| Nuclear transfer technique is important not only for the study of nuclear-cytoplasm interaction, but also has extensive applications in human clinics and animal husbandry. In recent years, the technique of nuclear transfer of somatic cell is developing very rapidly. The fibroblast 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 in vitro maturation, the removal of nucleus from matured oocyte, the injection of fibroblast cell into perivitelline space of enucleated oocyte, the fusion of enucleated oocyte-fibroblast cell complexes, the action and culture of reconstructed embryos, etc.Some conclusions were drawn from the experiment as follows:1. The usable cumulus-oocyte (COCs) was collected from slaughtered goat ovaries during the breeding and nonbreeding seasons by surface-follicles cutting. The average total number of COCs recovered per ovary was 4.01(4011/3980) and 0.49(198/405), respectively. There is significant different between the numbers of COCs collected from the two seasons. The results show that more COCs can be recovered by surface-follicles cutting during the breeding season than the nonbreeding seasons.2. To compare effects of different mediums on the maturation rates of oocytes, the COCs were matured in different mediums as follows: 1 M199+lIU/ml hCG+1ug/ml 17β -E2+10mg/ml BSA; II M199+1IU/ml hCG+1ug/ml 17β-E2+10%HGS; III M199+1IU hCG+lug/ml 17 β -E2+1096FBS, the maturation rates of oocytes in group I , group II , group III were 88. 8%, 75% and 27.9%separately, the results indicated that the maturation rate of oocytes in group I was higher than that in group II and group III .3. The diameter of follicles has an effect on the maturation rate of the goat oocyes. The maturation rate of the goat oocyes from the follicleswith diameter 2< <5mm and 6mm< was 88% (142/160) and 3.5% (3/80).4. The cumulus cells have a important effect on the maturation of oocytes that the maturation rates of oocytes having three layers of cumulus, half naked and naked oocyes were 89. 4%(161/180), 46. 7%(28/60) and 0(0/30).5. Usable goat COCs recovered by surface-follicles cutting from breeding seasons and nonbreeding seasons were matured in vitro. The percentage of maturation were 85. 9% (1818/2140) and 35. 5% (71/200), respectively. The results show that COCs recovered from breeding seasons were better than from nonbreeding seasons.6. The system of culturing fibroblast cell was established. Cells of P1-P10 were used as donor cells. The fibroblast cells that were used as donor cells were induced to enter G0 stage of the cell cycle when they were cultured in the medium, the serum of which was decreased from 10% to 0. 5%.7. The enucleation and injection of fibroblast 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.8. Two electric pulses (1.5KV/cm, 40usec each, 0. 5sec interval) were applied. It was the optimal parameter of electrofusion for the oocyte-fibroblast cell complexes.9. When reconstructed embryos were activated by ionomycin for 4min, the percentage of cleavage of reconstructed embryos was 18% (54/301). And the proportion of reconstructed embryos developing to the morula stage was 0(0/54). When reconstructed embryos were activated by ionomycin for 4min and then by 6-DMAP for 5h, the percentage of cleavage of reconstructed embryos was 19. 15% (91/475). And the proportion of reconstructed embryos developing to the morula stage was 8.8% (8/91). When reconstructed embryos were cultured in A23187 for 4min following activated by CHX+CD for Ih, then CHX 4h, the percentage of cleavage of reconstructed embryos were 21. 16%(111/525); And the proportion of reconstructed embryos developing to the morula stage were 6. 3%(7/lll). The percentage of cleavage of goat-goat and goat-...
|
PDF Full Text Request