Study On Improving Efficiency Of Nuclear Transplantation In Sheep

In this paper,the maturation, cryopreservation and activation of sheep oocytes in vitro were indicated and analysised the effect of nuclear transfer (NT) and embryo culture methods on the developmental efficiency of homogeneous and heterogeneous reconstructed embryos. The studies included several following researches: (1) different additives such as progesterone added in oocyte maturation culture medium were studied the effect of those additives on the maturation rate of sheep oocytes. (2) different concentration of fucose were optimized on the effect of the sheep oocytes cryopreservation, so enough oocytes can be provided for the sheep NT. (3) the effect of intracytoplasmic injection of sperm extraction on the cleavage rate of the sheep oocytes and the developmental rate of the blastocysts were studied and compared with the traditional chemical activation method using ionomycin and 6-DMAP. (4) sheep and goat ears skin fibroblast cells were cultured in vitro and passaged and the effect of different concentration of insulin and EGF on the proliferation velocity of the sheep fibroblast cells were studied. Then these cells were used for NT manipulation, homogeneous and heterogeneous reconstructed embryos were produced. (5) the effect of different culture media on the developmental capability of parthenogenetic embryos, homogeneous or heterogeneous reconstructed embryos were studied . The results as following:1.Four groups with different combined additives ( 5μg/mL FSH+1.0 IU/mL LH+10% OCS; 5μg/mL FSH+1.0 IU/mL LH+10%FBS; 0.075 IU/mL HMG+OCS; 0.075 IU/mL HMG+10%FBS) were dissolved into the oocytes maturation media (OM) .This result indicated that OCS was more favorable than FBS for the sheep oocyte maturation. Addition of 5%～10% BFF was useful for the oocytes maturation. However, 20% BFF inhibited the maturation capacity of sheep oocytes When 0.1μg/mL, 0.25μg/mL, 0.5μg/mL, 1.0μg/mL and 2.0μg/mL progesterone were added in OM media ,the results demonstrated addition of 0.5μg/mL progesterone was the optimized concentration for in vitro maturation of sheep oocytes. Thus the followed compositive culture medium was proposed for sheep oocytes maturation in vitro: TCM199+10 mmol/L Hepes+0.38 mmol/L pyruvate sodium+ 25 mmol/L glutamine+1μg/mL 17β-E2+5μg/mL FSH+1.0 IU/mL LH+10%OCS+0.5μg/mL progesterone. 2. The effect of different cytopreservation media and different developmental stage of oocytes on the developmental rates were compared. The cytopreservation media had no significantly impact on the rate of oocyte cleavage and blastocyst,the results indicated that containing EG and 0.1 mol/L fucose in cyropreservation medium was suitable for freezing sheep oocytes cultured for 24 h;The blastocyst rate of reconstructed embryos using donor cells treated with serum starvation or the control was not different significantly. The cyropreserved oocytes could be used as recipients for NT and was not significantly effect on the developmental abilities of the reconstructed embryos.3. Sheep oocyte with 120 v/mm electric pulse activation for 3 times could get the higher blastocyst rate than the activation rate of 1 times or 2 times (P<0.05). Sperm extraction ( 5 ~6 mg/mL) was injected into sheep cytoplasm to active oocyte , three different doses(3pL,6 pL and 9 pL ) were selected , the results showed the two methods are not different significantly, but both were significantly different with the electric pulse method.4 . DMEM was suitable for sheep and goat cell culture. We cyropreserved sheep and goat cells by adding 10% DMSO after thawing and culturing 5~6 passages, the cells still maintained the same viability as the primary cells, the study used these cells as nuclear donors for sheep NT and sheep-goat inter-specific NT and obtained a high blastcyst rate. EGF and Insulin were added into DMEM medium for sheep cells culture. EGF and Insulin can both significantly improve the proliferation of sheep cells and can significantly cooperate with each other. But the effect of concentration of Insulin on the cell proliferation didn't differ with each other significantly.5. Three groups at different maturation times(15~17 h,19~21 h and 23~25 h) were compared . According to the experiment results, the recommended culture time of oocyte should be 19~21h for the both high rates of maturation and de-nucleation. The blastocyst rate between donor cells treated with serum starvation and the control were no difference significantly.6. Injecting sheep skin fibroblast cells nucleus into oocyte cytoplasm to reconstruct embryos and using Ionomycin and 6-DMAP to active it, the rate of oocytes fusion are higher than the active fusion results. 3-4 h interval between reconstructed and activate oocytes obtained a higher activation rate.7. The goat fibroblast cells were used as nucler donors and the ovine oocytes were used as the cell recipients. The activation methods used intracytoplasmic injection of sperm extraction or Ionomycin combined with 6-DMAP were applied to active the presumptive reconstructed embryos. The blastocyst rate between using donor cells treated with serum starvation and the control as nucler donors were no difference significantly. 8. The result showed that the blastocyst rate was improved when ovine homogeneous and heterogeneous reconstructed embyos were cultured in SOFaa +CR1aa(first cultured in CR1aa +BSA,then in SOFaa subsequentially). 2.0 and 4.0μg/mL progesterone were separately added into embryo culture medium,the percentage of blastocysts was higher than control and others concentration group.Co-cultured systems with oviduct epithelial cell or granular cells can improve the development of blastocysts. Changed co-cultured feed layer could significantly improved the development of ovine homogeneous reconstructed embryos.