Comparative assessment of development competence of zona-intact and zona-free cloned goat embryos produced by innovative micromanipulation tools

• A new approach of cloning (zona free) using micromanipulator tools is described.
• Using innovative microtools, 100% oocyte enucleation rate is achieved.
• The developmental rates of zona free embryos were higher than zona intact group.
• Micromanipulator is used, but doesn’t require fine skill like conventional method.

The present study demonstrated a new approach for production of zona free cloned embryos in goat wherein, instead of hand guided bisection applied under Hand-made cloning technique, we have adopted micromanipulator based enucleation using innovative microtools viz., Bisection blade and Aspiration pipette. Using bisection blade, oocyte was bisected into two halves and one half having polar body-MII chromosome was instantly discarded by aspiration pipette, resulted into 100% enucleation efficiency. This process did not require Hoechst staining for confirmation of enucleation. The electrofusion process was carried out using indigenously prepared electrodes for individual electrofusion of demicytoplasts-somatic cell triplet, which revealed significantly higher (P≤0.01) fusion rate (92.78±0.5%) as compared to conventional technique (64.61±0.93%) for zona intact embryos. The developmental rates of zona-free embryos were observed as significantly higher (P≤0.01) 2–4 cells (79.13±0.82 vs 29.42±0.55), 8–16 cells (66.68±01.02 vs 23.83±0.9), morula (31.93±1.12 vs 17.93±1.07), blastocysts (24.0±1.56 vs 7.79±0.98) and mean blastomere counts (210.3±2.3 vs 154.0±1.60) as compared to zona-intact embryos.