| Project/Area Number |
07556123
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Applied veterinary science
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| Research Institution | Yamaguchi University |
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Principal Investigator |
SUZUKI Tatsuyuki Department of Agriculture, Yamaguchi University Professor, 農学部, 教授 (00216409)
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| Co-Investigator(Kenkyū-buntansha) |
HUJIWARA Noboru Department of Agriculture, Kyushu University Professor, 農学部, 教授 (60150512)
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| Project Period (FY) |
1995 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1997: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1996: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | bovine / immature oocytes / ethylene glycol / torehalose / PVP / cryopreservation / transfer / calf / 卵母細胞 / GV期 / 体外受精 / 胚盤胞 / 二重蛍光染色 / 電顕観察 |
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| Research Abstract |
The objentives of this study was to evaluate in vitro fetilization and cleavage rates of frozen-thawed bovine oocytes at the germinal vesicle (GV) stage. In mouse oocytes, spindle microtubule reorganization after GV breakdown is particularly sensitive to cold and readily damaged by exposure to low temperatures, the damage becoming apparent only at the time of the first mitotic division. The effects of various permeating cryopprotective agents [1.8M ethylene glycol (EG), 1.3M ethylene glycol monomethylether (EME), and 1.6M 1,2-propanediol (PROH) and different concentration of trehalose (T) and polyvinylpyrrolidone (PVP) on post-thaw developmental capacity were examined. When bovine GV oocytes were frozen slowly in mixtures of 1.8M EG plus 5% PVP and 0.05M T,almost 80% developed to metaphase II ; 22.2% degenerated after in vitro maturation, and none of those that had been cryopreserved underwent parthenogenetic activation. The total fertilization rate was higher (p<0.05) for oocytes frozen in a mixture of 1.8M EG plus 0.05M T or 0.1M T than in a mizture of 1.8M EG with or without 0.2M T ; however, there was no difference in the number of normally fertilized or polyspermic oocytes that had been frozen in various cryoprotective solutions. No significant difference was observed in subsequent development using EG,EME,and PROH for GV oocytes. The addition of 0.05M or 0.1M trehalose to the freezing solution yielded significantly better cleavage and blastocyst rates than the solutions ocntaining 0.2M or no trehalose. For unfrozen controls, GV oocyte yielded significantly higher (p<0.01) cleavage and blastocyst rates compared with frozen-thawed GV oocytes. It was found that 5% PVP had a benefical effect compared with 10 or 20% concentrations for the development of blastocysts. Transfer of six blastocysts derieved from frozen-thawed GV oocytes into three recipient heifers resulted in three pregnancies and the birth of one set of twins and one singleton calf.
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