Reagents and Hormones
Tissue culture medium (TCM-199 with Earle’s Salts) was purchased from Gibco-BRL (Grand Island, NY). Equine pituitary LH (11.5 NIH LH-S1 U/ml; Bethesda, MD) and porcine pituitary FSH preparations were obtained from Sigma Chemical Co. (St. Louis, MO). All other reagents and media supplements were of tissue culture grade and were purchased from Sigma. Fatty acid-free BSA was purchased from Boehringer-Mannheim (Indianapolis, IN). Low melting point agarose for embryo embedment was purchased from Bethesda Research Laboratories (Gaithersburg, MD). All other reagents for electron microscopy were purchased from Electron Microscopy Sciences (Fort Washington, PA). Prostaglandin F2a was obtained from Phar-macia & Upjohn (Lutalyse; Kalamazoo, MI). Folltropin was obtained from Vetrepharm Canada (London, ON, Canada).
In Vivo Embryo Production
For production of in vivo compact morulae (multiple ovulations, MO), Holstein donor cows were superovulated by i.m. administration of 400 mg Folltropin given in a series of decreasing doses over a 3- or 4-day period. Estrus was induced by i.m. administration of 25 mg prostaglandin F2a on the morning and evening of the third or fourth day of FSH treatment. Estrus detection was performed twice daily beginning 24 h after the first prostaglandin F2a injection. Donor cows were artificially inseminated 12 and 24 h after first standing estrus with semen from a proven Holstein sire. Embryos used in this study were recovered by nonsurgical uterine flushing of donor cattle on either Day 6 or Day 8 of the cycle (Day 0: first standing estrus). Based on evaluation at X60, a total of five grade 1 compact morulae were identified, fixed in McDowell’s and Trump’s fixative (4% formaldehyde:1% glutaraldehyde), and held at 4°C until processed for transmission electron microscopy.