Objective: The invisibility of domestic oocyte nucleus in bright field currently forces operators to blindly aspirate nucleus out in oocyte enucleation, usually causing large cytoplasm losses and poor developmental competences of cloned embryos. Although fluorescent labeling of nucleus allows for nucleus localization, the involved photobleaching problems and barriers to the execution of enucleation process limit its online-application in oocyte enucleation. This paper reports a novel label-free oocyte enucleation method for precise removal of the nucleus with less cytoplasm loss.
Methods: The relative positions between the injection pipette and nucleus for complete removal of nucleus with less cytoplasm loss were determined through a finite element modeling of nucleus aspiration. To position injection pipette to the above positions relative to nucleus, the appropriate oocyte orientation and trajectory of injection pipette inside oocyte were derived according to the offline-calibrated 3-D distribution of nucleus and the simulated dynamic drift of nucleus that occurs as injection pipette is maneuvered inside oocyte. Finally, a robotic label-free precise enucleation procedure was established.
Results: The experimental results on more than 1000 porcine oocytes proved that this system is capable of reducing cytoplasm loss by 60% at the same level of enucleation success rate and almost doubling the cleavage rate of clone embryos in comparison to blind aspiration method.
Conclusions: The results prove that our method significantly improves the developmental competence of cloned embryos in comparison to manual enucleation method.
Significance: Our method is expected to improve the extremely low success rate of animal cloning in the future.