146 Quercetin supplementation during boar semen thawing and incubation improves the in vitro production of pig embryos

Abstract

Elevated levels of reactive oxygen species in the in vitro environment cause oxidative stress, which leads to membrane damage, decreased fertility, and morphological deformities of spermatozoa. Antioxidants, such as quercetin (a polyphenol flavonoid), are often supplemented to reduce the effects of oxidative stress on spermatozoa. Supplementing frozen-thawed boar semen with quercetin improves sperm forward progressive motility, viability and lipid peroxidation up to 10 h after thawing. However, the effects of fertilizing with quercetin-supplemented sperm are unknown. Therefore, the objective of this study was to determine the effects of supplementing quercetin (0.25, 0.50, 0.75 mM) during the thawing and incubation of frozen-thawed boar semen on oocyte fertilization characteristics (n = 400) and subsequent embryonic development (n = 1340) at 48 and 144 h for cleavage and blastocyst formation, respectively. Oocytes from aspired aspirated mature follicles (3-6 mm diameter) were obtained from a local abattoir and matured in medium 199 for 40 to 44 h at 38.5°C in an atmosphere of 5% CO₂. Fertilization was performed using pooled frozen-thawed semen from 3 different boars, and co-incubation of the sperm (2 × 10⁵ sperm mL⁻¹) and oocytes (30 oocytes/well) lasted for 6 to 8 h at 38.5°C in an atmosphere of 5% CO₂. Data were analysed using ANOVA with the main effects including treatment, well and replicate. Chi-squared analysis was used to determine percentages of embryos reaching the different developmental stages for each treatment. There were no differences in penetration rates and male pronuclear formation between treatment groups; however, supplementation of 0.25 (18.18 ± 10.63%), 0.50 (20.93 ± 9.89%) and 0.75 mM (18.07 ± 12.02%) quercetin significantly decreased (P < 0.05) polyspermic penetration rates compared with no supplementation (40.00 ± 11.34%). Embryos produced from frozen-thawed boar sperm supplemented with 0.25 and 0.50 mM quercetin had a significantly higher percentage (P < 0.05) of embryos reaching the 2-cell stage of development by 48 h after IVF (75.00 ± 7.89%, 68.75 ± 2.23%, respectively) compared with 0.75 mM quercetin supplementation (64.62 ± 3.88%) and no supplementation (62.97 ± 4.11%). Supplementation of 0.25 (44.12 ± 6.23%), 0.50 (43.75 ± 7.02%) and 0.75 mM (43.08 ± 2.98%) quercetin to the sperm significantly increased (P < 0.05) the percentage of embryos reaching the blastocyst stage of development by 144 h after IVF compared with no supplementation (28.27 ± 8.07%). These results indicate that supplementing frozen-thawed boar semen with quercetin decreases the incidence of polyspermic penetration and improves early embryonic development in pigs.