Effect of Antioxidant D-Aspartic Acid and Thawing Rate on the Freeze-Thawing Process of Ram Semen

The study was conducted to determine the influence of D-aspartic acid (D-Asp) as antioxidant supplement and thawing rates on ram sperm motility, membrane integrity, abnormality, viability, mitochondria activity, malondialdehyde and antioxidant activities, and total antioxidant capacity after freezing-thawing process. Semen samples from five mature rams (3-4 years old) were diluted with extenders (1.5% soybean lecithin, 7% glycerol) containing no supplements (control) and D-Asp (5, 10, and 15 mg/L) and cryopreserved. Frozen straws were thawed at water bath temperatures at 37 ˚C for 30s and at 60 ˚C for 6s. Addition of 10 mg/L of D-Asp improved significantly progressive motility, average path velocity, and straight-line velocity percentages after freeze-thaw (P<0.05). Plasma membrane integrity, mitochondria activity, viability, total antioxidant capacity, and glutathione peroxidase were higher in group receiving 10 mg/L in comparison to other treatments (P<0.05). Meanwhile, total abnormality significantly decreased at this concentration (10 mg/L) in comparison to the level of 15 mg/L and control group (P<0.05). Our results revealed that malondialdehyde level was lower in group receiving 10 mg/L D-Asp compared to other treatments (P<0.05). There were no significant interactions between concentrations of D-Asp and thawing protocols for any semen samples. Also, significant improvement in sperm viability and mitochondria activity were observed at 37 ˚C to 30 s thawing method (P<0.05). In overall, results of the present study demonstrate that addition of D-Asp at level of 10 mg/L have beneficial effect on quality of post-thawed ram semen cryopreserved in an extender. Therefore, this antioxidant in the suitable dose can be recommended as an additional component of ram freezing extender. The 60 ˚C to 6s thawing procedure is not an appropriate replacement for 37 ˚C to 30 s.