Effect of blastomere sex and fluorescent labelling on the development of bovine chimeric embryos reconstituted at the four-cell stage

The development rate of bovine chimeric embryos reconstituted at the 4-cell stage is relatively low. If chimerism is to be used as an approach in prod ucing transgenic livestock, it is important to investigate whether this rat e is affected by the sex of the blastomeres being combined and if all blast omeres survive equally well. In Experiment 1, blastomeres from 4-cell stage embryos were inserted into surrogate zonae pellucidae either in pairs to r econstitute 4-cell chimeras, or as the original sets of four to make handle d controls. The development of chimeras with one pair of blastomeres labell ed with PKH26-GL was also investigated. The rate of development into blasto cysts, was similar in chimeras with unlabelled blastomeres (23%) and in tho se in which one pair of blastomeres was labelled (26%) and was lower (P < 0 .001) than in the handled and lVF control groups (43 and 58%, respectively) . Labelled cells were distributed approximately evenly between ICM and trop hoblast. In Experiment 2, the effect of sex differences between pairs of bl astomeres in chimeras was investigated; chimeras were reconstituted from pa irs of blastomeres taken from 4-cell embryos in which the remaining pair wa s sexed by PCR. No significant differences according to the sex of constitu ent blastomeres were detectable (mixed sex, 27%; males, 24%; females, 2 1 % ; P > 0.05). These results suggest that, in addition to the negative effect s of micromanipulation, factors other than the sex of the blastomeres are i nvolved in the reduced rate of development of chimeric bovine embryos. They also confirm the usefulness of PKH26-GL labelling for tracking the progeny of cleaving bovine blastomeres at least to the blastocyst stage. Mol. Repr od. Dev. 60: 202-207, 2001. (C) 2001 Wiley-Liss, Inc.