DOSE- AND TIME-DEPENDENT EFFECTS OF EPICATECHIN ON BOVINE SPERMATOZA IN VITRO

The purpose of this study was to determine the effect of epicatechin (EPI) on the motility, viability, and reactive oxygen species (ROS) production of bovine spermatozoa in the presence of various EPI doses during different time periods (Time 0 h, 2 h, 6 h, 24 h). Semen samples were cultivated in physiological saline solution containing 200, 100, 50, 10, 5 and 1 mu M/L of EPI dissolved in 0.5% DMSO. Spermatozoa motility was determined using the HTM IVOS and CASA (Computer Assisted Semen Analyzer) system. The cell viability was measured by the MTT (metabolic activity) assay and chemiluminescence was evaluated to quantify the ROS generation. Initial (0 h) viability was significantly improved (P<0.05) in experimental samples supplemented with the lowest concentration of EPI (1 mu M/L). Motility after 2 h of cultivation showed significant increase (P<0.05) values only at concentration 100 mu M/L. In the case of ROS production and even spermatozoa viability after 2 h of cultivation was not observed significant differences. Prolonged cultivation (6 h) showed significant improvements in all evaluated parameters in the case of concentrations ranging between 1 and 50 mu M/L EPI (P<0.001 in concentration 5 mu M/L for viability; P<0.01 in concentrations 10 and 50 mu M/L for motility, viability; and ROS production even in concentration 5 mu M/L; P<0.05 in concentrations 1 and 5 mu M/L for motility and ROS production). 24 h of cultivation confirmed the protective effect of EPI in experimental groups after comparison with the control group with significant differences (P<0.001) for motility, as well as for viability and ROS production in concentrations ranging between 1 and 50 mu M/L. Higher concentrations of EPI (100 and 200 mu M/L) also showed significant changes (P<0.01) in values of viability and ROS production.