Caprine blastocyst formation following intracytoplasmic sperm injection and defined culture

Abstract

Experiments were undertaken to develop intracytoplasmic sperm injection (ICSI) to produce caprine embryos out of the normal breeding season. Oocytes were obtained from 2-6 mm ovarian follicles at slaughter. Selected oocytes with two to four layers of cumulus cells were incubated in 1 ml of H-TCM199 supplemented with 10 micrograms each of oFSH and bLH (NHPP, 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 transferred into 75 microliters of freshly prepared maturation medium under paraffin oil and a mixture of 5% O2, 5% CO2 and 90% N2. Approximately 26 h after recovery oocytes were denuded by incubation with hyaluronidase (100 IU/ml) and pipetting and held at 38.5 degrees C for 90 min. Spermatozoa frozen in egg yolk extender were thawed in a 37 degrees C water bath for 15 s. Motile fractions were selected by swim-up, then incubated for 90 min in TALP with 10 micrograms heparin/ml. Each oocyte was positioned with its first polar body at 6 or 12 o'clock by a holding pipette. Sperm (1 microliter) were added to 10 microliters medium containing 10% polyvinylpyrrolidone. A sperm cell was aspirated into a pipette, and then injected head-first into the cytoplasm of an oocyte maintained in H-TCM199 + 20% FBS at 37 degrees C. Injected oocytes were transferred to HM and, after 90 min, cultured in 50 microliters of BSA-free synthetic oviduct fluid plus polyvinyl alcohol, citrate and non-essential amino acids. Results demonstrate that caprine blastocysts can be produced outside the breeding season by the use of frozen-thawed semen and injection of sperm cells with broken tails into ova followed by culture in defined medium.