| Hand made cloning is a new nuclear transfer method which develops in these years. The whole process doesn't need micromanipulation, needs low technology, saves running time, and cuts down cost, so this method becomes more and more welcome. Because the embryos are removaled zona, blastomeres are ease to separate with each other before pyknosis, so embryos culturing is the top-drawer step in all the process. In this study, glass needle was used to make WOW at the bottom of the four-well dish, and the zona-free embryos were cultured in the WOW.Experiment 1: The bovine zona-free parthenogenic activated embryos were cultured in the WOW in order to investigate the WOW made in this method whether affected the embryos development or not. The zona were removalled in 3 states: The zona being digested before putting in ionomycin , the zona being digested before putting in 6-DMAP and the zona being digested before putting in SOFaa. The zona were removalled in different states can check the zona-free embryos sensitive challenge to the ionomycin and 6-DMAP. Culturing embryos with different numbers in each group (5,15,30) in the WOWs in order to check the different on the embryos development. The results showed that cleavage rate of the three groups (81.1%,78.9% and 78.9%) had no significant differences between zona-intact and control (P>0.05).The blastocyst rate of the group of zona being digested before putting in 6-DMAP (31.5%) had no significant differences among the group of zona being digested before putting in SOFaa (P>0.05), and the other two groups and control (P > 0.05). The groups of zona being digested before putting in ionomycin didn't develop to blastocyst. The number of cells had no significant difference between the group of zona being digested before putting in 6-DMAP and the group of zona being digested before putting in SOFaa, and had no significant difference compared to control (P>0.05). There is no differences on the development of embryos culturing embryos with different numbers in each group (5,15,30). Conclusion: the WOW made in this method suited culture zona-free embryos; Using the ionomycin and 6-DMAP to activate zona-free oocytes, it needed cut down the ionomycin's density and(or) action time, and there were no influence to zona-free embryos with 2 mmol/L6-DMAP; WOW culture method could culture a few embryos, and elevate the embryos'development. Experiment 2: Collecting 853 oocytes, and calculating the maturing rate. The donor cells were digested different degree(the surface were slick and sentus), and the reconstructed embryos were cultured in microdrop to verify whether different in fusion rate , cleavage rate, blastocyst rate and No. of blastocyst cells or not. The reconstructed embryos were cultured in WOW, and every 15 embryos were cultured in one gab of the four-orifice, and statistic the cleavage rate, blastocyst rate and No. of blastocyst cells. Result: the maturing rate were low(51.93%); The cells which the surface were sentus were used to donor cells, the fusion rate of reconstructed embryos(79.9% ) had difference with the ones(64.2%) witch the surface of donor cells were slick(P<0.05), and there were no different in cleavage rate, blastocyst rate and No. of blastocyst cells with each other(P>0.05). Using WOW culturing reconstructed embryos, the blastocyst rate were higher compared with the ones cultured in microdrop, and had differences(P<0.05). There were no difference in cleavage rate and No. of blastocyst cells. Conclusion: when choosing the donor cells, the cells which the surface were sentus and the diameter were about 25μm were better and it could elevate the fusion rate; Using WOW culture method could elevate the blastocyst rate of reconstructed embryos.
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