Azita Afzali - PhD Student of Reproductive Biology, Faculty of Medicine, Tehran University of Medical Sciences
Hassan Nazari - Research Institute of Animal Embryo Technology, Shahrekord University
Abolfazl Shirazi - Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahrekord University
Ebrahim Ahmadi - Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahrekord University

Visible oocyte nucleus in hand-made cloning of domesticated species via induction of metaphase plate protrusion improve the efficacy of this process by increasing the number of oocytes for enucleation and attain the cytoplasts with the least amount of lost cytoplasm. For this purpose, the present investigation was carried out use of microtubule stabilizing (paclitaxel and cytochalazin B) in comparison to depolarizing (demecolcine) chemical agents for induction of metaphase plate protrusion in in vitro matured bovine oocytes which makes it easy to remove the maternal chromosomes for nuclear transfer (NT) in hand-made cloning process. After in vitro maturation of bovine oocytes and remove the zona pellucida, oocytes were incubated in Medium 199 supplemented with 20% FBS containing 0.5 μg/ml demecolcine, 1 μg/ml paclitaxel, or 1 μg/ml cytochalazin B for 90 min at 39°C for induction of protrusion of metaphase plate and subsequent removing the protruded cytoplasm under a stereomicroscope using finely drawn hand-made pipettes. This stage is a prerequisite for production of reconstructed embryo with somatic cells. After statistical analysis, the results of this study indicated that the maximum protrusion of division spindle was occurred with demecolcine (83.01 vs. 52.6 and 45.23 for cytochalazin B and paclitaxel, respectively) (P<0.05). This result has been achieved by considering the less impression of these compounds on development of the reconstructed produced embryos. Therefore, it can be concluded that demecolcine is a good chemical agent with desired instill of spindle apparatus protrusion to use in production of SCNT derived embryos with hand-made cloning method.

Hand-made cloning, Demecolcine,Paclitaxel, Cytochalazin B, Bovine