The effect of recipient oocyte volume on nuclear transfer in cattle

Abstract

This study compared the developmental potential of bovine nuclear transfer embryos with varying amounts of cytoplasm. Embryos formed from single cytoplasts fused to blastomeres by a single electrical pulse or from double cytoplasts using a double electrical pulse resulted in reconstituted embryos containing 75% and 150% of the original oocyte volume. No differences in fusion, cleavage, or development rates to blastocysts were observed between the groups. Mean cell numbers 2 days after fusion were significantly lower in single‐cytoplast clones. Cell numbers of resulting blastocysts were likewise significantly lower in single‐cytoplast clones. Embryos formed by fusion of blastomeres with single cytoplasts using a single electrical pulse or from double cytoplasts using either a single or a double pulse resulted in reconstituted embryos containing 50%, 100% and 100% of the original oocyte volume. Again, no differences in fusion or cleavage rates were observed between groups, but the development to blastocysts at day 7 was significantly higher in double cytoplasts constructed with one fusion pulse than in single cytoplasts (P< 0.05). Mean cell numbers 2 days after fusion were significantly lower in single‐cytoplast clones (P< 0.05), but at the blastocyst stage, no statistically significant differences in cell numbers were observed. The results of this study show that cytoplasmic volume plays a role in the development of nuclear transfer embryos. When using crude enucleation methods such as oocyte bisection, normal cytoplasmic volumes can be achieved by fusing double cytoplasts with embryonic blastomeres. Mol. Reprod. Dev. 50:185–191, 1998. © 1998 Wiley‐Liss, Inc.

Development of Human Cloned Blastocysts Following Somatic Cell Nuclear Transfer with Adult Fibroblasts

“…Increasing the amount of cytoplasm removed during the enucleation process has an impact on the development potential of nuclear transfer embryos [44,45]. Anecdotally, removal of the first polar body with either technique appeared to increase the amount of cytoplasm removed.…”

Effect of Culture Media on In Vitro Development of Cloned Mouse Embryos

“…In the manipulation control embryos, a certain volume of oocyte cytoplasm was removed, while in NT embryos the spindle surrounded by an amount of cytoplasm rich in actin was removed. It has been demonstrated that the amount of spindle-surrounding cytoplasm should be restricted to a minimum in order not to affect the developmental potential of NT embryos (Peura et al, 1998) and that chemical enucleation could be beneficial (Baguisi and Overström, 2000).…”

“…A study in bovine NT embryos (Lavoir et al, 1997) revealed a twofold higher incidence of multinucleation in embryos reconstructed with differentiated oogonia versus embryos reconstructed with blastomere nuclei. Another possible explanation is that enucleation can coincide with a high amount of actin removal, which could interfere with proper cytokinesis (Peura et al, 1998). It has also been put forward that aberrant culture conditions can increase the rate of multinucleation.…”

Developmental Competence of HMC Derived Bovine Cloned Embryos Obtained from Somatic Cell Nuclear Transfer of Adult Fibroblasts and Granulosa Cells

“…The first published attempt to exclude micromanipulators was the bovine zona-free embryonic cell nuclear transfer method [3], which was, subsequently, slightly modified [4] for carrying out somatic cell nuclear transfer. We also made an attempt to use the Handmade Cloning (or HMC TM ) method, which is a 'zona-free' method of somatic cell nuclear transfer [5] in cattle.…”