine, and 1, 2, and 3 mM for vitamin E). A sperm sample without antioxidants was also examined, and Andromed was used as a control. Straws were frozen in liquid nitrogen vapor. After storage for 10 days, the frozen straws were thawed at 37 ºC in a water bath for 30 s and evaluated for sperm motility, viability, acrosome integrity, membrane integrity, mitochondrial activity, lipid peroxidation, and DNA methylation. The results showed that the supplementation of 5 mM glutathione in iso-osmolality of the tris-based semen extender improved plasma membrane damage and increased the pregnancy rate compared to the control group (P < 0.05). Furthermore, adding 5 mM glutathione or 6 mM cysteine to the semen extender decreased the lipid peroxidation of the thawed semen (2.76 and 2.80 nmol/mL, respectively). These results were significantly better than those of the other groups (P < 0.05). Nevertheless, an increased concentration of 3 mM of vitamin E in the semen extender increased the MDA concentration (3.78 nmol/mL) in comparison to those obtained with concentrations of 1 mM (2.99 nmol/mL) and 2 mM (3.31 nmol/mL) of vitamin E (P < 0.05). The highest motility rate was 56.10 ± 0.30 % with a viability rate of 58.00 ± 0.45 %, which was achieved with 5 mM glutathione. These results were significantly higher than those of the other treatments (P < 0.05) but lower than those of the control group, which had a motility rate of 68.70 ± 0.470 % and a viability rate of 67.30 ± 0.56 %. In addition, the supplementation of 5 mM glutathione and 6 mM of cysteine did not show a significant difference in sperm DNA methylation compared to fresh sperm (P > 0.05). In summary, this study demonstrated that the addition of 5 mM glutathione to the diluent with iso-osmolality of Anglo-Nubian goat semen has a good effect on frozen semen quality.