312 THE EFFECTS OF ECG AND ESTRADIOL ON CANINE OOCYTES DURING IN VITRO MATURATION

Abstract

The effects of eCG and estradiol (E2) were evaluated on canine oocytes during in vitro maturation (IVM). Canine ovaries were obtained from routine ovariohysterectomies, and oocytes with one or more intact cumulus layers and a consistently dark and smooth cytoplasm were collected by mincing the ovaries in TL HEPES warmed to 38°C. Oocytes were cultured for 72 h at 38.5°C in 5% CO₂ in humidified air in medium containing TCM 199, 2.92 mM calcium lactate, 2.0 mM pyruvic acid, 4.43 mM HEPES, 10% fetal bovine serum, and 1% Pen/Strep solution (12166 GibcoBRL, Grand Island, NY, USA) and with no added hormones (control), 2 ng/mL E2, 10 IU/mL eCG, or both E2 and eCG. At the end of culture, oocytes were removed and denuded by aspiration in a medium containing 50% v/v Trypsin-EDTA 10X solution (T-4174Sigma, St. Louis, MO, USA) and 50% v/v PBS with 3 mg mL⁻¹ polyvinyl alcohol. Denuded oocytes were fixed in 3.7% paraformaldehyde for 1 h and then transferred to a 1.9 mM Hoechst 33342 solution in glycerol. Nuclear status was observed under UV light and recorded. Chi-square analysis was performed to determine statistical significance where P < 0.05. Oocytes cultured with eCG were observed as having a significantly larger percentage of retained germinal vesicles (GV) at the end of culture (78%) compared to those cultured with E2 (62%) or in the control (66%). Addition of E2 with eCG was observed to increase the GV retention percentage (84%); however, it was not significantly greater than treatment with eCG alone. The eCG- and eCG with E2-treated oocytes also showed cumulus cell mass expansion where the cumulus oocyte complex diameter greatly increased and became ‘fluffy’ in appearance. The control and E2-supplemented media were the only treatments where MII oocytes were observed (5% and 9%, respectively), and the percentages of meiotic resumption for these treatments (15% and 17%, respectively) were significantly greater than those for eCG-(8%) and eCG with E2 (2%)-treated oocytes. The percentage of oocytes in which there was no observable DNA at the end of culture remained relatively constant between all four treatment groups and was also average compared to most reports of canine IVM in the literature. These data suggest that it is possible eCG promotes cumulus cell expansion and acts as a meiotic inhibitor for canine oocytes in vitro. The addition of E2 appears to augment the inhibitory effect of eCG, but in culture alone did not significantly increase the percentage of oocytes that resumed meiosis compared to the control. The authors would like to acknowledge Genetic Savings and Clone for making this work possible.

Table 1 
Nuclear status of oocytes after culture