| At present, SCNT have still many severe problems: the low efficiency, high rate of abortion, abnormal development of cloned animal and so on. However, maybe these are due to somatic cell differentiation, the treatment methods of synchronization and the incomplete reprogramming of nucelus of reconstructed embryo. The reprogramming of nucleus of reconstructed embryos was divided into two procedures: its' morphological remodeling and epigenetic modification. Remodeling was controlled by MPF, and had directly influence on its correct reprogramming, embryos quality and the helth of cloned animals. The reprogramming is a process of many genetic modifications. At present, there is no a definite biochemical standard for evaluating genomic reprogramming. This experiment study the nucleus remodeling and development of reconstructed embryos, by prolonging the interacted time between its nucleus and cytoplasm, adding caffeine into NCSU-23, carrying out subculture and synchronization of granulosa cell, and in purpose for improving the reprogramming and efficiency of in vitro production of embryos. This study had been divided into five experiments, its results as follow:Experiment 1: Effect of time of activation-delayed on development of NT embryos. After culture for 0,1.5,3,4.5h, NT embryos were activated by one pulse of the electric field intensity of 1.3 KV·cm-1 and the duration of 80μs, whose development was observed. The purpose of this experiment was to study influence of prolonging the time that MPF acted on nucleus on development of constructed embryos. The experimental result showed that as the cleavage rate of NT embryos, the rate of 4-8 cell embryos and more than 8-cell embryos was concerned, the Oh group was significantly lower than the 1.5h,3h and 4.5h group(P＜0.05), and there were no significant differences among the 1.5h,3h and 4.5h group(P＞0.05), but the 3h group was better than the 1.5h and 4.5h group. Therefore, activation-delayed can promote the development of NT embryos, but the best developmental effect of NT embryos was at 3h. After culturing for 3h, reconstructed embryos were activated after culture for 3h, and its developmental effect was best.Experiment 2: The effect of caffeine on morphological remodeling of nucelus and development of reconstructed embryos. This experiment included three aspects:(1) Observation of the appearance of PCC of reconstructed embryos. After culture in NCSU-23 with or without 2.5mM caffeine for 0h,1.5h,3h和4.5h, reconstructed embryos were fixed for 48h in 25%(v/v) acetic acid in ethanol at room temperature and stained with 1%(w/v) orcein in 45%(v/v) acetic acid. The experimental result showed that the change of nucelus of reconstructed embryos occurred: swelling, nuclear envelope breakdown(NEBD) and premature chromosome condense (PCC) by turn, known as morphological remodeling of nucleus.(2) The effect of caffeine morphological remodeling of nucleus of reconstructed embryos. After culture in NCSU-23 with or without 2.5mM caffeine for 0h,1.5h＞3h和4.5h, NT embryos were stained (the method was the same as (1)), and NEBD and PCC was observed. The experimental result showed that there was no the appearance of NEBD at Oh, and the appearance rate of approximately 11% from 1.5h to 4.5h in group without caffeine, and the appearance rate of NEBD step quickly up from Oh to 1.5h, and dropped quickly out from 1.5h to 4.5h in group with caffeine; the appearance rate of PCC went up from Oh to 3h, and decreased from 3h to 4.5h in two groups, but the appearance rate of PCC of the group with caffeine was significantly higher than the one without caffeine (P＜0.05). The analyses indicated that the peak of remodeling of nucleus is at 3h after culture, but the appearance rate of PCC of NT embryos cultured in NCSU-23 with caffeine was higher than without caffeine(57.14%vs37.93%). Therefore, caffeine can promote remodeling of nucleus obviously.(3) Effect of NCSU-23 with or without caffeine on development of NT embryos. NT embryos were cultured in NCSU-23 with or without 2.5mM caffeine, and the rate of cleavage, 4-8cell and more than 8-cell embryos was observed and evaluated. The experimental result showed that the cleavage rate was 46.77% (29/62), 39.13% (27/69), respectively. There were no significant differences between two groups (P＞0.05). The rate of 4-8cell embryos was 30.65% (19/62), 15.94% (11/69), respectively. There were significant differences between two groups (P＜0.05). The rate of more than 8-cell embryos was 12.90% (8/62), 5.8% (4/69), respectively. There were significant differences between two groups (P＜0.05). Therefore, caffeine can't improve the cleavage rate of NT embryos, but it can promote development of NT embryos.Experiment 3: The development of NT embryos with primary granulosa cell was compared with the one with serial subculture. According to somatic cell, this experiment was divided into three groups: primary culture, serial subculture 4 times and 7 times. After SCNT, the development of NT embryos was assessed by the rate of fusion of 'enucleated oocyte-granulosa cell' couplets, cleavage, 4-cell, and more than 8-cell embryos. The purpose of this experiment was to study the influence of serial culture of granulosa cell on the fusion of couplet and developmental potential of NT embryos. The experimental result showed that the fused rate was 59.84% (73/122), 61.54% (72/117), 57.14% (68/119), respectively. There were no significant differences among three groups (P＞0.05). The cleavage rate was 45.12% (33/73), 41.67% (30/72), 45.59% (31/68) respectively. There were no significant differences among them (P＞0.05). The rate of 4-8cell embryos was 32.88% (24/73), 36.10% (26/72), 32.35% (21/68) respectively. There were no significant differences among them (P＞0.05). The rate of more than 8-cell embryos was. 13.70% (10/73), 11.11% (8/72), 11.76% (8/68) respectively. There were no significant differences among them (P＞0.05). Therefore, there is no effect of serial subculture of porcine granulosa cell on the fused rate of 'enucleated oocyte-granulosa cell' couplet and developmental potential of NT embryos.Experiment 4: Effect of the treatment methods of synchronization of granulosa cells G0/G1phase on development of NT embryos. According to the treatment methods, this experiment was divided into three groups: 80% confluence, high confluence (4 days) and serm starvation (3 days). After SCNT, the development of NT embryos was evaluated by the rate of fusion of 'enucleated oocyte-granulosa cell' couplets, cleavage, 4-cell, and more than 8-cell embryos. The purpose of this experiment was to study the influence of methods of synchronization of granulosa cells G0/G1phase on the fusion of 'enucleated oocyte-granulosa cell' couplet and developmental potential of NT embryos. The experimental result showed that the fused rate was 56.91% (70/123), 58.46% (76/130), 55.37% (57/121), respectively. There were no significant differences among three groups (P＞0.05). The cleavage rate was 44.29% (31/70), 46.05% (35/76), 42.10% (24/57) respectively. There were no significant differences among them (P＞0.05). The rate of 4-8cell embryos was 32.86% (23/70), 31.58% (24/76), 35.09% (20/57), respectively. There were no significant differences among them (P＞0.05). The rate of more than 8-cell embryos was 10.00% (7/70), 13.16% (10/76), 10.52% (6/57) respectively. There were no significant differences among them (P＞0.05). Therefore, there is no effect of the treatment methods of synchronization of granulosa cells G0/G1phase on the fusion of 'enucleated oocyte-granulosa cell' couplets and the development of NT embryos.In conclusion, after microinjection of porcine granulosa cell, delaying the time of activation can promote the development of NT embryos, but 3h is the best. After NT, reconstructed embryos were cultured in NCSU-23 with or without 2.5mM caffeine, and stained with with 1%(w/v) orcein in 45%(v/v) acetic acid. The changes of nucleus were observed: swelling, NEBD, and PCC occurred by turn, and the peak of PCC is at 3h after culture of reconstructed embryos. But caffeine improved the remodeling of nucleus and the development of NT embryos. The peak of PCC was the same as the best time of activation-delayed (3h), showing that after NT, the intact remodeling of nucleus was benefited to the development of NT embryos. However, there were no effect of serial culture and the methods of synchronization of G0/G1 phase on the fusion of 'enucleated oocytes', the remodeling and the development of NT embryos. Therefore, when the remodeling of nucleus and the development of NT embryos was regarded as standard of the reprogramming, caffeine can promote the reprogramming of nucleus of NT embryos, but there were no effect of serial culture and synchronization of G0/G1 phase on the reprogramming.
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