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| Improvement of Enucleation Method for Mouse(Mus musculus) Oocytes |
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Abstract In order to find a simple and effective method for enucleation in mouse(Mus musculus) oocytes, present study improved a enucleation method of metaphaseⅡ(MⅡ) oocytes under the condition of Piezo-electricity generation device assisted micromanipulation, and compared the enucleation efficiency and further development of nuclear transfer reconstructed embryo between improved and conventional enucleation method in sucrose-free or sucrose-containing enucleation manipulation solution. In addition, after enucleation by improved method, the further development of reconstructed embryo was compared in manipulation solution containing different serum concentration. The results demonstrated that the rates of visible nuclear of MⅡ oocytes were 98.4%(184/187) and 98.7 % (153/155) in manipulation solution without sucrose and with 3% sucrose, respectively. By adjusting the object lens distance to visualize spindle region of MⅡ oocyte, there was no significant difference (P＞0.05). In enucleation manipulation solution without and with sucrose, by using improved enucleation method, that was enucleation pipette pass through the zona pellucida, aspirated the MⅡ spindle region and pulled out from oocytes, the enucleation rates were 84.6% and 88.2% respectively. The enucleation rates by using conventional enucleation method in these manipulation solutions were 83.1% and 88.6%, respectively, there was no significant differences between them (P＞0.05). However, the oocyte cytoplasm extracted was significant less in improved method than that in conventional method, indicating the advantage of improved method. After enucleated using improved method in manipulation solution without sucrose, the reconstructed embryo developmental rates of 8-cell embryo, morula and blastocyst were the highest among these groups. The developmental rates of morula(16.3%) and blastocyst(5.8%) by using improved method in manipulation solution without sucrose were significantly higher than in manipulation solution with sucrose(5.1% and 0), and conventional method in manipulation solution without or with sucrose(3.3% and 1.2%)(P＜0.05). Moreover, blastocysts were obtained only using improved method in manipulation solution without sucrose. The blastocysts rate in manipulation solution containing 20% fetal bovine serum(FBS) group was significantly higher than 10% FBS group (10.9% vs 4.9%)(P＜0.05). Conclusion was that oocytes enucleated by improved methods in manipulation solution containing 20% FBS was effective condition for supporting the development of reconstructed embryos. Present study made a helpful exploration in improvement of somatic cell nuclear transfer efficiency by improving the nuclear visualization and enucleation methods for mouse oocytes.
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Received: 09 June 2014
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