The use of demecolcine for enucleation of bovine oocytes

Nuclear transfer requires removing all genetic materials associated with the chromosomes of recipient oocyte. This Study was designed to further explore the pharmacological role of Demecolcine (DM) on assisting enucleation in animal somatic cell nuclear transfer. The in vitro matured bovine oocytes were incubated with DM at different concentration, or at a fixed DM concentration for additional different hours, then the oocytes extrusion cones rate (ECR) were determined in the inverted microscope. The highest ECR (61.90%) was measured from the treatment with 0.5μg/mL demecolcine. The time-dependent manner of the development of the extrusion cones, in 2hr groups were significantly higher (P<0.05) than in Ohr, O.5hr, Ihr and 2.5hr groups. The highest ECR were found in vitro matured 18hr groups (73.86%), which was significantly higher (P<0.05) than that observed in 14hr, 16hr groups, and 20hr groups. However, the Granualar cell existence during maturation can influence on PR and embryos development rate. The in vitro matured 18hr bovine oocytes with granualar cell were added to DM, which were significantly higher (P<0.05) than that of control groups. Meanwhile, we evaluated the effects of DM on the cleavage rate of in Vitro matured oocytes. The results showed that the IVM medium with or without DM, the IVF embryos rate of cleavage, of blastocysts, and average cell number of blastocysts between the two groups were not significantly different from each other. This simple, chemically assisted method to remove maternal chromosomes makes it possible to produce a large number of nuclear-transferred eggs and to efficiently produce cloned bovines.