Porcine somatic cell nuclear transfer (NT) has been successfully performed, but its efficiency remains quite low. In this study, we improvised on the enucleation method to enhance the development of NT embryos. Initially, an experiment was performed to determine the location relationship between the metaphase plate and the first polar body, where the results showed that the metaphase plate may frequently be displaced during the varying period of maturation process. When the metaphase plates were removed using the 'blind' enucleation method, the enucleation rate was affected by the maturation time; however, when the spindle view system was used, an enucleation rate of 100% was achieved. In the next experiment, these two methods were used to construct embryos: the fusion efficiency was significantly higher (p < 0.01) in the spindle view system group and the development rates of the reconstructed embryos were significantly higher in the spindle view system group compared with the 'blind' enucleation group (p < 0.01). An average of 174 (141-210) cloned embryos from the spindle view system group were transferred into five surrogate pigs and one piglet was delivered at 114 days after embryo transfer by caesarean section. DNA analysis confirmed that the piglet was genetically identical to the male donor pig. We showed that enucleation by the spindle view system is the another new technique compare the handmade cloning method [Theriogenology 2007: 68, 1104] to promote the development of the reconstructed embryos, and that a full-term cloned pig could be produced using this method.

The objective of our study was to establish the feasibility of experimental protocols for cloning sika deer. We performed auxiliary enucleation to improve the efficiency of nuclear transfer operation by optimizing the demecolcine concentration to induce cytoplasmic protrusions in the sika deer oocytes. In the present study,we had studied the impact of different demecolcine concentrations on cytoplasmic protrusions and enucleation rates. We determined that 95.9% of the sika deer oocytes formed cytoplasmic protrusions when treated for 1 h with 0.8 μg/ml demecolcine. The lowest observed rate of protrusion was 19.3% after overnight treatment with demecolcine. When the oocytes aged or had a poor cumulus expansion, they exhibited a significant decrease in the ability to form cytoplasmic protrusions. The rates of enucleation (94.9% vs 85.8%, p < 0.05), cell fusion (84.6% vs 70.1%, p < 0.05) and blastocyst formation (15.4% vs 10.9%, p < 0.05) using demecolcine auxiliary enucleation were significantly higher than those after blind enucleation. These results demonstrated that sika deer oocytes could be enucleated quickly and effectively using demecolcine auxiliary enucleation, which could enhance the enucleation rate, cell fusion rate and blastocyst rate of cloned embryos in vitro.