The cryoprotective effects of amino acids supplementation on cooled and post-thaw Markhoz bucks semen quality

Oxidative damage to sperm resulting from reactive oxygen species generated by the cellular components of semen is one of the main causes for the decline in motility and fertility of sperm during the freeze-thawing process. This study has been designed to evaluate the effects of glutamine, proline, Basal Medium Eagle (BME) and creatine on cooled/rewarmed and frozen-thawed Markhoz bucks spermatozoa. A total number of 42 ejaculates were collected twice a week from 4 mature bucks using an artificial vagina. Only semen samples with motility of more than 70% and sperm concentrations higher than 3 × 109 sperm/ml were used for cryopreservation. After initial evaluation, the collected ejaculates were pooled together to minimize individual variation. Twelve pooled ejaculates were included in the study. The pooled semen samples were diluted 1:4 before cooling/rewarming (part 1) and freezing/thawing (part 2) with Tris-Citrate-Froctose-Yolk extender containing creatine (2.5, 5 or 7.5 mM), glutamine (5 mM), proline (5 mM) and BME (10%). Control extender was not supplemented with amino acids and creatine. To determine semen quality characteristics, the rates of motility, progressive motility, viability, total abnormality and acrosome and membrane integrity were evaluated. The obtained results indicated that the addition of creatine (2.5 mM), glutamine (5 mM), proline (5 mM) or BME (10%) significantly increase (p < 0.05) sperm parameters (motility, progressive motility, viability, acrosome and membrane integrity) and decrease total abnormalities compared to control extender. However, extenders containing creatine (2.5 mM) or creatine (2.5 mM) plus BME (10%) presented significantly higher values (p < 0.05) than other treatments. In conclusion, the results presented in this study show that using antioxidants can improve the sperm characteristics of spermatozoa before and after freezing.