The mammalian blastocyst comprises an inner cell mass (ICM) and a trophectoderm cell layer. In this study the allocation of blastomeres to either cell lineage was compared between murine, porcine and bovine blastocysts. Chemical permeation of trophectoderm cells by the Ca2+ ionophore A23187 in combination with DNA-specific fluorochromes resulted in the differential staining of trophectoderm and ICM. Confocal microscopy confirmed the exclusive permeation of trophectoderm and the internal localisation of intact ICM cells in bovine blastocysts. Overall, differential cell counts were obtained in approximately 85% of the embryos assessed. Mean (±SEM) total cell numbers were 72.2 ± 3.1 and 93.1±5 for in vivo derived murine (n = 41) and porcine (n = 21) expanded blastocysts, respectively. Corresponding ICM cell number counts revealed ICM/total cell number ratios of 0.27 and 0.21, respectively. Comparison of in vivo (n = 20) and in vitro derived bovine embryos on day 8 (n = 29) or day 9 (n = 29) revealed a total cell number of 195.25±9.9, 166.14±9.9 and 105±6.7 at the expanded blastocyst stage with corresponding ICM/total cell ratios of 0.27, 0.23 and 0.23, respectively. While total cell numbers differed significantly among the three groups of bovine embryos (p<0.05), the ICM/total cell ratio did not. These results indicate that a similar proportion of cells is allocated to the ICM among blastocysts of genetically divergent species.

Blastocyst development, total cell number and allocation to inner cell mass (ICM) and trophectoderm (TE) lineages was compared among day 9 hatched blastocysts from four culture treatments in a two-factor design. Two modified commercial media (KSOM and SOF) were used in atmospheres with two oxygen concentrations (5% and 20% O2). No significant effect of medium on development was found, but 20% O2 increased hatching (p < 0.05). There were more cells in hatched blastocysts cultured in KSOM than in SOF (181 vs 136, respectively; p < 0.0001); however, ICM/total cell ratio was not affected by medium. There was a trend suggesting that the proportion of cells allocated to ICM was lower in hatched blastocysts cultured under 5% O2 compared with 20% O2 (0.323 vs 0.380, respectively; p < 0.1). No significant interactions between medium type and oxygen concentration were found. These results indicate that culture components used in this study may affect cell proliferation without altering cell allocation, and that oxygen concentration may play a role in allocation of cells to ICM and TE lineages.