IMPROVED YIELDS OF BOVINE BLASTOCYSTS FROM IN-VITRO PRODUCED OOCYTES .2. MEDIA AND COCULTURE CELLS

Three experiments were conducted with 9,472 oocytes, aspirated from ovaries of slaughtered cows (7,318 cleaved zygotes), to compare some media and co-culture cells for in vitro maturation of oocytes and culture of embryos. The endpoint was development to expanded blastocysts at 9.0 d after exposure of matured oocytes to sperm. In the first experiment, oocytes were matured in TCM-199 and co-cultured in TCM-199 or Menezo's B2 (B2), with granulosa or buffalo rat liver (BRL) cells. The type of co-culture cell had no effect on the proportion of oocytes or cleaved zygotes that developed to expanded blastocysts, but 31% of cleaved zygotes developed to expanded blastocysts in B2 compared with 21% in TCM-199 (P<0.001), and embryos developed faster in B2. In the second experiment, oocytes were matured in TCM-199, Ham's F-10 (F-10), or B2, then all were co-cultured in B2 with BRL cells. The proportion of cleaved zygotes that developed to expanded blastocysts was 28%, 40%, and 8% after maturation in TCM-199, F-10, or B2, respectively (P<0.001). In the third experiment, presumptive zygotes were vortexed to remove cumulus at 15 hr after exposure to sperm and classified as having dense even cytoplasm or thin and/or uneven cytoplasm. Then zygotes were co-cultured with BRL or bovine oviduct epithelial (BOE) cells. There was little difference in the proportion of cleaved zygotes that developed to expanded blastocysts after coculture with BRL (51%) or BOE cells (54%), but embryos developed somewhat faster with BOE than with BRL cells. Presumptive zygotes with even cytoplasm, compared to presumptive zygotes with uneven cytoplasm, had higher cleavage rates (85% vs 76%, P<0.001) and higher rates of development of cleaved zygotes to expanded blastocysts (59% vs 44%, P<0.001). In these experiments, the best combination for growing embryos in vitro was F-10 maturation medium, B2 embryo culture medium, and BOE cells for co-culture.