Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia.
Avelane Clinic, Krčméryho 2B, 949 01 Nitra, Slovakia.
Molecules. 2019 Sep 5;24(18):3226. doi: 10.3390/molecules24183226.
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 ( < 0.001), mitochondrial activity ( < 0.001), but increased the generation of ROS ( < 0.001), as well as oxidative damage to proteins ( < 0.001), DNA ( < 0.001) and lipids ( < 0.001). EPI supplementation, particularly at a concentration range of 50-100 μmol/L, resulted in higher preservation of the spermatozoa vitality ( < 0.001). Furthermore, 50-100 μmol/L EPI were significantly effective in the prevention of oxidative damage to sperm proteins ( < 0.001), lipids ( < 0.001) and DNA ( < 0.01 in relation to 50 μmol/L EPI; < 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 ( < 0.001) and mitochondrial activity ( < 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 ( < 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.
表儿茶素（EPI）是一种天然类黄酮，具有抗菌、抗炎和抗癌特性。此外，该分子还具有强大的活性氧（ROS）清除和金属螯合特性。在这项研究中，我们评估了 EPI 逆转 ROS 介导的牛精子活力、存活、DNA 完整性和氧化谱改变的效率。在第一个实验中，精子从新鲜精液中洗出，并在 6 小时的体外培养中暴露于亚铁抗坏血酸（FeAA）存在下的 12.5 μmol/L EPI、25 μmol/L EPI、50 μmol/L EPI 和 100 μmol/L EPI 中。在第二个实验中，将精液分成等分试样，并使用含有 12.5 μmol/L EPI、25 μmol/L EPI、50 μmol/L EPI、100 μmol/L EPI 或不含补充剂的商业精液 extender 冷冻保存。使用计算机辅助精子分析评估精子运动能力，使用代谢活性测试研究细胞活力。使用发光法定量 ROS 产生，使用染色质弥散试验评估 DNA 片段化。在培养结束时制备细胞裂解物，以评估蛋白质羰基和丙二醛的浓度。暴露于 FeAA 导致精子运动能力显著降低（<0.001）、线粒体活性（<0.001），但增加 ROS 的产生（<0.001），以及蛋白质（<0.001）、DNA（<0.001）和脂质（<0.001）的氧化损伤。EPI 补充，特别是在 50-100 μmol/L 浓度范围内，导致精子活力更高（<0.001）。此外，50-100 μmol/L EPI 可显著有效预防精子蛋白质（<0.001）、脂质（<0.001）和 DNA（<0.01 与 50 μmol/L EPI 相比；<0.001 与 100 μmol/L EPI 相比）的氧化损伤。在冷冻保存的精子中，给予 50-100 μmol/L EPI 导致精子运动能力（<0.001）和线粒体活性（<0.001）更高。ROS 产生、蛋白质羰基数量、脂质过氧化以及氧化 DNA 损伤均显著降低，特别是在存在 100 μmol/L EPI 的样品中（<0.001）。我们的结果表明，EPI 可以作为一种有效的抗氧化剂，可防止精子受到氧化损伤，从而保持其活力和功能。然而，它在生殖技术中实现更高受精率的潜力需要得到验证。
