Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Area of Science:

• Embryology
• Reproductive Biology
• Molecular Biology

Background:

• Cytoplasmic microinjection is crucial for genetic modification and gene function studies in embryos.
• Conventional microinjection methods are inefficient in livestock zygotes due to dark cytoplasm and elastic membranes.
• Existing techniques face challenges in visualizing and penetrating the plasma membrane of bovine, ovine, and porcine embryos.

Purpose of the Study:

• To develop and validate an efficient microinjection protocol for livestock zygotes.
• To adapt microinjection techniques for cattle, sheep, and pig embryos.
• To improve the delivery of substances into the cytoplasm of one-cell embryos.

Main Methods:

• A laser creates a hole in the zona pellucida of the zygote.
• A blunt-end glass micropipette is inserted through the hole.
• Plasma membrane integrity is compromised by aspirating cytoplasmic content, followed by injection of the desired solution.

Main Results:

• The protocol achieved 100% efficiency in delivering solutions into bovine and ovine zygotes.
• Minimal embryo lysis was observed using this method.
• Normal blastocyst development rates were maintained in treated embryos.

Conclusions:

• This laser-assisted microinjection technique is highly effective for livestock zygotes.
• The method overcomes limitations of conventional techniques in species with challenging zygote characteristics.
• The protocol supports efficient genetic manipulation and functional studies in preimplantation embryos.