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KMID : 0900920020260030245
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Korean journal of Animal Reproduction
2002 Volume.26 No. 3 p.245 ~ p.252
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In vitro Development of Somatic Cell Nuclear Transferred Bovine Embryos Following Activation Timing in Enucleated and Cryopreserved MII Oocytes
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Kim Eun-Young
Lee Young-Jae
Park Se-Young
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Abstract
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This study was to evaluate the in vitro survival of bovine enucleated MII (eMII) oocytes according to minimum volume cooling (MVC) freezing method and activation timing, and their in vitro development after somatic cell nuclear transfer (SONT). in vitro matured bovine oocytes for 20 h were stained with 5mutextrm{g}/mell Hoechst, and their 1st polar body and MII plate were removed by enucleation micropipette under UV filter. Also, eMII oocytes were subjected to activation after (group II) and before (group III) vitrification in 5 {mu}{textrm}{m} ionomycin added CRlaa medium for 5 min. For vitrification, eMll oocytes were pretreated with EG10 for 5 min, exposed to EG30 for 30 sec and then directly plunged into LN_2. Thawing was taken by 4-step procedures at 37^{circ}C. Survived eMII oocytes were subjected to SONT with cultured adult bovine ear cells. Reconstructed oocytes were cultured in 10 mutextrm{g}/mell of cycloheximide and 2.5 mutextrm{g}/mell of cytochalasin D added CRlaa medium for 1 h, and then in 10 mutextrm{g}/mell of cycloheximide added CRlaa medium for 4 h. Subsequently, the reconstructed oocytes were incubated for 2 days and cleaved embryos were further cultured on cumulus-cell monolayer drop in CRlaa medium for 6 days. Survival rates of bovine vitrified-thawed eMII oocytes in group II (activation after vitrification and thawing) and III (activation before vitrification) were 81.0% and 84.9%, respectively. Fusion rates of cytoplasts and oocytes in group II and III were 69.0% and 70.0%, respectively, and their results were not different with non-frozen NT group (control, 75.2%). Although their cleaved rates (53.4% and 58.4%) were not different, cytoplasmic fragment rate in group II (32.8%) was significantly higher than that in group III (15.6%)(P<0.05). Also, subsequent development rate into >morula in group II (8.6%) was low than that in group III(15.6%). However, in vitro development rate in group III was not different with that in control (24.8%). This result suggested that MVC method was appropriate freezing method for the bovine eMII oocytes and vitrified eMII oocytes after pre-activation could support in vitro embryonic development after SONT as equally well as fresh oocytes.
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KEYWORD
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Enucleated bovine oocytes, MVC freezing, Activation timing, Somatic cell nuclear transfer, In vitro development
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