163 IN VITRO CULTURE OF PORCINE OOCYTE-GRANULOSA CELL COMPLEXES

Abstract

The objective of this study was to investigate the effects of porcine follicular fluid (PFF) and insulin-like growth factor-1 (IGF-1) on the growth of porcine oocyte-granulosa cell complexes (OGC) in vitro, in an effort to improve meiotic and developmental competence in vitro. Porcine OGC were manually dissected from early antral follicles of diameter 400 to 700 μm, and intact oocytes with undamaged granulosa cells were selected for culture. Between 3 and 5 OGC were combined in 50-uL droplets and cultured for 12 days in M199 medium supplemented with PVP, oestradiol, FSH, transferrin, L-ascorbic acid, and insulin. The OGC were cultured at 38.5°C in a humidified atmosphere of 5% CO₂ in air for 12 days, and the oocytes were matured for 44 h. Oocyte diameter, exclusive of the zona pellucida, was measured on day 0 and day 12 of culture. Control group was cultured in the absence of PFF or IGF-1. The experiment was divided into 2 parts. In part 1, treatment groups were cultured with 2.5, 5.0, or 7.5% (all v/v) PFF. Control OGC grew as spheres that were formed by granulosa cells. In treatment groups, the granulosa cells spread and grew on the bottom of dishes. When oocyte diameter was measured after 12 days of culture, no significant difference among groups was observed (104.07, 103.96, and 104.27 μm at the 3 PFF concentrations used; control: 104.03 μm). Similarly, the survival rate of oocytes did not differ significantly among groups. However, survival rate fell somewhat in the group treated with PFF (control: 65%; tests: 58.87, 58.33, and 50.83% at the 3 PFF concentrations used). The maturation rates of oocytes in the control was significantly higher than those of the treatment groups [25.83% (control) v. 14, 11.67, and 3.67% (the 3 treatment groups); P < 0.05]. Thus, the first conclusion is that supplementation of culture medium with PFF did not enhance the development of porcine OGC in vitro. In part 2 of the experiment, treatment groups were cultured with 10, 50, or 100 ng mL^–1 IGF-1. The percentages of OGC showing antrum formation were 80, 80, 100, and 100% in groups treated with 0, 10, 50, or 100 ng mL^–1 IGF-1, respectively. Average oocyte diameter was 94.16 to 94.58 μm just after OGC collection. However, the average diameter of oocytes cultured for 12 days with 50 or 100 ng mL^–1 IGF-1 was significantly higher than that of the control or 10 ng mL^–1 groups [108.88 and 108.31 μm (50 and 100 ng mL^–1 IGF-1) v. 105.98 μm and 106.67 μm (0 and 10 ng mL^–1 IGF-1); P < 0.05]. The maturation rate of oocytes grown with 10 and 50 ng mL^–1 IGF-1 was higher than those of the other 2 groups [30 and 40.6% (10 and 50 ng mL^–1 IGF-1, respectively) v. 25 and 26% (0 and 100 ng mL^–1 IGF-1, respectively); P < 0.05]. Thus, the second conclusion is that 50 ng mL^–1 IGF-1 improves the growth and maturation of porcine oocyte-granulosa cell complexes in vitro.