Effects of enucleation method on in vitro and in vivo development rate of cloned pig embryos

Enucleation is a crucial procedure for mammalian somatic cell nuclear transfer (SCNT), especially for domestic animal cloning. Oocytes of domestic animals such as pigs and cattle contain dark lipid droplets that hinder localization and removal of the nucleus. Using an oocyte enucleation technique that can obtain a high enucleation rate but has minimal negative effects on the reprogramming potential of oocyte for cloning is beneficial for enhancing the outcome of SCNT. In this study, we compared the pig cloning efficiency resulting from blind aspiration-based (BA-B) enucleation and spindle imaging system-assisted (SIS-A) enucleation, and compared the pig SCNT success rate associated with BA-B enucleation and blind aspiration plus post-enucleation staining-based (BAPPS-B) enucleation. SIS-A enucleation achieved a significantly higher oocyte enucleation success rate and tended to obtain a higher in vivo full term development rate of SCNT embryos than BA-B enucleation. BAPPS-B enucleation also obtained significantly higher in vitro as well as in vivo full term development efficiency of cloned porcine embryos than BA-B enucleation. These data indicate that SIS-A and BAPPS-B enucleation are better approaches for pig SCNT than BA-B enucleation.