Effects of α-tocopherol and Ascorbic Acid on Equine Semen Quality after Cryopreservation

This study investigated the effects of ascorbic acid and α-tocopherol supplementation on semen quality parameters of equine thawed-frozen semen. Semen was divided in seven different treatments in a final concentration of 100 × 10⁶ sperm/mL by using Gent extender containing no supplements (control) and the following supplements with three different concentrations: α-tocopherol (0.5, 1, and 2 mM) and ascorbic acid (0.45, 0.9, and 1.8 g/L). After thawing, all samples were maintained at 37°C, while analyses were performed at 0, 60, and 120 minutes. Evaluation of viability and acrosome status (using Pisum sativum agglutinin conjugated to fluorescein isothiocyanate and propidium iodide), mitochondrial membrane potential (5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolyl carbocyanine iodine JC-1]), membrane lipid peroxidation (LPO; C₁₁-BODIPY^581/591), and stability of the plasmatic membrane (merocyanine 540 and Yo-Pro-1) of each sample was determined by flow cytometry. Relative to the control group, supplementation with α-tocopherol improved (P ≤ .05) postthaw membrane LPO, yet the higher concentrations of ascorbic acid (0.9 and 1.8 g/L, respectively) showed a negative effect on membrane LPO. Neither antioxidant significantly increased (P > .05) the acrosome integrity and mitochondrial membrane potential of frozen-thawed spermatozoa, although supplementation with α-tocopherol and ascorbic acid (0.9 and 1.8 g/L, respectively) had a positive effect on membrane integrity and stability (P ≤ .05). For all semen parameters, the lower concentration of ascorbic acid (0.45 g/L) did not show significant differences (P > .05) compared with the control. In conclusion, α-tocopherol seems to be an efficient antioxidant for reducing the oxidative stress provoked by cryopreservation, decreasing lipid peroxidation on equine spermatozoa.