| Although the Mammals Cloning by somatic cell nuclear transfer (SCNT) has become a common technology in recent years, the efficiency of nuclear transfer is low and has been estimated to be less than 5% of oocytes used . Moreover, nuclear transfer is a complex procedure that requires considerable technical skills. The procedures of enucleation and donor nucleus injection mainly depend on micromanipulation, which is technically demanding, time-consuming, inherently invasive, and clearly damaging to cytoplast spatial organization. These factors hampered the widespread application of traditional nuclear transfer (TNT). As an alternative to tranditional mechanical enucleation procedure, the development of induced enucleation (IE) might provide us a possibility to produce bulks of enucleated oocytes with less time and work intense, as well as a minimal decrease in the oocyte cytoplasmic volume and protein/molecular content. In induced enucleation nuclear transfer (INT) procedure, cytoplasts are activated previously and usually in TⅡphase when used as donor nuclei recipients. While MⅡcytoplasts and delayed activation of MⅡcytoplasts are thought to be important for the cloning of mice and other species by allowing an increased opportunity for restore or recruitment of some chromatin-associated factors which are important for nuclei reprogramming. This study combined the demecolcine-induced enucleation with reverse-order nuclear transfer (RNT) protocol to optimize the procedure of induced enucleation reverse-order nuclear transfer (IRNT). So this study did some experiments using ICR mouse oocytes:1. To optimize the enucleation protocols of mouse oocytes, the study examined the effects of the enucleation methods of abradeding the zona pellucida (AZP) or sticking the zona pellucida (SZP) straightly, sucrose treatment, concentration of cytochalasin B (CB) and inner diameter of pipettes on enucleation efficiency,and the effects of the concentration of CB on parthenogenetic embryos removed partial cytoplasm from 1/12 to 1/8. The results showed that (1) there were no significant differences between supplemented 3% sucrose group and control group on manipulating time and enucleation rate achieved by AZP or SZP (P>0.05); (2) 5, 10 and 20μg/mL CB treatments had no significant effects on manipulating time (P>0.05). AZP had no significant different enucleation rates among 5, 10 and 20μg/mL CB groups(P>0.05); however, in SZP, the enucleation rates of 5 and 10μg/mL CB treatments were significantly higher than that of 20μg/mL (P<0.05); (3) the cleavage rate of parthenogenetic embryos had no significant difference between various groups (P>0.05). Blastocyst rate of 20μg/mL CB group was significantly lower than that of other groups (P<0.05); (4) the enucleation rates of 10, 15 and 20μm inner diameter of pipettes were no significant different in AZP (P>0.05); the enucleation rates of 10 and 15μm inner diameter of pipettes were higher than that of 20μm in SZP (P<0.05). The enucleation rates achieved by AZP was significantly higher than that achieved by SZP (P<0.05). The results indicate that the optimized protocol combining AZP, 10μg/mL CB with 15μm inner diameter of pipettes significantly improves the efficiency of enucleation, and can be used for nuclear transfer on mouse.2. To promote the application of induced enucleation (IE) on somatic cell nuclear transfer (SCNT), the study examined the effects of two activating methods and oocytes age on IE rate and second PB2 rate by demecolcine-induced enucleation using ICR mouse oocytes . The results showed that: (1) activated by 7% ethanol or 10mM SrCl2, the IE rate of oocytes collected at 20～21 h after hCG administration was very significantly higher than other groups(P<0.01), and there were no significant differences between ethanol groups and SrCl2 groups on IE rate of oocytes collected at the same time (P>0.05); (2) the second PB2 rate of oocytes activated by 10mM SrCl2 had no significant difference at different time after hCG administration (P>0.05), however, activated by 7 % ethanol , the maximum second PB2 rate (88.7%) were obtained when oocytes were collected at 20～21 h after hCG administration, and there were no significant differences between ethanol groups and SrCl2 groups on the second PB2 rate of oocytes collected at the same time (P>0.05). The results indicate that both 7% ethanol and 10mM SrCl2 can be used for demecolcine-induced enucleation on ICR mouse, and oocytes collected from ICR mouse at 20～21 h after hCG administration were favoriate to demecolcine-induced enucleation (ethanol, 62.6%; SrCl2 ,56.8%).3. Experiments optimized the timing and the duration of DC treatment in IRNT, examined the effects of IRNT, INT and TNT on the developmental potential of NT embryos. The results showed that: demecolcine treatment begun immediately after electrical pulse or after activation by ethanol had no significant effects on the efficiency of IRNT (P>0.05); the efficiency of IRNT was significantly improved by treatment 60 min after activation by ethanol compared to treatment 40 min (P<0.05); the cleavage rates had no significant difference between IRNT, INT and TNT (P>0.05), but the blastocyst rate was significantly improved in IRNT compared to INT and TNT (P<0.01); the reconstructed rate of NT embryos differ very significantly between IRNT, INT and TNT (P<0.01); In summary, the present results suggest that the RNT protocol can significantly improve the developmental potential of NT embryos derived from DC-induced enucleated cytoplasts. IRNT protocol is an alternative to traditional NT protocol.
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