53 Effect of anthocyanin supplementation in bovine pre-implantation embryonic development during in vitro maturation of oocytes

Abstract

Oocyte protection against reactive oxygen species (ROS) during in vitro maturation (IVM) may play a decisive role in pre-implantation embryonic development. For instance, anthocyanins have shown greater antioxidant effects than vitamins C and E. The objective of this study was to determine the anthocyanin supplementation level that influences quantity and quality of bovine blastocysts development during IVM. Cumulus–oocyte complexes (COC) were recovered from 185 abattoir ovaries in 6 sessions and classified (Grade 1 and 2) for maturation. Oocytes were in IVM in commercial medium (Vitrogen®) supplemented with anthocyanin (pelargonidin chloride) at different concentrations: 0 (control), 1, 10, 20, and 40 μM, in droplets of 70 μL with 12 to 15 COC at 38.5°C, 5% CO₂ and 90% humidity for 22 to 24 h. Sperm selection was performed by Percoll gradient method (45/90%) with centrifugation at 600 × g for 6 min. The final concentration for IVF was 2 × 10⁶ sperm mL⁻¹. A total of 462 oocytes were used in the experiment (6 replicates). Presumptive zygotes were in vitro cultured (IVC) in commercial medium (Vitrogen) in droplets of 70 µL with 12–15 zygotes at 38°C, 5% CO₂, and 90% humidity until the blastocyst stage (Day 7 of culture). The cleavage (Day 2), morulae (Day 4), and blastocyst (Day 7) rates were measured during IVC. The data were processed with non-parametric tests (Kruskal–Wallis test with independent samples, P < 0.05) using IBM SPSS Statistics 2.0 for Windows. The results in the control group of cleavage, morulae, and blastocyst rates were 67.3, 27.0, and 22.1%, respectively. Although, numerically, anthocyanin at 1 μM resulted in a higher blastocyst rate (28.8%) and anthocyanin at 10 μM resulted in a greater number of blastocysts of advanced stages (65.0%), anthocyanin supplementation during IVM did not influence the quantity and quality of bovine blastocyst development (P > 0.05). In conclusion, the supplementation of anthocyanin to the maturation medium did not affect in vitro development of bovine embryos. Complementary studies at the cellular and gene expression level may be required.