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http://dx.doi.org/10.14397/jals.2013.47.6.167

Selection and Synchronization of Recipient Cytopalsts for Improving the Efficiency of Porcine Somatic Cell Nuclear Transfer  

Kim, Tae-Suk (Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University)
Park, Sang-Hoon (Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University)
Lee, Mi-Ran (Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University)
Kim, So-Young (Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University)
Eun, Hye-Ju (Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University)
Baek, Sang-Ki (Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University)
Ko, Yeoung-Gyu (Animal Genetic Resources Station, National Institute of Animal Science, Rural Development Administration)
Kim, Sung-Woo (Animal Genetic Resources Station, National Institute of Animal Science, Rural Development Administration)
Sung, Hwan-Hwoo (Animal Genetic Resources Station, National Institute of Animal Science, Rural Development Administration)
Lee, Joon-Hee (Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University)
Publication Information
Journal of agriculture & life science / v.47, no.6, 2013 , pp. 167-181 More about this Journal
Abstract
The development of embryos reconstructed by nuclear transfer (NT) is dependent upon numerous factors. Central to development is the quality and development of the recipient cytoplast. Typically oocytes at either AI/TI or MII are potential candidates used as recipient cytoplasts for NT, because they contain active MPF, which causes NEBD and PCC in the transferred nucleus and may be essential for nuclear reprogramming. In vitro maturation of porcine oocytes generally spend longer times (44-48h), progress asynchronously from immature stage and also present insufficient MPF for NEBD and PCC. Here, we have developed three stages establishment of in vitro porcine oocyte maturation to select good quality of oocytes used as recipient cytoplasts for NT. Firstly, oocytes were assessed by the activity of glucose-6-phosphate dehydrogenase (G6PD) before in vitro oocyte maturation. Positively BCB stained oocytes reached to MII (67.0% vs. 58.4%) and produced more parthenogenetic blastocysts (50.2% vs. 29.6%) as compared with negatively BCB stained oocytes (P < 0.05). Secondly, positively BCB stained oocytes were treated with $5{\mu}g/ml$ CHXM to block meiotic progression for synchronizing the cell cycle of oocytes for 16 hr. All of oocytes (130/130) were efficiently synchronized at GV stage by treatment with CHXM, whereas most of oocytes (w/o CHXM) were passed through GV stage (80/176). In addition, retrieval from treatment of CHXM could reversibly induce meiotic resumption and progresses synchronously in porcine oocytes. However, there was no difference between CHXM treated and non-treated group (21.9% vs. 22.3%) in the developmental rate to the blastocyst stage of parthenogenetic embryos. Following treatment of 5 mM caffeine (a phosphatase inhibitor) for 12 hr, all of oocytes were significantly increased the activity of MPF (P < 0.05). Finally, a piglet was produced from NT embryos reconstructed by using non-caffeine treated recipients.
Keywords
Nuclear transfer; Recipient cytoplasts; Brilliant cresyl blue (BCB); Synchronization of cell cycle; CHXM; Caffeine;
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