Development of in vitro embryo production systems for red deer ( Cervus elaphus) : Part 2. The timing of in vitro nuclear oocyte maturation

Abstract

The time course of in vitro red deer nuclear oocyte maturation was determined. Ovaries were obtained at slaughter and oocytes were aspirated from follicles greater than 2 mm in diameter. Oocytes with compact cumulus cells were matured in 50 μl microdrops (10 per drop) under mineral oil containing TCM 199 supplemented with 0.33 mM pyruvate, 10 μg LH and FSH, 1 μg oestradiol and 10% foetal bovine serum. Oocytes were matured at 39 °C and 5% CO 2 in air. At 3 h intervals (0–27 h) oocytes were removed from incubation, cumulus expansion scored and removed, and fixed oocytes in ethanol:acetic acid (3:1) for 48 h. Oocytes were stained with lacmoid (1%) and nuclear maturation assessed. Oocytes were arrested in the germinal vesicle (GV) stage at aspiration and up to 6 h of incubation. The nuclear membrane began to disperse after 6 h and by 10.6±0.6 h of incubation 75% of the oocytes exhibited germinal vesicle breakdown (GVBD). The mean time for 50% of the oocytes to reach metaphase one (MI) and metaphase two (MII) was 11.7±0.4 and 24.8±0.9 h, respectively. Cumulus oophorus were tightly compacted at aspiration and did not begin expansion until 12 h of culture. Full expansion was complete by 18 h of culture. Corona radiata cells did not begin expansion until 15 h and were fully expanded by 24 h. Results indicate that in vitro red deer oocyte maturation follows a similar time course of nuclear maturation as reported for bovine and ovine oocytes.