Cloned sheep blastocysts derived from oocytes enucleated manually using a pulled pasteur pipette.

Area of Science:

• Reproductive Biology
• Developmental Biology
• Biotechnology

Background:

• Somatic cell nuclear transfer (SCNT) is crucial for cloning and regenerative medicine.
• Efficient enucleation of oocytes (cytoplasts) is a bottleneck in SCNT, requiring specialized skills and equipment.
• Existing enucleation methods are often slow and costly, limiting SCNT applications.

Purpose of the Study:

• To develop an efficient, high-throughput, and simplified manual oocyte enucleation method for SCNT.
• To assess the feasibility and efficiency of using pulled Pasteur pipettes for oocyte enucleation.
• To compare the developmental potential of SCNT embryos derived from manually enucleated oocytes with standard methods.

Main Methods:

• Pulled fine-tipped Pasteur pipettes (50-60 μm inner diameter) were used for manual oocyte enucleation under a stereomicroscope.
• Two manual enucleation techniques were employed: blind bisection and oriented removal of the chromosome-containing protrusion.
• Oocytes were treated with demecolcine to induce metaphase II chromosome extrusion prior to enucleation.

Main Results:

• Manual enucleation achieved oocyte survival rates of 81.4-91.5%, comparable to standard methods.
• High throughput was demonstrated, with 80-120 oocytes enucleated in 10 minutes, significantly faster than conventional techniques.
• Embryonic development rates of SCNT embryos from manually enucleated oocytes were comparable to those from standard enucleation methods.

Conclusions:

• A simple, rapid, and cost-effective manual oocyte enucleation technique using pulled Pasteur pipettes is established.
• This method significantly improves throughput for SCNT, making it more accessible.
• The developed technique yields SCNT embryos with developmental potential comparable to existing, more complex methods.