| Summary: | 碩士 === 國立中興大學 === 畜產學系 === 88 === Nuclear or embryo cloning is a powerful tool for large scale production of identical multiplets containing the same genetic materials from single embryos. The size and cell cycle stages of the nuclear donor might play a critical role on the efficiency of this technology. Series experiments were conducted to investigate the effect of blastomere diameters and cell cycle stages on the developmental competence of nuclear transfer rabbit embryos using the karyoplast derived from 16- or 32-cell stage embryos. All the single blastomere and the reconstructed zygote were cultured in Ham's F-10 medium supplemented with 10% pregnant rabbit serum at 38C and 5% CO2 in air. In experiment 1, the size (diameter) of blastomeres was measured using an image analysis software (Optimas 5.2). The average diameter of blastomeres from 16-cell stage embryos was twice as those from 32-cell stage (51 vs. 27 μm). Large blastomeres (LB, diameter ≧51 or 27μm) and small blastomeres (SB, diameter 51 or 27μm) from 16 and 32-cell embryos were transferred to the enucleated MII oocyte and the in vitro developmental competence of the reconstructed zygote was evaluated. No significant difference in the cleavage rate (≧3 divisions) of single SB and LB derived from either stage embryos was found (52 vs. 50% for 16-cell blastomeres, 28 vs. 17% for 32-cell blastomeres). When 16-cell blastomeres were used as nuclear donors, similar activation (52-61%) and developmental rates (morula/blastocyst stage, 18%) were also observed in both SB and LB transplants. However, cleavage rates (≧3 divisions) were higher in the SB than in the LB transplant groups (73 vs. 55%, P<0.05). For the enucleated oocyte receiving karyoplasts derived from 32-cell embryos, a significantly better cleavage (43 vs. 6%) and developmental rates (14 vs. 0%) were observed in the LB transplant group. In experiment 2, 16- and 32-cell blastomeres were obtained by immediate separation of the embryo divided from previous embryonic stages, i.e., the 8- and the 16-cell embryos, respectively. The separated blastomere for nuclear donors were treated by demicolcine (for 10 hr, DEM), a microtubule polymerization inhibitor, or/and aphidicolin (for 6 hr, APH), a DNA synthesis inhibitor, to synchronize the cell cycle stage of the donor nucleus at the G1 or S phase, respectively. Regardless of the embryonic stage of donor cells, the fusion rate of the transplant was 83% in the control group (without DEM treatment, G1/-DEM) which was significantly higher (p<0.05) than DEM treatment (G1/+DEM) and DEM plus APH (S/+DEM+APH) combined treatment groups (65 and 66%,). Rates of development were also higher in the control group (91%) than in DEM (85%) treated and S/+DEM+APH treated transplants (75%). In conclusion, it appeared that the size of blastomeres did not affect their own development in vitro. When small 16-cell blastomeres were used as nuclear donors, cleavage and developmental rates of the reconstructed zygote were improved. However, the developmental competence was compromised when 32-cell blastomeres, especially the small ones, were used. Furthermore, control of the cell cycle stage of the donor nucleus in the G1 phase was beneficial for their subsequent development of the nuclear transplant rabbit embryo compared to that controlled in the S-phase.
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