| The technique of nuclear transfer is of great significance to scientific study. In recent years, the technique of nuclear transfer of somatic cell is developing very rapidly. The oviducal epithelial cell from goats was used for nuclear transfer and a relevant procedure of nuclear transfer was made in this experiment, the procedure includes the production of holding and injection pipette, the culture of oocytes for in vitro maturation, the removal of nucleus from matured oocyte, the injection of oviducal epithelial cell into perivitelline space of enucleated oocyte, the fusion of enucleated oocyte-oviducal epithelial cell complexes, the activation and culture of reconstructed embryos, etc.In this experiment, ovaries of goats were collected in slaughterhouse, cumulus-oocyte complexes (COCs)were recovered from the ovaries, the COCs were transferred into the drop of maturation medium in Petri dish overlaid with paraffin oil, and they were cultured at 38 C in a 5% CO2 in air atmosphere. To compare effects of different mediums on the maturation rates of oocytes, the cumulus-oocyte complexes were matured in different mediums as follows: I M199+10%EGS+liu/ml HCG+lug/ml 17- 0 -E2; II M199+10%EGS+liu/ml HCG; III M199+10%FCS+liu/ml HCG+lug/ml 17- -E2, the maturation rates of oocytes in group I , group II and group HI were 80.85?(^ 51.47%$) 63.86% separately, the results indicated that the maturation rate of oocytes in group I was higher than that in group II(p<0.01) and group III (p<0.05).After the maturation of oocytes, the enucleation and injection of oviducal epithelial cell into perivitelline space were operated in the Petri dish using the point-beveled pipette. After the enucleation, oocytes were stained with aceto-orcein, the enucleation rate was 74.42%.The oviducal epithelial cells that were used as donor cells were induced to enter GO stage of the cell cycle when they were cultured in the medium, the serum of which was decreased from 10% to 0.5%.The enucleated oocyte-oviducal epithelial cell complexes were fused with different DC pulse, the distance between the two electrodes was three times the diameter of oocytes. When the pulse voltage was 30V 40V, 50V and 60V , the fusion rates were 29.17%, 44.83%, 50.79% and 46.3% separately. When the pulse width was 40us, 50us and 60us, the fusion rates were 30.23%, 48.48% + 43.55% separately. The resultsindicated that there was no obvious difference among the fusion rates when the pulse voltage was 40 V ,50 V and 60 V ,or the pulse width was 50us and 60us , but these rates were higher than the fusion rate when the pulse voltage was 30V(p<0.05) or the pulse width was 40us(p<0.05).The reconstructed embryos were activated by ionomycin or ionomycin+6-DMAP, the elevation rates were 20.45% and 35.85% separately, the results indicated that the elevation rate of reconstructed embryos in the later group was higher than that in the former group, but they were both higher than that in the control group obviously.The reconstructed embryos were cultured in different culture systems as follows: I M199+15%FCS; II M199+15%FCS+GOE Feeder. The development rates of 4-cell stage embryos in group I and II were 23.81% and 28.3% separately, the development rates of morulas were 0% and 9.43% separately, the results indicated that there was no obvious difference between the development rates of 4-cell stage embryos in group I and II ,but the development rate of morulas in group II was higher than that in group I (p<0.05).Some conclusions were drawn from the experiment as follows :(1) If EGS and 17-3 -E2 were added to the medium, the maturation rate of oocytes can be improved.(2) The enucleation and injection of oviducal epithelial cell into perivitelline space were operated in the culture dish using the point-belved pipette, which would make the time for operation decreased and be helpful to the development of the embryos.(3) The fusion rate was influenced by DC pulse voltage and pulse width. When the distance between the two electrodes was three...
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