| Comparing with electrical activation, intracytoplasmic direct injection was a better choice in bovine somatic cell nuclear transfer (SCNT), because it is easy, fast, and do not need expensive electrofusion apparatus, which may hinder its large-scale application. Somatic cell cloning is a complex and easier technology. In this research, we studied effects of ovary storage, enucleation methods and embryo culture medium on the preimplantation development of clone bovine embryos, to optimize the procedures of nuclear transfer. Evaluation of embryo quality is very important, both in the sense of clinical or theoretical. In this study, we established methods of karyotyping and differential staining of bovine preimplantation embryos, which derived from the methods reported previously and from the actual situation in our laboratory. The main results were as follows:1. Condition of ovary preservation on development of preimplantation SCNT bovine embryosThe experiment includes two parts, the first part focus on ovarian storage length. Mainly set up three groups (2h,9h,21h), with 2h group as the control, they all storaged at 20℃in PBS. Compared with the control,9h group did not decrease the maturation rate (64.55% vs. 70.41%, P>0.05), cleavage rate (65.92% vs. 66.11%, P>0.05) and rate of blastocyst (34.09% vs. 37.20%, P>0.05). When ovarian preservation time reached 21h, the maturation rate (42.47% vs. 70.41%, P<0.05), the cleavage rate (40.44% vs. 66.11%, P<0.05) and blastocyst rate (13.60% vs. 37.20%, P<0.05) were significantly lower than the control group and the 9h group. The results showed that too long period of ovarian storage will affect bovine oocyte maturation and early development of SCNT embryos, while not too long preservation time (9h) will not affect the early embryo formation rate.The second part designed three test groups of different storage temperature (4℃, 20℃,37℃) and a control group, they all storaged 9h in PBS. Compared with the control group,37℃save significantly reduced oocyte maturation rate (47.36% vs. 70.41%, P <0.05), while 4℃and 20℃did not (55.30% vs. 70.41%,64.55% vs. 70.41%, P>0.05). In the cleavage rate and blastocyst rate,20℃group was significantly higher than 4℃group (65.92% vs. 46.95%,34.09% vs. 25.78%, P<0.05),4℃group was significantly higher than 37℃group (46.95% vs. 24.46%,27.58% vs. 15.56%, P<0.05), both 4℃and 37℃group were significantly different from control group. The results shows that too high or too low are not suitable for ovary preservation, and at room temperature or slightly below room temperature is more suitable for the preservation of ovarian.1. Effects of enucleation method on development of preimplantation SCNT bovine embryosEnucleation method had significant effect on early development of SCNT bovine embryos, fluorescence assisted enucleation method can significantly improve the embryos reconstructed rate (83.35% vs. 59.09%, P<0.05); UV irradiation 10s or more, significantly reduced embryo cleavage rate and blastocyst rate, but 5s following irradiation group compared with the blind group, their cleavage rate (62.44% vs. 66.93%, P>0.05) and blastocyst rate (30.63% vs.33.57%, P> 0.05) had no difference. In conclusion, the use of fluorescent light under 5s assisted enucleation method was better for this experiment, because this method not only simplifies the operation, but also improve the utilization of oocytes.3. Embryo culture medium on development of preimplantation SCNT embryosRandomly selected embryos were cultured with KSOM or SOF, we found in KSOM group the blastocyst rate was significantly higher than SOF group (38.24% vs. 30.63%, P <0.05). The results showed KSOM is a more suitable medium for early development of SCNT embryos. However, there was no significant difference between the cleavage rate of two medium (64.21% vs. 62.44%, P> 0.05).4. Karyotyping of SCNT bovine embryosIn this study, we compared the chromosome number of embryos derived from blind and fluorescence assisted enucleation method. The proportion of abnormal chromosomes (72.33%) of UV radiation over 10s group was significantly higher than the other two groups (39.17% and 42.50%), while the other two groups were not significantly different. By analysising the overall chromosome mutation of SCNT embryos, we found that among the abnormal chromosome, mixoploidy (17.95%) and polyploidy (12.82%) were often more than haploid (7.69%). This protocol came from the traditional methods of classical genetics, with certain modifications, we successfully established the karyotyping methods of clone embryo in our laboratory.5. Differential staining of cloned bovine embryosEmbryos that completed the activation were randomly divided into two groups and cultured independly with mSOF and KSOM, then to do differential staining. Trophoblast cells (59.87 vs. 74.83, P<0.01) and total cell number (81.59 vs. 100.16, P<0.01) were significantly different, but there has no significant difference between the inner cell mass (P>0.05). We based on the trial recently reported, with certain modifications, we initially established the differential staining method in the laboratory.
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