Developmental Potential of Rhesus Monkey Embryos Produced by in Vitro Fertilization

This study was an examination of the developmental potential of in vitro fertilization (IVF)-produced rhesus monkey embryos that were cultured in medium alone or cocultured with various cell types. End points were the quality and yield of embryos attaining the expanded or hatched blastocyst stage. A total of 96 IVF-produced embryos were cryopreserved and thawed, and 90 embryos were considered intact and suitable for culture. These embryos were placed into one of five treatment groups consisting of four different cell supports and medium alone. Two primary cultures (bovine oviductal cells [bOVID] and bovine cumulus cells [bCUM]) and two established cell lines (Vero cells and buffalo rat liver cells [BRL]) were utilized for coculture of embryos. Embryos were cultured for up to 14 days, and growth curves were established for all embryos that expanded and/or hatched. The developmental rate for embryos classified as viable varied substantially; in number of days to reach a given stage, early morulae ranged from Days 3 to 9 post-insemination, morulae from Days 4 to 9, blastocysts from Days 6 to 11, expanded blastocysts from Days 7 to 12, and hatched blastocysts from Days 9 to 15. On the basis of developmental curves, 30% of the embryos were arrested upon thawing or shortly after. Of the remaining embryos classified as viable, developmental efficiencies to the hatched blastocyst stage for the various treatments were 1) bOVID, 33%; 2) bCUM, 15%; 3) Vero cells, 9%; 4) BRL, 45%; and 5) medium alone, 8%. The nuclei of the inner cell mass (ICM) and trophoblastic cells of those embryos that hatched (14 of 90) were differentially stained and cell counts were determined. The total cell number per embryo ranged from 177 to 1674, with the percentage of ICM per embryo varying from 12 to 68%. While some coculture treatments produced a significantly greater number of expanded and hatched blastocysts compared to medium alone, there was no correlation between cell counts of hatched blastocysts and any treatment group. Therefore, while cocultures improved the developmental efficiency of rhesus monkey embryos compared to culture in medium alone, the majority of embryos that reached the hatched blastocyst stage in all treatments showed a well-developed ICM, suggesting that embryo quality was not improved with coculture. In conclusion, BRL were found to be beneficial for the development of rhesus monkey hatched blastocysts of good quality while providing the convenience of a rigorously controlled established cell line for coculturing.