322 MEIOTIC COMPETENCE OF CANINE OOCYTES EMBEDDED IN COLLAGEN GELS

Abstract

The low meiotic competence of canine oocytes cultured in vitro is a major obstacle to the in vitro production of canine embryos. The objectives of the present study were to examine meiotic competence of oocytes embedded in collagen gels and to investigate the effects of timed exposure of the oocytes embedded in collagen gels to hormone supplements on the nuclear maturation. Ovaries were collected from 17 bitches at various stages of the estrous cycles by ovariohysterectomy following anesthesia at local veterinary practices. Only non-degenerate COCs were collected and then suspended in TCM-199 supplemented with 5% fetal calf serum. Only oocytes with diameter >110 μm were selected and used for this study. In the first experiment, the effect of embedding in collagen gels of COCs on their meiotic competence was tested. Half of selected oocytes were embedded in 0.3 mL of collagen gels (3 to 4 COCs per gel) as described by Yamamoto et al. (Yamamoto K et al., 1999 Theriogenology 52, 81–89). The COCs with or without collagen gels were cultured in a dish containing 2.5 mL of TCM-199 supplemented with 0.1 IU mL⁻¹ HMG and 10 IU mL⁻¹ hCG (3 to 4 COCs per dish) for 72 h at 38.5°C in a humidified atmosphere of 5% CO₂ in air. In the second experiment, the effect of removal of hormonal supplements from maturation medium on nuclear maturation in vitro was examined. At 24 and 48 h after the start of culture, the COCs embedded in collagen gels were cultured in TCM-199 without HMG and hCG for 48 and 24 h, respectively. As a control, the COCs embedded in collagen gels were cultured with hormone supplement for 72 h. After 72 of maturation culture, the oocytes were fixed, stained with Hoechst 33342 and examined for the meiotic stage of the oocytes using a fluorescence microscope. Data were analyzed by ANOVA. The proportion of oocytes that resumed meiosis was significantly higher (P < 0.05) in the COCs with collagen gels than in the control COCs without collagen gels (50.6 v. 26.5%). Significantly more oocytes reached metaphase I to metaphase II stage (MI/II) in the collagen gels culture than in the control culture (P < 0.05; 27.4 v. 8.3%). The proportion of collagen embedded-oocytes that resumed meiosis was significantly higher (P < 0.05) in COCs cultured with hormone supplements for 24 h than in COCs cultured for 48 h (59.1 v. 30.4%) but not different from COCs exposed for 72 h (41.9%). Moreover, there were no significant differences of MI/MII rates (22 to 24%) among the three treatment groups. These observations indicate that embedding of COCs in collagen gels enhances the meiotic competence of canine oocytes, but removal of hormone supplement from maturation medium does not improve the ability of the oocytes to reach MI/MII stage.