CB处理和胞质去除对山羊孤雌胚体外发育的影响

为优化山羊核移植方案，实验研究了细胞松弛素B(cytochalasin-B，CB)处理和胞质去除对山羊孤雌胚体外发育的影响。将山羊MⅡ期卵母细胞用7.5 μg/mL CB分别处理5、10、15、20和30 min(实验Ⅰ)，处理后分别去除1/4～1/2的细胞质(实验Ⅱ)，并在每个实验中设对照组(不做任何处理)，孤雌激活后，用碘化丙啶和Hoechst 33258 对囊胚ICM细胞及TE细胞进行双染色，分别记录内细胞团(ICM)和滋养层(TE)细胞数。结果表明, 实验组Ⅰ及对照组均获得了较高的卵裂率(分别为76.83%～85.50%和86.50%)及较高的囊胚期发育率(分别为57.40%～62.20%和59.80%)，囊胚细胞数及ICM细胞数在各组间无统计学差异(P＞0.05)；实验Ⅱ，随着去除胞质比例的1增大孤雌胚的卵裂率(75.76%～16.07%)、囊胚率(38.67%～6.67%)、囊胚细胞总数(107.15～63.67)及ICM百分率(26.32%～19.37%)呈下降趋势，超过1/3时孤雌胚的发育力显著降低(P <0.05)。实验结果指出，(1)7.5 μg/mL CB在30 min以内对山羊孤雌胚体外发育力无不利影响；(2)胞质去除量控制在1/3以下对孤雌胚的发育影响不大。

Effects of Cytochalasin-B and Cytoplasm Removal on In vitro Parthenogenetic Embryos of Goat

To optimize the protocols of nuclear transfer, effects of cytochalasin-B(CB) treatment and cytoplasm removal on parthenogenetic embryos of goat were observed. MetaphaseⅡ(MⅡ)stage goat oocytes were treated with 7.5 μg/mL CB for 5、10、15、20 and 30 min, respectively (groupⅠ), removal of cytoplasm from 1/4 to 1/2(group Ⅱ)and with nothing(control). Differential staining of inner cell mass (ICM) and trophectoderm (TE) cells of blastocysts (Day 5~7) was performed with propidium iodide and Hoechst 33258, and the numbers of ICM and TE cells were recorded respectively. The groupⅠand control showed higher cleavage rate(83%~ 85.50%,86.50%)and blastocyst rate(57.40%~62.20%,59.80%)than that of group Ⅱ, the numbers of total cells and ratio of ICM cells had no significantly difference between various groups(P＞0.05). However, the cleavage rate(75.76%~16.07%), blastocyst rate(38.67%~6.67%), numbers of total cells(107.15~63.67)and the ratio of ICM cells(26.32%~19.37%)decreased with increasing removal proportion of cytoplasm, and embryonic developmental ability significantly decreased when the removal proportion went beyond 1/3. These results suggest that(1)The treatment of 7.5 μg/mL CB for 30 min has no significant effect on oocyte development of goat embryo in vitro;(2)The volume of removal of cytoplasm under 1/3 has no significant effect on development of goat embryo in vitro.