212 EFFECTS OF VASCULAR ENDOTHELIAL GROWTH FACTOR ON THE EARLY DEVELOPMENT AND POLYSPERMY RATE OF OVINE EMBRYOS PRODUCED IN VITRO

Abstract

To investigate the effect of vascular endothelial growth factor (VEGF) on the early development and polyspermy rate of ovine embryos in vitro, 2 experiments were conducted with human recombinant VEGF₁₆₅ supplemented to the media during maturation, fertilization, and culture in vitro, respectively. Ovaries were collected from ewes at a local slaughterhouse. All oocytes surrounded by a multilayer of cumulus cells were collected and rinsed 3 times in maturation medium (control medium and treatment medium, respectively). A total of 100 oocytes in each group were cultured in 4-well plates (Nunc) containing 800 μL of maturation medium at 38.5°C in an atmosphere of 5% CO₂ with saturated humidity. Four replicates of each experiment were conducted. Statistical analyses were conducted by ANOVA with SPSS 12.0 software (SPSS Inc., Chicago, IL, USA). Data are expressed as means, and P < 0.05 was considered significant. In Experiment 1, to investigate the effect of VEGF on the early development of ovine embryos in vitro, VEGF was used at 5 ng mL^–1 (treatment group A) and 10 ng mL^–1 (treatment group B) in maturation medium (TCM-199 + BSA), HSOF fertilization medium, and SOF culture medium. The results showed that the maturation rate was increased significantly (P < 0.01), from 75.76% in the control treatment to 83.98 and 80.23% in treatment group A and treatment group B, respectively. The cleavage rate was increased from 75.85% in the control group to 79.39% in treatment group A (P > 0.05). The development rates of morulae (45.03%) and blastocysts (23.54%) in treatment group A were significantly higher (P < 0.01) than those in the control group (38.94 and 18.09%, respectively). In addition, the development rates of blastocysts in treatment group B (21.05%) were lower than those in treatment group A (P > 0.05) and higher than those in the control group (P > 0.05). In Experiment 2, to investigate the effect of VEGF on the polyspermy rate of ovine embryos in vitro, 5 ng mL^–1 of VEGF was used in TCM-199 + BSA maturation medium in this experiment. The results showed that the fertilization rate after 18 h of IVF was increased significantly (P < 0.01), from 75.75% in the control group to 83.86% in the treatment group, and that the polyspermy rate was decreased significantly (P < 0.01), from 12.64% in the control group to 7.68% in the treatment group. These results indicate that VEGF significantly improved the maturation and fertilization rates of ovine oocytes and, consequently, the rate of embryo development in vitro, especially when the medium was supplemented with 5 ng mL^–1 of VEGF. The VEGF obviously decreased the polyspermy rate and bated the phenomenon of polyspermy in the process of ovine oocyte IVF.

The present study was supported by the National Natural Science Foundation of China (No. 30371035).