Epicatechin (EPI) is a natural flavonoid with antibacterial, anti-inflammatory and anti-cancer properties. Furthermore, the molecule exhibits powerful reactive oxygen species (ROS) scavenging and metal-chelating properties. In this study, we assessed the efficiency of EPI to reverse ROS-mediated alterations to the motility, viability, DNA integrity and oxidative profile of bovine spermatozoa. For the first experiment, spermatozoa were washed out of fresh semen and exposed to 12.5 μmol/L EPI, 25 μmol/L EPI, 50 μmol/L EPI and 100 μmol/L EPI in the presence of ferrous ascorbate (FeAA) during a 6 h in vitro culture. For the second experiment, the ejaculates were split into aliquots and cryopreserved with a commercial semen extender supplemented with 12.5 μmol/L EPI, 25 μmol/L EPI, 50 μmol/L EPI, 100 μmol/L EPI or containing no supplement. Sperm motility was assessed using the computer-aided sperm analysis and the cell viability was studied with the metabolic activity test. ROS production was quantified using luminometry, and DNA fragmentation was evaluated using the chromatin dispersion test. Cell lysates were prepared at the end of the culture in order to assess the concentration of protein carbonyls and malondialdehyde. Exposure to FeAA led to a significantly reduced sperm motility (p < 0.001), mitochondrial activity (p < 0.001), but increased the generation of ROS (p < 0.001), as well as oxidative damage to proteins (p < 0.001), DNA (p < 0.001) and lipids (p < 0.001). EPI supplementation, particularly at a concentration range of 50-100 μmol/L, resulted in higher preservation of the spermatozoa vitality (p < 0.001). Furthermore, 50-100 μmol/L EPI were significantly effective in the prevention of oxidative damage to sperm proteins (p < 0.001), lipids (p < 0.001) and DNA (p < 0.01 in relation to 50 μmol/L EPI; p < 0.001 with respect to 100 μmol/L EPI). In the case of the cryopreserved spermatozoa, the administration of 50-100 μmol/L EPI resulted in higher sperm motility (p < 0.001) and mitochondrial activity (p < 0.001). ROS production, the number of protein carbonyls, lipid peroxidation as well as oxidative DNA damage were found to be significantly decreased particularly in samples cryopreserved in the presence of 100 μmol/L EPI (p < 0.001). Our results suggest that EPI could behave as an effective antioxidant which may prevent oxidative insults to spermatozoa, and thus, preserve their vitality and functionality. Nevertheless, its potential to achieve higher fertilization rates in reproductive technologies needs to be validated.
Keywords: antioxidants; bulls; cryopreservation; epicatechin; ferrous ascorbate; oxidative stress; spermatozoa.