Cloned Sheep Blastocysts Derived from Oocytes Enucleated Manually Using a Pulled Pasteur Pipette

The potential applications of a simplified method of somatic cell nuclear transfer (SCNT) that is improved in both efficiency and throughput is considerable. Technically, a major step of SCNT is to produce large pools of enucleated oocytes (cytoplasts) efficiently, a process that requires considerable micromanipulation skill and expensive equipment. Here, we have developed an efficient and high-throughput method of manual oocyte enucleation using a simple device, a pulled Pasteur pipette, that can be connected to standard zona-free method of embryo reconstitution. Common Pasteur pipettes were pulled on a flame to produce finely drawn pipettes with inner diameters approximately less than half the oocyte diameter (similar to 50-60 mu m), and slightly larger than cytoplasmic protrusion (similar to 20-30 mu m) that was induced after demecolcine treatment of MII-stage oocytes. Oocyte manipulation was performed under a stereomicroscope by either bisecting the oocyte into two approximately equal demioocytes (blind manual enucleation), or by positioning the oocytes so that the cytoplasmic extrusion that contains the MII chromosome mass is removed with the minimum amount of cytoplasm (oriented manual enucleation). The survival rate of the manually enucleated oocytes was 81.4-91.5%, comparable to standard zona-free method of oocyte enucleation (>95%). A total of 80-120 oocytes could be enucleated in 10 min, which was considerably higher than standard zona-free enucleation method. In vitro development rates of cloned embryos derived from manually enucleated cytoplasts with varying cytoplasmic volumes (50%, 95%, and 100%) was comparable, and embryonic developmental rates of the two latter groups were at least as good as standard zona-free method. The manual method of oocyte enucleation described here can be learned and mastered for simple, fast, and cheap production of cloned embryos with comparable efficiency to other available methods.