Quercetin reduces the in vitro production of H2O2 during chilled storage of rabbit spermatozoa

• Production of H2O2 can lead to oxidative stress and reduce sperm fertility.
• We examine the effect of two antioxidants on rabbit sperm during chilled storage.
• The addition of 1 mM, 6 mM or 12 mM of methionine does not affect the level of H2O2.
• Quercetin protects sperm against oxidative stress by reducing H2O2 production.

Reactive oxygen species, such as hydrogen peroxide, H2O2, can reduce sperm quality during storage. This study evaluated the effect of methionine and quercetin on rabbit sperm quality during liquid storage over 96 h. Semen was collected from adult bucks (n = 4) and pooled following evaluation. In Experiment 1, pooled ejaculates were diluted with a Tris extender supplemented with methionine (1, 6 or 12 mM), quercetin (50 or 200 μM) or no antioxidant (control) and then subdivided for storage at 5 °C or 15 °C. Sperm quality was assessed by CASA (total motility [TM]) and flow cytometry (viability, acrosome integrity and H2O2 production) at 0, 48, 72 and 96 h. Experiments were replicated three times. Motility was significantly higher in control samples and lowest following dilution with 200 μM quercetin, irrespective of storage temperature. Storage at 15 °C improved viability and acrosome integrity compared with 5 °C, but produced significantly more H2O2 at 72 and 96 h in sperm diluted with methionine or no antioxidant. Quercetin-supplemented spermatozoa exhibited lower levels of H2O2 at both storage temperatures for all incubation times (P < 0.05). In Experiment 2, the concentration of quercetin (0, 25, 50, 100 and 200 μM) was investigated with additional quality parameters; lipid peroxidation and DNA integrity. All concentrations of quercetin reduced H202 and lipid peroxidation during storage at 15 °C, but were not beneficial for TM, viability, acrosome or DNA integrity. Only supplementation with 100 and 200 μM quercetin resulted in similar H202 levels at 5 °C and 15 °C (P > 0.05). Overall, quercetin-supplementation to sperm medium provided protection against oxidative stress in 15 °C-stored rabbit spermatozoa over 96 h.