This study was conducted to determine the additive effects of exogenous growth factors during in vitro oocyte maturation (IVM) and the sequential culture of nuclear transfer (NT) embryos. Oocyte maturation and culture of reconstructed embryos derived from bovine granulosa cells were performed in culture medium supplemented with either epidermal growth factor (EGF) alone or a combination of EGF with insulin-like growth factor-I (IGF-I). The maturation rates of oocytes matured in the presence of EGF or the EGF + IGF-I combination were significantly higher than those of oocytes matured in the presence of only fetal calf serum (FCS) (P < 0.05). The developing NT embryos showed no significant differences in fusion, cleavage or blastocyst rates among the culture groups (P > 0.05). IGF-I alone or in combination with EGF in sequential embryo culture medium significantly increased the ratio of inner cell mass (ICM) to total blastocyst cells (P < 0.05). Our results showed that the addition of growth factors to IVM and sequential culture media of cloned bovine embryos increased the ICM without changing the total cell number. These unknown and uncontrolled effects of growth factors can alter the allocation of ICM and trophectoderm cells (TE) in NT embryos. A decrease in TE cell numbers could be a reason for developmental abnormalities in embryos in the cloning system.

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.

Studies with intact preimplantation mouse embryos and some types of chimaeric aggregates have shown that the most advanced cells are preferentially allocated to the inner cell mass (ICM) rather than the trophectoderm. Thus, differences between 4-cell and 8-cell stage embryos could contribute to the tendency for tetraploid cells to colonise the trophectoderm more readily than the ICM in 4-cell tetraploid[harr ]8 cell diploid chimaeras. The aim of the present study was to test whether 4-cell stage embryos in 4-cell diploid[harr ]8-cell diploid aggregates contributed equally to all lineages present in the E12.5 conceptus. These chimaeras were compared with those produced from standard aggregates of two whole 8-cell embryos and aggregates of half an 8-cell embryo with a whole 8-cell embryo. As expected, the overall contribution of 4-cell embryos was lower than that of 8-cell embryos and similar to that of half 8-cell stage embryos. In the 4-cell[harr ]8-cell chimaeras the 4-cell stage embryos did not contribute more to the trophectoderm than the ICM derivatives. Thus, differences between 4-cell and 8-cell embryos cannot explain the restricted tissue distribution of tetraploid cells previously reported for 4-cell tetraploid[harr ]8-cell diploid chimaeras. It is suggested that cells from the more advanced embryo are more likely to contribute to the ICM but, for technical reasons, are prevented from doing so in simple aggregates of equal numbers of whole 4-cell and whole 8-cell stage embryos.

In a previous study of mouse tetraploid[harr ]diploid chimaeric blastocysts, tetraploid cells were found to be more abundant in the trophectoderm than the inner cell mass (ICM) and more abundant in the mural trophectoderm than the polar trophectoderm. This non-random allocation of tetraploid cells to different regions of the chimaeric blastocyst may contribute to the restricted tissue distribution seen in postimplantation stage tetraploid[harr ]diploid chimaeras. However, the tetraploid and diploid embryos that were aggregated together differed in several respects: the tetraploid embryos had fewer cells and these cells were bigger and differed in ploidy. Each of these factors might underlie a non-random allocation of tetraploid cells to the chimaeric blastocyst. A combination of micromanipulation and electrofusion was used to produce two series of chimaeras that distinguished between the effects of cell size and ploidy on the allocation of cells to different tissues in chimaeric blastocysts. When aggregated cells differed in cell size but not ploidy, the derivatives of the larger cell contributed significantly more to the mural trophectoderm and polar trophectoderm than the ICM. When aggregated cells differed in ploidy but not cell size, the tetraploid cells contributed significantly more to the mural trophectoderm than the ICM. In both experiments the contributions to the polar trophectoderm tended to be intermediate between those of the mural trophectoderm and ICM. These experiments show that both the larger size and increased ploidy of tetraploid cells could have contributed to the non-random cell distribution that was observed in a previous study of tetraploid[harr ]diploid chimaeric blastocysts.