206 The effects of quercetin supplementation during oocyte maturation of the in vitro production of pig embryos

Oocyte viability can be reduced during in vitro maturation due to oxidative stress and a lack of antioxidant presence in the environment. Additionally, oxidative stress is a culprit in the relatively low success rate of in vitro fertilisation (IVF) in pigs. Quercetin is a natural antioxidant dietary flavonoid that has the ability to suppress free radical formation. The purpose of this study was to determine the effects of the quercetin supplementation during oocyte maturation on IVF embryonic development success rates in pigs. Oocytes were matured for 40–44 h supplemented with quercetin (0, 0.25, 0.5, 0.75 mM; Sigma-Aldrich Co.), then fertilised using frozen-thawed boar semen (1.0 × 10⁵ sperm cells/mL) and co-incubated for 6–8 h followed by embryo culture for 144 h. After maturation oocytes were evaluated for cumulus cell expansion (n = 300, r = 6) under a stereomicroscope (scale 0–4) then denuded to determine reactive oxygen species (ROS) production (n = 135) by measuring the fluorescent intensity from the oxidation of 2′,7′-dichlorodihydrofluorescein diacetate. After IVF, zygotes were fixed, permeabilised, and stained with bisBenzimide H 33342 trihydrochloride and evaluated for penetration, polyspermy, and pronucleus formation rates (n = 480), and embryos were evaluated for cleavage and blastocyst formation (n = 599) at 48 h and 144 h post-IVF, respectively. Cumulus cell expansion score percentages were determined and analysed using the chi-squared test. Fluorescent intensities of individual oocyte were analysed using a GLM and means were compared using l.s.d. after adjusting the value of the control group to 1. The IVF and embryo development data were reported as the percent observed/drop and mean percentages using a GLM and were analysed with differences being compared using Tukey’s test. The average cumulus cell expansion score at the end of maturation when no quercetin was supplemented was 2.78 ± 0.15. As quercetin supplementation increased, cumulus cell expansion decreased (1.33 ± 0.11 for 0.75 mM quercetin). Supplementation of 0.25 mM quercetin decreased (P < 0.05) ROS levels (0.52 ± 0.12) but 0.75 mM quercetin increased (P < 0.05) ROS levels (1.73 ± 0.11) compared with no quercetin supplementation. Supplementation of quercetin decreased (P < 0.05) oocyte penetration rates, increased (P < 0.05) polyspermic oocytes and pronuclear formation. Quercetin supplementation during oocyte maturation also reduced embryonic development success through a decrease (P < 0.05) of cleavage and blastocyst formation rates compared with no quercetin supplementation (78.69 ± 0.02 and 31.18 ± 0.03, respectively). Based on these results, it appears that quercetin supplementation to the oocyte maturation media in concentrations greater than 0.25 mM are detrimental to the in vitro production of pig embryos and could be toxic. Supplementation of 0.25 mM quercetin appears to not be any different from not supplementing quercetin to the maturation media, except for reduction in ROS concentrations in the oocyte.