Chemically Assisted Handmade Enucleation of Porcine Oocytes

The purpose of our work was to find an efficient and reliable chemically assisted procedure for enucleation of porcine oocytes connected to the handmade cloning (HMC) technique without the potentially harmful chromatin staining and ultraviolet (UV) irradiation for cytoplast selection. After 41–42 h in vitro maturation, porcine oocytes were incubated with 0.4 µg/mL demecolcine for 45 min. Subsequently, the cumulus cells were removed and zonae pellucidae were partially digested. Oocytes with extrusion cones or oocytes only with polar body (PB) were subjected to oriented bisection. Less than half of the cytoplasm with the extrusion cone or adjacent to the PB was removed with a microblade. The remaining putative cytoplasts, containing the major part of the cytoplasm, were used as recipients for reconstruction with porcine fetal fibroblasts as nuclear donors. The overall efficiency achieved with chemically assisted enucleation was higher compared to oriented bisection without demecolcine incubation (90 ± 3% vs...