ブタ生体回収由来ガラス化胚の生存性の改善
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概要
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An efficient cryopreservation protocol for porcine morulae was investigated with three types of vitrification having different cooling rates (Exp. 1). Survival of embryos vitrified after removal of cytoplasmic lipid droplets was also examined by means of the minimum volume cooling (MVC) method (Exp. 2). In Exp. 1, the morula stage embryos were vitrified with a 0.25 ml plastic straw (ST-method), gel loading tip (GLT-method) and the MVC-method, respectively, and stored in liquid nitrogen after which they were warmed in sucrose solutions with cryoprotectants being subsequently removed in a stepwise manner. In Exp. 2, morulae were centrifuged with 7.5 μg/ml cytocharasin B at 12000 × g for 20 min to polarize the cytoplasmic lipid droplets that were then removed from the embryos by micromanipulation (delipation). Both those delipated at the morula stage and the intact embryos at the morula to blastocyst stages were vitrified by the MVC-method. In vitro survival of the vitrified embryos was assessed in both experiments by culturing in NCSU-23 + 10% FCS for 48 h. In vitro developments of vitrified embryos after warming to blastocysts were 20% (6/30) for the ST-method, 39% (18/46) for the GLT-method, and 60% (26/43) for the MVC-method. Embryo survival was further improved by vitrification after delipation (95%, 35/37) compared to intact vitrified morulae (24/42, 57%, P<0.001) and blastocysts (23/31, 74%, P<0.05). Moreover, the number of cells in blastocysts (92 ± 25) derived from the delipated-vitrified morulae was comparable to those derived from intact control non-vitrified embryos (103 ± 31). Our results demonstrate that vitrified porcine morulae have the highest survival when using the MVC-method in conjunction with delipation.
- 日本繁殖生物学会の論文
- 2004-08-01
著者
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牛島 仁
Chiba Prefectural Animal Experimental Station
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吉岡 裕樹
Department of Life Science, Meiji University
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江崎 律子
Department of Life Science, Meiji University
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TAKAHASHI Keiji
Chiba Prefectural Animal Experimental Station
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KUWAYAMA Masashige
KATO Ladies Clinic
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NAKANE Takashi
Chiba Prefectural Animal Experimental Station
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NAGASHIMA Hiroshi
Department of Life Science, Meiji University
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YOSHIOKA Hiroki
Department of Diagnostic Radiology and Nuclear Medicine, Gunma University Graduate School of Medicin
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Yoshioka Hiroki
Department Of Diagnostic Radiology And Nuclear Medicine Gunma University Graduate School Of Medicine
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USHIJIMA Hitoshi
Chiba Prefectural Animal Experimental Station
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ESAKI Ritsuko
Department of Life Science, Meiji University
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Yoshioka Hiroki
Department Of Life Science Meiji University
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Esaki Ritsuko
Department Of Life Science Meiji University
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Kuwayama M
Kato Ladies Clinic
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Nagashima Hiroshi
Department Of Life Science Meiji University
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Nagashima Hiroshi
Department of Environmental and Civil Engineering, University of Okayama
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