Studies on development of a simplified rapid freezing method for preservation of farm and small animal eggs

一种简易快速冷冻方法保存农畜及小动物蛋的研究

• 批准号: 60560281
• 负责人: MIYANOTO Hajime
• 金额: $ 1.15万
• 依托单位: Kyoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1985
• 资助国家: 日本
• 起止时间: 1985 至 1986
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-60560281/
• 关键词: Pig oocytes; Cooling; Mouse embryos; Liquid nitrogen vapour; Rapid freezing; Glycerol; 平衡時間

1. When pig oocytes at the germinal vesicle stage without or with 0.5 M sucrose were cooled at 0.5゜C/min to 0, 10, 20 and 30゜C (control), the proportion of oocytes matured to metaphase II during culture was 0, 0, 16-21 and 46-52%, respectively. After oocytes were cooled 3゜C/min to 20゜C, none reached metaphase II during culture. Addition of 0.5 M sucrose and cryoprotectants (DMSO, glycerol and ethylene glycol) had no apparent effect on survival of cooled oocytes. These results show that pig oocytes were all killed when cooled to temperatures of 10 or 0゜C. In conclusion, pig oocytes appear to be extremely sensitive to cooling and there are differences between species in the responses of oocytes to cooling.2. Mouse morulae were frozen rapidly to -196゜C in the presence of glycerol by a two-step procedure; the embryos were transferred directly from -7゜C after seeding into liquid nitrogen vapour at -170--180゜C and then into liquid nitrogen 10-15 min later. Suitable conditions for the survival of embryos frozen with liquid nitrogen vapour were found to be: 2 M glycerol, 2 M propylene glycol, 2 M ethylene glycol; 5-30 min equilibration time at 0゜C; 3-60 holding time in liquid nitrogen vapour; dilution of glycerol with sucrose out of the frozen-thawed embryos; morula and early blastocyst stage embryos. Relatively high survival rates (69-74%) were obtained after rapid freezing by liquid nitrogen vapour. The present freezing procedure provides a quick and easily performed technique for preserving mouse embryos with a minimum of specialized equipment and makes it possible to freeze a large number of embryo samples in a short time. The present liquid nitrogen vapour method, therefore, may be applied to the embryo preservation of other species.

1. 将未添加或添加0.5 M蔗糖的猪胚泡期卵母细胞以0.5ºC/min的速度冷却至0、10、20和30ºC（对照组）时，培养过程中成熟至中期II的卵母细胞比例分别为0、0、16-21和46-52%。将卵母细胞冷却3 C/min至20 C后，培养过程中没有卵母细胞达到中期II。添加0.5 M蔗糖和低温保护剂（DMSO、甘油和乙二醇）对冷却卵母细胞的存活无明显影响。这些结果表明，当冷却到10或0 C时，猪卵母细胞全部死亡。综上所述，猪卵母细胞对冷却表现出极强的敏感性，且不同物种的卵母细胞对冷却的反应存在差异。在甘油存在下，通过两步程序将小鼠桑葚胚快速冷冻至-196˚C；胚在-170—180 C播种到液氮蒸汽后，直接从-7 C转移到液氮中，10-15分钟后再转移到液氮中。液氮蒸汽冷冻胚胎的适宜条件为：2 M甘油，2 M丙二醇，2 M乙二醇；在0 C下的5-30分钟平衡时间；在液氮蒸气中保温时间3-60；用蔗糖从冻融胚胎中稀释甘油；桑葚胚和早期囊胚期胚胎。液氮蒸气快速冷冻后存活率较高（69-74%）。本方法提供了一种快速、简便的保存小鼠胚胎的技术，只需最少的专用设备，可以在短时间内冷冻大量胚胎样本。因此，本发明的液氮蒸汽法可应用于其它物种的胚胎保存。

项目成果

宮本元: 哺乳類動物卵子研究会誌. 2. 59-62 (1985)

Hajime Miyamoto：《哺乳动物卵母细胞研究会杂志》2. 59-62 (1985)。

Hajime MIYAMOTO: "Effects of equilibration time to dimethylsulfoxide prior to freezing on the survival of mouse embryos frozen two-step method" Japanese Journal of Animal AI Research. 7. 76-79 (1985)

Hajime MIYAMOTO：“冷冻前二甲基亚砜的平衡时间对冷冻两步法小鼠胚胎存活率的影响”日本动物人工智能研究杂志。

H.MIYAMOTO: Journal of Experimental Zoology.

H.宫本：实验动物学杂志。

Hajime MIYAMOTO: "The importance of equilibration time to glycerol prior to freezing in the cryopreservation of mouse embryos" Japanese Journal of Zootechnical Science. 57. 250-256 (1986)

Hajime MIYAMOTO：“小鼠胚胎冷冻保存前甘油平衡时间的重要性”《日本动物科学杂志》。

Hajime MIYAMOTO: 日本畜産学会報. 57. 250-256 (1986)

Hajime MIYAMOTO：日本动物科学会通报 57. 250-256 (1986)。