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Reactive oxygen species generators affect quality parameters and apoptosis markers differently in red deer spermatozoa

¹ Biology of Reproduction Group, National Wildlife Research Institute (IREC), CSIC-UCLM-JCCM, and Institute for Regional Development (IDR), Ciencia y Tecnología Agroforestal, ETSIA, University of Castilla-La Mancha, Avenida de España s/n, 02071 Albacete, Spain² Laboratorio de Teriogenología, CONICET-Universidad Nacional del Comahue, 8303 Río Negro, Argentina³ Regional Center of Animal Selection and Reproduction (CERSYRA), JCCM, Valdepeñas, 13300 Ciudad Real, Spain⁴ Molecular Biology, University of León, 24071 León, Spain

Correspondence should be addressed to F Martínez-Pastor; Email: felipe.martinez{at}uclm.es

Fe²⁺/ascorbate, hydrogen peroxide (H₂O₂), and hypoxanthine/xanthine oxidase (XOD) are commonly used for inducing oxidative stress on spermatozoa. A comparative study of these agents was carried out on thawed spermatozoa from red deer. First, we tested a high, medium, and low concentration of each agent: 100, 10, and 1 µM Fe²⁺ (hydroxyl radical generator); 1 mM, 100, and 10 µM H₂O₂; and 100, 10, and 1 mU/ml XOD (superoxide and H₂O₂ generator), incubated at 37 °C for 180 min. Intracellular reactive oxygen species (ROS; H₂DCFDA) increased with dose and time similarly for the three systems at each concentration level. Motility and mitochondrial membrane potential (_m) were considerably decreased by H₂O₂ (1 mM and 100 µM) and XOD (100 and 10 mU/ml). Only 1 mM H₂O₂ reduced viability. The antioxidant Trolox (10 µM) reduced intracellular ROS, but could not prevent the H₂O₂ or XOD effects. In a second experiment, YO-PRO-1 and M540 were used as apoptotic and membrane stability markers respectively. Only H₂O₂ increased the proportion of apoptotic and membrane-destabilized spermatozoa. Catalase added to XOD prevented _m loss, confirming that H₂O₂ was the causative agent, not superoxide. In a third experiment, caspase activation was tested using the (FAM-VAD-FMK) probe. Viable spermatozoa with activated caspases could be detected in untreated samples, and only H₂O₂ increased their proportion after 60 min. There were important differences between ROS generators, H₂O₂ being the most cytotoxic. Although H₂O₂ and XOD caused _m dissipation, this was not reflected in increasing apoptotic markers.