Abstract

In vitro embryo production (IVP) is a highly demanded technique in the animal breeding industry. However, the conventional protocol based on in vitro fertilization (IVF) still shows low success rates in horses. For this reason, intracytoplasmic sperm injection (ICSI) is currently the most widely used method. Unlike in other species where IVF has long been established and yields higher success rates than ICSI, equine sperm capacitation remains a significant challenge. A repeatable IVF protocol was only published in 2022, which requires prolonged sperm capacitation (22 hours at 38 ºC), and not all ejaculates can withstand this process. Given the growing interest in applying IVF to equine reproduction and the known involvement of reactive oxygen species (ROS) in sperm capacitation, this study aimed to enhance sperm capacitation in fresh semen by inhibiting intracellular antioxidant systems, using three specific inhibitors. The study compared the previously described capacitation protocol (CAP) with a non-capacitating control (CTR) and with CAP supplemented with the inhibitors, through four independent experiments: - Experiment 1: Capacitation in the presence of 3-amino-1,2,4-triazole (3-AT), a catalase inhibitor. - Experiment 2: Capacitation with LCS-1, an inhibitor of superoxide dismutase 1 (SOD1). - Experiment 3: Capacitation with L-Buthionine-(S,R)-Sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase, a key enzyme in glutathione (GSH) synthesis. - Experiment 4: Assessment of capacitation progression with a combination of the three inhibitors used in the previous experiments. In experiments 1-3, sperm samples were incubated for 22 hours at 38 ºC and evaluated at 0, 3, and 22 hours. In experiment 4, samples were incubated for 24 hours and analyzed every 3 hours. The datasets include flow cytometry data on sperm viability, mitochondrial membrane potential, membrane lipid scrambling, intracellular calcium and ROS levels, and protein tyrosine phosphorylation. Additionally, they include kinetic sperm parameters analyzed by computer-assisted sperm analysis (CASA).