| Experment 1.It is important the nuclear transfer process that oocyte mature.This experiment was desgined:①w e added different diameter size bovine liquor folliculi(BFF)to mature fluid in vitro.10% FBS mature fluid was for control group.Our test contained the diameter > 8 mm 10% cow liquor folliculi (BFF), 2～8 mm 10% BFF and 5% > 8 mm BFF + 2～8 mm 5% BFF.After mature 18h removes the ovocyte the granular cell and compared t mature rate and activated oocytes the rate of blastocyst rate.The result indicated that, Between the control group and the experimental group does not have the remarkable difference (P>0.05)in the maturing rate.Blastocyst rate(43.40%)with > 8 mm 10% BFF is remarkable higher than control group. Mix group blastocyst rate is also remarkably higher than control group.So BFF can effectively enhance the development rate of bovine parthenogentic blastocyst.②Effect of BFF on reconstructed embryos.Tests①research to discover.Because mucoitin with big BFF does not favor to carry on the reconstructed embryos operation.Therefore reduced > 8mm BFF. Take 10% FBS as control group, the experimental group is 5% BFF (2～8 mm) + 5% FBS, 5% (> 8 mm 2.5% +2～8 mm 2.5%,Mix) + 5%FBS and 10% BFF (2～8 mm) respectively.We used matured oocytes to reconstruct embryos after mature 18h. 5% (> 8 mm 2.5% + 2～8 mm 2.5%,The mix) + 5% FBS pouch embryo rate (26.56%) and other groups are more remarkable than the difference. The development rate of reconstructed embryos in 5% (>8 mm 2.5% + 2～8 mm 2.5%,mix) group and 5% FBS group is higher significantly than others group is treated (p<0.05).The BFF compares FBS to be able to enhance The development of bovine parthenogentic blastocyst and reconstructed embryos.Experment 2.First time has conducted the preliminary research using the immunity histochemistry method to the bovine oocytes mature processβ-tubulin change process. The result discovered using Nikon Corporation TE 300,oocytes matured for0, 6h cannot see beta theβ-tubulin distribution, 12h has the weak fluorescence, 14hβ-tubulin revolves around the chromatin, fluorescence relative weak, the 16h fluorescence is strong, explained the bovine ovocyte bursts in 14～16h GVBD and discharges the first polarbody. As the negative comparison, the ovocyte has not been able to see the fluorescence.Experment 3.Enucleation of the oocyte is one of the key steps in NT.We researched whether sucrose and demecolcine (DM) treatment is applicable.①Working concentrations of sucrose 0-10%s (v/v) the sucrose incubated 1h.Three oocytes was revealed in seventy-four only with 3% group.②Matured oocytes were activated 5 min with ionomycin (Ion). Adding 0, 0.4, 0.5, 0.6μg.mL-1DM in the media SOFaa in 1.5 h. The oocytes were induced enucleation by DM.③Activated with Ion for 5 min and incubated 0.5,1,1.5,2h respectively to induce enucleation .The same we didn't find induced enucleation by DM the phenomenon.④Matured oocytes were put in DM with 0.4, 0.5, 0.6 mg.mL-1 for 2h.we found that the majority of the treated bovine oocytes formed a small transparent bud into the perivitelline space.Media with 0.5μg.mL-1 is remarkably higher than other two groups (P <0.05), but has not revealed nucleus in control group.⑤Put matured oocytes in DM for incubated 0.5h, 1h, 2h respectively. It is the highest with 2h (76.54%)than 0.5h and 1h group(P <0.05).⑥The revealed enucleation by different matured time of bovine oocytes were investigated.Adding 0.5μg.mL-1DM in the media incubated 2h and 18h matured oocyted is remarkable higher than 14h and 16h group (P <0.01), compareed not the remarkable difference with the 20h mature group.⑦Adding DM in matured culture media during the mature process was investigated. Granular cell was removed 2h later. We found that increased DM in mature 18h was able to enhance it to revealed enucleation efficiency compared with other groups and enhance the development rate of bovine blastocyst.The development rate of bovine fibroblast reconstructed embryos in 18h group and 16h group is higher significantly than 14h group.The result indicated that using the sucrose cannot achieve the ideal revealed nucleus the effect. At the same time DMcan not induces enucleation the goal. The first time we found DM can be used to revealed enucleation.Experment 4.The bovine reconstructed embryos activated by Ion + 6-DMAP and ethyl alcohol + 6-DMAP procedures were investigated. The result indicated that in Ion + 6-DMAP group, the cleavage rate of oocytes in ion activated for 5min was significantly higher than those in other treatments and the rate of blastocyst was significantly higher than others groups(p﹤0.05); In the ethyl alcohol + 6-DMAP group, reconstructed embryos by activated in the 5min is obvious, 5min group compared with 3min, 7min and 10min,the cleavage rate of 5min group is remarkable higher than others groups(p﹤0.05). the cleavage rate reaches 56.53%, the rate of blastocyst is 8.51%. These results indicate that bovine reconstructed embryos can be activated effectively by Ion + 6-DMAP and ethyl alcohol + 6-DMAP, but ion + the 6-DMAP group is better than ethyl alcohol + 6-DMAP.Experment 5.Effect of the donor cell on development of reconstructed embryos.①Donor cell by serum starvation with 0.5% is hungry 0d, 1～3d, 4～6d, 7～9d respectively. the cleavage of ratereconstructed embryos is not differentnot (P>O. 05).The rate of blastocyst is better in 1～3d group.②Cells passaged for 1-3 generations were used for nucear transfer, morula rate is 32.06%,significantly higher than that of 0,4-6,7-9 generation groups(p﹤0.05).③a fter thawing,cells were incubated for 24h,starvated for 1-3d, morula rate of reconstructed embryo has no difference among 2,4 and 6 generation after thawing,but nucear transfer effect is better than 8 generation after thawing (p﹤0.05).Experment 6.To identify the reconstructed embryos by miemsatellite DNA.By sequence amplification of microsatellite DNA,the reconstructed embryos is completely consistent donor cell 11 STR the position spot PAGE genotype. |
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