Recently, we have reported the restored developmental ability of porcine oocytes denuded at 24 h (DO24s) by fusion with ooplasmic fragments produced from cumulus oocyte complexes (COCs) by "centri-fusion" method (The 103rd SRD meeting). In the present study, further experiments were carried out in order to realize different contribution of brownish- and transparent-fragments (B and T, respectively). The B-fragments have more mitochondria than the T-ones. Both fragments without chromosomes (cytoplasts) were produced from matured COCs by serial centrifugation and selected. The DO24s were fused with 2 cytoplasts by electro-fusion according to the following design; Groups of (1) DO24s (control), (2) 2B-fragment-fused, (3) 2T-fragment-fused and (4) 1B+1T-fragment-fused DO24s. The fused oocytes were fertilized in vitro and subsequently cultured for 6 days. Sperm penetration and female pronucleus formation rates were significantly higher in Groups 2 and 4 than those of control group; however, those in Group 3 were not different (P<0.05) from those in the other groups. Male pronucleus (MPN) formation, normal fertilization, and blastocyst rates in Groups 2-4 were significantly higher (P<0.05) than those in control group. Blastocyst cell number was not different between experimental groups. In conclusion, B- and T-cytoplasts equally contribute to MPN formation and developmental ability of DO24s to the blastocyst stage. B-cytoplasts might contribute much to sperm penetration than T-cytoplasts.
Supported by Monbukagakusho scholarships.
