Culture of blastomeres from in vitro-matured,fertilized, and cultured bovine embryos

A culture system was devised to study the differentiation of bovine blastomeres. Blastomeres (2–13 per well) from embryos produced by in vitro maturation, fertilization, and culture of oocytes obtained from slaughterhouse ovaries were cultured for 10 days in 24-well culture plates on feeder layers in blastomere culture medium (BCM: equal parts tissue culture medium 199 and low-glucose Dulbecco's modified Eagle's medium with 10% fetal bovine serum). Ovine embryonic fibroblasts and STO cells were superior to bovine and mouse embryonic fibroblasts as mitotically inactivated feeder cells. Over five studies in which four blastomeres from an embryo were added to each culture well, an average of one colony per well formed from the blastomeres. The colonies continued to grow throughout the culture period, and most colonies resembled trophectoderm in their cellular characteristics, although some cultures contained a mixture of trophectoderm and endoderm. When the number of blastomeres cultured in each well was varied from 2–8, the number of colonies formed was proportional to the number of blastomeres added. Blastomeres from day 5 and day 6 embryos produced fewer colonies than did those from day 4 embryos, perhaps as a result of differentiation and tighter blastomere adhesion resulting in damage during their separation. The absence of serum did not alter the number of colonies formed. A number of growth factors, including LIF, OM, PDGFα, and FGF4, had no effect on the number of colonies, the size of colonies, or their alkaline phosphatase staining score beyond that provided by the feeder layer or serum when present. Blastomeres did not form colonies in the absence of feeder layers. Mol. Reprod. Dev. 48:238–245, 1997. © 1997 Wiley-Liss, Inc.