353 EFFECTS OF CULTURE DURATION AND CUMULUS CELL REMOVAL ON CANINE OOCYTE IN VITRO MATURATION AND FERTILIZATION USING VASAL SPERM

Abstract

The objective of this study was to test the hypothesis that in vitro maturation (IVM) and fertilization (IVF) rates of canine oocytes could be improved by increasing culture duration or decreasing/increasing cumulus cell contact with the oocytes when using sperm retrieved from the vas deferens. The canine oocyte is ovulated at the germinal vesicle stage, and maturation of the oocyte occurs in the oviduct and requires up to five days. Therefore, an increase in the culture duration may cause an increase in oocyte nuclear maturation. Canine ovaries and testes were collected from a local clinic, placed in warm saline solution, and transported to the laboratory. Two distinct experiments were carried out, one involving IVM (M-II) after cumulus cell removal at 72 h and 96 h for nuclear maturation evaluation, and the second experiment the same but continued up to IVF. The oocytes were recovered from the ovaries by mincing them in warm Medium-199 with Hanks salts, L-glutamine, and HEPES (GIBCO, Grand Island, NY, USA; Invitrogen Co., Carlsbad, CA, USA). Canine oocytes with a dark cytoplasm and at least 2 layers of cumulus cells were cultured in Medium-199 supplemented with Earle's salts, 2200 mg mL⁻¹ sodium bicarbonate, 25 mM HEPES, 2 mM sodium pyruvate, 5 µg mL⁻¹ progesterone, 100 ng mL⁻¹ epidermal growth factor, 10 IU mL⁻¹ human chorionic gonadotropin (HCG), 5 µg mL⁻¹ insulin, 0.50 mM epinephrine, 10% estrus bitch serum, 0.01 mM nonessential amino acids, and 20 µg mL⁻¹ gentamicin. The oocytes were cultured for 72, 96, 120, or 144 h at 38.5°C in 5% CO₂ in humidified air. The cumulus cells were removed after either a 72- or 96-h culture period. For the first 48 h, the cumulus–oocyte complexes were cultured in the modified Medium-199 containing 10 IU mL⁻¹ HCG and then cultured in the same medium free of HCG. The oocytes were denuded by pipetting, stained with Hoechst 33342, and examined for nuclear maturation. ANOVA was used for statistical analysis of the data. The IVM rate (MII) was significantly higher (P < 0.05) at 72 and 96 h compared to 48, 120, and 144 h (15.1% and 16.9% vs. 6%, 12.4%, and 9.1%, respectively). The removal of cumulus cells at 72 h and 96 h resulted in 17.9% (43/240) and 14.8% (35/236) IVM rate (MII), respectively (P > 0.05). The sperm motility index (SMI = motility percentage × sperm activity grade) was significantly higher in sperm retrieved from the vas deferens (vasal sperm) compared to epididymal and testicular sperm (259 vs. 95 and 19.2, respectively, P < 0.05). The mature oocytes were inseminated by vasal sperm following in vitro hyperactivation with HEPES solution supplemented with 3 mg mL⁻¹ bovine serum albumin. The IVF rates of the oocytes following 72 and 96 h of maturation in vitro were 48.2% and 40%, respectively (P > 0.05). Sperm penetration was significantly higher at 96 h compared to 72 h, and the number of sperm heads inside the ooplasm was 3.2 for the 72 h group vs. 4.8 for the 96 h group (P < 0.05). In conclusion, increasing the IVM culture period beyond 72 h did not increase the oocyte maturation rates, and increasing the culture time to 96 h without cumulus cells present increased the rate of sperm penetration.