In vitro development of horse oocytes reconstructed with the nuclei of fetal and adult cells.

Abstract

This study investigated the basic conditions required for the production of horse embryos by the transfer of the nuclei of fetal and adult fibroblast cells to enucleated oocytes. Cumulus-oocyte complexes were recovered from abattoir ovaries and matured in vitro in groups of 20-30 for 28-30 h in tissue culture medium 199 containing 20% v:v fetal bovine serum in coculture with equine oviduct epithelial cells. Fetal fibroblast cells (FFC) were derived from a 32-day-old Thoroughbred x Pony fetus, and adult skin fibroblast cells (SFC) were obtained from subdermal biopsies recovered from a 4-yr-old female Pony. The rates of fusion between the recipient cytoplasm with either FFC or SFC were significantly greater when the cells were treated with a combination of direct current (DC) pulses and Sendai virus rather than with DC pulses alone (81%-82% vs. 49%-57%, P < 0.05). There were no differences in the rates of nuclear reprogramming between FFC and SFC (88% vs. 84%), but the rate of cleavage of the resulting embryos to the 2-cell stage was higher when FFC were used (53%) than when SFC were used (35%). Blastocysts were obtained from oocytes reconstructed with both types of donor cells and after culture in vitro for 6-7 days, but the overall proportion of blastocysts produced was very low in both cases (FFC, 4%; SFC, 7%). These results demonstrate a very limited potential for in vitro development of horse embryos after nuclear reprogramming following the transfer of nuclei from either fetal or adult fibroblasts into recipient enucleated oocytes.