| 1. In this study, Fibroblasts derived from Siberian tiger(Panthera tigris altaica)ear were cultured by tissue culture either with or without over-night cold digestion, and compared the effect of different culture medium, serum concentration, and different concentration of EGF and insulin on the growth of the 7th generation of Siberian tiger fibroblasts(STFCs). The results demonstrated that fibroblasts could be successfully derived from Siberian tiger ear with two tissue culture methods, DMEM/F-12, DMEM/F-12 supplemented with 20% FBS were suitable for STFCs, DMEM/F-12 with 10% FBS supplemented with 1ng/mL EGF, 10ng/mL EGF could significantly improve the growth of STFCs, while 5μg/mL, 50μg/mL INS could not.2. In this study, we cultured Siberian tiger fibroblasts in vitro, analyzed their biological characteristics, chromosomes and cell cycles. The results indicated that Siberian tiger ear fibroblasts can be successfully obtained by tissue culture either with or without overnight cold digestion. The cultured cells were typical fibroblasts with normal morphology, growth curve and chromosome quantity; G0/G1 percentage increased and S percentage decreased with the confluence of cells. Proportions of cells in G0/G1 and S phases were significantly different between 40%-50% , 80%-90%, and 95%-100% confluence; there was no distinct difference between 80%-90% and 95%-100% confluence. Cells at the same density (80%-90% confluence) were treated with or without 0.5% serum, GO/G1 rate of the former was higher than the later, but the difference was not significant. The GO/G1 proportion of 95%-100% confluence was slightly higher than serum starving (80%-90% confluence), but no significant difference was found. Therefore, Siberian tiger fibroblasts cultured in vitro can be used as donor cells for nuclear transfer and treatment with serum starvation is not necessary. By the frequency of G0/G1 cells, serum starvation can be replaced by contact inhibition when the fibroblasts are at least 80% confluent.3. Different oestrus cattle serum(OCS) at different oestrus stage (OCSD0,OCSD3, OCSD5,OCSD7) were added into oocyte culture mediums and the results indicated when the OCSD0 or OCSD3 were added, the rate of oocyte cleavage were significantly higher than the controlled group, when OCSD5 or OCSD7 were added, no significantly difference were seen with the controlled group.The different combination of 30ng/mL EGF,50μg/mL insulin and 50μg/mL insulin+30ng/mL EGF were added into oocyte culture medium correspondingly with 20% serum and the oocyte cleavage rate in 30ng/mL EGF were significantly higher than the controlled group ,which were not significantly higher than the 50μg/mL insulin group and the 50μg/mL insulin+30ng/mL EGF group.0.1μg/mL progesterone were added into oocyte culture mediums which obtained 20% FBS or 20% OCSD0, and the results indicated that the rate of oocyte cleavage were significantly lower than the controlled group. Hemolytic OCSD3 were added into oocyte culture mediums, and the rate of oocyte cleavage were significantly lower than the controlled group.4. Bovine oocytes were actived by ionomycin and 6-DMAP ,the percentage of cleavage and blastocysts were 79% and 20%,this method can be used in nuclear transfer.Rabbit oocytes were separately actived by 7% ethanol,7% ethanol and 6-DMAP, ionomycin and 6-DMAP, their percentage of cleavage are separately 75.6%,76.3%,77.8%; the percentage of blastocysts were separately 18.4%,14.3%,18.6%,the difference was not significant. The three methodsall can effectively active rabbit oocytes.5.The effect of OCS(D0),EGF,insulin,progesterone and OCS(Dn) on the development of the blastocyst were studied and the results indicated that the rate of oocyte cleavage and the blastocyst were not significantly different when added the FBS or OCS ; the rate of oocyte cleavage and the blastocyst were significantly high when added EGF,insulin and progesterone correspondingly, in which the oocyte cleavage rate reached 87.9% when the progesterone were added and the blastocyst rate reached 61.3% when the insulin were added; the rate of oocyte cleavage were no much different when the different period OCS were added, but when added the different period of OCSD0,OCSD3,OCSD5,OCSD7, the blastocyst rate were significantly higher than the controlled group , the blastocyst rate reached 65.8% when the OCSD7 were added.6. In this study, we compared the treatment methods of different donor nucleus and different delayed activation time on the effects of the developmental competence of embryos reconstructed by directly injecting the donor nuclei into enucleated cytoplasts. Siberian TFC treated with different confrequency (40-50%, 80-90%, 95-100%) or serum starvation (0.5% serum for three days) were used as donor nuclei, there was no significant difference of cleavage and blastocyst development rates of the cloned embryos among the four donor cell treatment groups. 40-50% confrequency group got the lowest cleavage rate while 95-100% confrequency group got the highest; the blastocyst developmental rate was lowest in serum starvation group while highest in 95-100% confrequency group. In the same cell confrequency, the cleavage and blastocyst rates were lower in serum starvation group compared to control group, but there was no statistic difference. Next we studied the different time interval (0.5h,1h,2h,3h)between nucleus injection and reconstructed oocyte activation on the effects of cleavage and blastocyst developmental rates of cloned embryos. The results showed that the cleavage rate was increased when the activation time was delayed. Althouh there was no statistic difference between the different time interval on the effects of blastocyst development, an increased blastocyst development current can be observed with prolonging time interval. So we can conclude that delayed activation and prolonging the nucleus-cytoplast activity time in a suitable range can improve the developmental competence of cloned embryos.7. Determined by cell agglutination assay, the titers of 4 week-immuned mouse serum is 1:256. Using Siberian TFC immuned mouse serum as first antibody and fluorescence marked rabbit anti-mouse IgG as the second antibody, The Siberian TFC and BFC are dyed in situ and observed under fluorescence microscope. Results show that the immuned mouse serum is special for Siberian TFC. While using Siberian TFC immuned mouse serum as first antibody and fluorescence marked rabbit anti-mouse IgG as the second antibody, the surface antigene of the reconstructed Siberian tiger embryos in different periods is detected. Results show that the dye is not obvious before morula stage but after morula stage the dye is obviously observed, while there is no obvious dye in bovine matured oocytes. So we can conclude that the genetic materials of the reconstructed embryos derive from donor Siberian TFC and the genes of Siberian TFC are expressed in the cloned embyos.8. Utilized the theory of when the 3'oligas are not paired, the reactivity of PCR cann't be started, we design 3'primer in the loci where there is significant oligas'difference of mitochondria genome between bovine and Siberian tiger. After PCR amplification, the change of mitochondria in bovine-tiger heter-cloned embryos were detected and analysed by Bio-software DNASTAR. The results show that the mitochondria derived from Siberian tiger can not be detected in the bovine-tiger heter-cloned blastocysts.
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