Producing of calf by transfer of IVF embryos derived from frozen-thawed immatured (GV) bovine oocytes

通过移植来自冻融未成熟 (GV) 牛卵母细胞的 IVF 胚胎来生产小牛

• 批准号: 07556123
• 负责人: SUZUKI Tatsuyuki
• 金额: $ 1.28万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07556123/
• 关键词: 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

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.

本研究的目的是评价牛卵母细胞在生发泡(GV)阶段的体外受精率和卵裂率。在小鼠卵母细胞中，GV破裂后的纺锤体微管重组对寒冷特别敏感，很容易受到低温的破坏，这种损害只有在第一次有丝分裂时才变得明显。研究了不同渗透性冷冻保护剂[1.8M乙二醇(EG)、1.3M乙二醇单甲醚(EME)、1.6M 1，2-丙二醇(PROH)]以及不同浓度的海藻糖(T)和聚乙烯吡咯烷酮(PVP)对解冻后胚胎发育能力的影响。牛GV卵母细胞在1.8mEG+5%PVP+0.05mT的混合液中缓慢冷冻时，近80%发育到中期II期，22.2%体外成熟后退化，所有冷冻后的卵母细胞均未发生孤雌激活。1.8mEG+0.05mT或0.1mT混合液中冷冻的卵母细胞总受精率高于1.8mEG+0.2MT或不加0.2MT组，但在不同冷冻保护液中冷冻的正常受精卵和多精卵数无显著差异。在随后使用EG、EME和PRH的GV卵母细胞的发育中，没有观察到显著的差异。在冷冻液中添加0.05M或0.1M的海藻糖，其卵裂率和囊胚率显著高于添加0.2M或不含海藻糖的冷冻液。对于未冷冻的对照组，GV卵母细胞的卵裂率和囊胚率显著高于冷冻解冻的GV卵母细胞(p&lt；0.01)。与10%和20%浓度的PVP相比，5%的PVP对囊胚的发育有较好的效果。将来自冷冻解冻的GV卵母细胞的6个囊胚移植到3头受体小母牛体内，结果3次怀孕，生下1对双胞胎和1头单胎小牛。

项目成果

鈴木 達行: "野生動物の家畜化と改良" 養賢堂出版, 247 (1996)

铃木达之：《野生动物的驯化与改良》养剑堂出版社，247（1996）

Suzuki T and others: "Fertilization and development of frozen thawed germinal vesicle bovine oocytes by a one step dilution method in vitro" Cryobiology. 33. 515-524 (1996)

Suzuki T 等人：“体外一步稀释法冷冻解冻生发囊牛卵母细胞的受精和发育”冷冻生物学。

Otoi T,Tachikawa S,Kondo S and Suzuki T.: "In vitro Fertilization and development of immature and mature bovine oocytes cryopreserved by ethlene glycol with sucrose." 32. 455-460 (1995)

Otoi T、Tachikawa S、Kondo S 和 Suzuki T.：“乙二醇和蔗糖冷冻保存的未成熟和成熟牛卵母细胞的体外受精和发育”。

Suzuki T and others: "The influence of polyvinylpyrrolidone on freezing of bovine blastocysts following biopsy" Cryobiology. 32. 505-510 (1995)

Suzuki T 等人：“聚乙烯吡咯烷酮对活检后牛囊胚冷冻的影响”冷冻生物学。

鈴木 達行: "一段階浸漬法による牛凍結融解卵子の体外受精と発生" バイオインダストリー. 9. 40-43 (1992)

Tatsuyuki Suzuki：“一步浸泡法进行牛冻融卵的体外受精和发育” Bioindustry. 9. 40-43 (1992)。