ROS generators affect quality parameters and apoptosis markers differently in red deer spermatozoa

19 Fe/ascorbate, hydrogen peroxide (H2O2) and hypoxanthine/xanthine oxidase (XOD) are commonly used for 20 inducing oxidative stress on spermatozoa. A comparative study of these agents was carried out on thawed 21 spermatozoa from red deer. First, we tested a high, medium and low concentration of each agent: 100, 10 and 22 1 μM Fe (hydroxil radical generator); 1 mM, 100 and 10 μM H2O2; and 100, 10 and 1 mU/mL XOD 23 (superoxide and H2O2 generator), incubating at 37 ◦C for 180 min. Intracellular ROS (H2DCFDA) increased 24 with dose and time, and similarly for the three systems at each concentration level. Motility and mitochondrial 25 membrane potential (∆ψm) were considerably decreased by H2O2 (1 mM and 100 μM) and XOD (100 and 26 10 mU/mL). Only H2O2 1 mM reduced viability. The antioxidant Trolox (10 μM) reduced intracellular ROS, 27 but could not prevent H2O2 or XOD effects. In a second experiment, we used YO-PRO-1 and M540 as 28 apoptotic and membrane-stability markers, respectively. Only H2O2 increased the proportion of apoptotic and 29 membrane-destabilized spermatozoa. Catalase added to XOD prevented∆ψm loss, confirming that H2O2 was 30 the causative agent, not superoxide. In a third experiment, caspase activation was tested using the FLICA 31 probe. Only H2O2 increased the proportion of viable spermatozoa with activated caspases. There were 32 important differences between ROS generators, being H2O2 the most cytotoxic. Although H2O2 and XOD 33 caused ∆ψm dissipation, it did not reflect on increasing apoptotic markers. We hypothesize that a 34 subpopulation of spermatozoa may lack apoptotic markers. 35 Page 2 of 32 [email protected] Manuscript submitted for review to Reproduction