Effects of quercetin supplementation in cooling extender on sperm quality of stallions.

BACKGROUND: Oxidative stress from excessive reactive oxygen species (ROS) during cooling impairs equine sperm quality. Quercetin, a flavonoid with antioxidant properties, has been proposed to reduce oxidative damage in cooled semen. AIMS/OBJECTIVE: This study aimed to identify a suitable quercetin concentration among those tested for cooled equine semen and evaluate its effects on sperm kinematics, membrane integrity, and oxidative stress parameters. METHODS: Four experiments were conducted. Experiment 1 (n=8 ejaculates, 4 stallions) evaluated quercetin at 0, 20, 30, or 45 µg/mL at 5°C for 24 h. Experiment 2 (n=42 ejaculates, 21 stallions) evaluated the selected concentration (20 µg/mL) in a larger population and included flow cytometry assessments. Experiment 3 (n=20 ejaculates, 10 stallions) evaluated quercetin effects in good and poor coolers. Experiment 4 (n=10 stallions) assessed quercetin under arachidonic acid-induced oxidative stress by measuring lipid peroxidation and H₂O₂ production. RESULTS: Among the concentrations tested, 20 µg/mL was selected for subsequent experiments because it did not negatively affect sperm kinematics or membrane integrity, whereas 30 and 45 µg/mL reduced sperm motility in a dose-dependent manner. Quercetin did not improve sperm function during conventional cooling, regardless of cooling tolerance profile. However, it significantly reduced lipid peroxidation and hydrogen peroxide production under severe oxidative stress. CONCLUSIONS: Quercetin at 20 µg/mL did not improve sperm function during conventional cooling but reduced lipid peroxidation and hydrogen peroxide production under severe oxidative stress. These findings suggest that quercetin may be beneficial under elevated oxidative challenge during equine semen handling and preservation.