In Vitro Development of Porcine Nuclear Transfer Embryos Reconstructed by Intracytoplasmic Microinjection of Cumulus Cell Nuclei into In Vitro Matured Oocytes

Porcine nuclear transfer embryos were reconstructed by intracytoplasmic nuclear injection of cumulus cell nuclei into recipient oocytes matured in vitro by two types of method (NCSU23-based and TCM199-based methods). Remodelling of the transferred nuclei and in vitro development of the reconstructed embryos were investigated. Intracytoplasmic nuclear injection did not induce activation of the recipient oocytes regardless of the type of maturation method (NCSU23-based: 61/61, 100%, TCM199-based: 49/50, 98%). When the reconstructed embryos were examined 1 h after nuclear injection many of them (NCSU23-based: 22/29, 76%; TCM199-based: 19/23, 83%) possessed condensed chromosomes or a chromosome array resembling the maternal metaphase plate. The proportion of the reconstructed embryos possessing condensed chromosomes arranged in a scattered fashion showed a tendency to increase, when they were examined 2-5 h after nuclear injection. Reconstructed embryos were electrically activated either 2-2.5 h or 3.5-4 h after nuclear injection, followed by fixation/staining to examine formation of pronucleus-like nuclei (pseudo-nuclei). Regardless of the nuclear injection/activation interval tested, 68% (36/53) of the reconstructed embryos developed pseudo-pronuclei ranging in number between 1 and 4. When the reconstructed embryos were cultured for 7 days to assess their developmental competence, 5 (5/98) to 11% (12/111) developed to blastocysts regardless of the type of the recipient oocyte and nuclear injection/activation intervals. These results show that intracytoplasmic injection of porcine cumulus cell nuclei achieves a high rate of nuclear remodelling and that the reconstructed embryos are capable of developing into blastocysts.