Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 2. The timing of in vitro nuclear oocyte maturation.
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 2mm in diameter. Oocytes with compact cumulus cells were matured in 50 microl microdrops (10 per drop) under mineral oil containing TCM 199 supplemented with 0.33 mM pyruvate, 10 microg LH and FSH, 1 microg oestradiol and 10% foetal bovine serum. Oocytes were matured at 39 degrees C and 5% CO(2) in air. At 3h 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 6h of incubation. The nuclear membrane began to disperse after 6h and by 10.6+/-0.6h 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+/-0.9h, respectively. Cumulus oophorus were tightly compacted at aspiration and did not begin expansion until 12h 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 24h. Results indicate that in vitro red deer oocyte maturation follows a similar time course of nuclear maturation as reported for bovine and ovine oocytes.
Berg
D K
DK
Reproductive Technologies Group, AgResearch Ruakura, Private Bag 3123, Hamilton, New Zealand. debra.berg@agresearch.co.nz
Thompson
J G
JG
Asher
G W
GW
D000818:Animals; D002467:Cell Nucleus / Q000502:physiology*; D002478:Cells, Cultured; D003670:Deer / Q000196:embryology*; D005260:Female; D008297:Male; D008677:Metaphase; D009685:Nuclear Envelope / Q000648:ultrastructure; D009865:Oocytes / Q000502:physiology / Q000648:ultrastructure*; D006080:Ovarian Follicle / Q000166:cytology; D010053:Ovary / Q000166:cytology / Q000502:physiology; D013997:Time Factors; D020858:Tissue and Organ Harvesting; D015044:Zona Pellucida / Q000502:physiology / Q000648:ultrastructure