CAPRINE BLASTOCYST FORMATION FOLLOWING INTRACYTOPLASMIC SPERM INJECTION AND DEFINED CULTURE

Experiments were undertaken to develop intracytoplasmic sperm injectio n (ICSI) to produce caprine embryos out of the normal breeding season. Oocytes were obtained from 2-6 mm ovarian follicles at slaughter. Sel ected oocytes with two to four layers of cumulus cells were incubated in 1 ml of H-TCM199 supplemented with 10 mu g each of oFSH and bLH (NH PP, NIDDK, NICHD, USDA) and 20% fetal bovine serum (FBS) in a thermos (38.5 degrees C) for 4.5 h during transportation. Then, oocytes were t ransferred into 75 mu l of freshly prepared maturation medium under pa raffin oil and a mixture of 5% O-2, 5% CO2 and 90% N-2. Approximately 26 h after recovery oocytes were denuded by incubation with hyaluronid ase (100 IU/ml) and pipetting and held at 38.5 degrees C for 90 min. S permatozoa frozen in egg yolk extender were thawed in a 37 degrees C w ater bath for 15 s. Motile fractions were selected by swim-up, then in cubated for 90 min in TALP with 10 mu g heparin/ml. Each oocyte was po sitioned with its first polar body at 6 or 12 o'clock by a holding pip ette. Sperm (1 mu l) were added to 10 mu l medium containing 10% polyv inylpyrrolidone. A sperm cell was aspirated into a pipette, and then i njected head-first into the cytoplasm of an oocyte maintained in H-TCM 199+20% FBS at 37 degrees C. Injected oocytes were transferred to HM a nd, after 90 min, cultured in 50 mu l of BSA-free synthetic oviduct fl uid plus polyvinyl alcohol, citrate and non-essential amino acids. Res ults demonstrate that caprine blastocysts can be produced outside the breeding season by the use of frozen-thawed semen and injection of spe rm cells with broken tails into ova followed by culture in defined med ium.