Abstract
This was a study on the developmental ability of porcine embryos reconstructed by microinjection of a nucleus of cultured (non-starving culture) cumulus cells directly into the cytoplasm of oocytes matured and enucleated in vitro. Cumulus-oocyte complexes (COCs) derived from slaughterhouse ovaries were matured for 22 h in a modified NCSU23 (mNCSU23) medium supplemented with pFF, cysteine, PMSG, hCG and EGF, and then for another 22 h in the same medium without the hormones. In the meantime, cumulus cells were freed from some COCs and cultured in MEM supplemented with 10% FCS. After 44 h of culture, the smaller cumulus cells (10-12 μm) were injected directly into the cystoplasm of enucleated oocytes. The cell membrane of cumulus cells was broken mechanically prior to the injection. After incubation for 30 min in mNCSU23 supplemented with 4 mg/ml BSA and 0.1 mg/ml cysteine, reconstructed oocytes were activated by exposure to 200 μM thimerosal for 10 min followed by incubation in 8 mM dithiothreitol for 30 min. Oocytes with an intact plasma membrane were then cultured in mNCSU23 supplemented with BSA. The development of reconstructed embryos and the number of nuclei in them were monitored after 48 h and 6 days of culture. As a control group, oocytes were activated parthenogenetically and cultured as described above. The percentage of cleavage in the nuclear transfer (NT) group after 48 h of activation was significantly lower than that in the control group (36.4% vs 83.7%, P<0.05), but the rates of blastocyst were similar in the NT and control groups (5.5% vs 10.9%). The mean numbers of cells in a blastocyst were 19.3 and 30.3 for NT and control groups, respectively. These results indicate that the reconstructed embryos prepared in the system described can develop to the blastocyst stage.
