Japanese Journal of Veterinary Research;Volume 28, Number 1-2

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IN VITRO CULTURE OF FROZEN AND THAWED MOUSE OVA

KANAGAWA, Hiroshi

Permalink : http://hdl.handle.net/2115/2182
JaLCDOI : 10.14943/jjvr.28.1-2.12

Abstract

Seventy mouse ova at the 4--8-cell stages were collected at room temperature from mice treated with pregnant mare's serum gonadotrophin and human chorionic gonadotrophin. Seventy ova were divided into 5 groups, and each group was placed in a 0.5 ml plastic straw with 0.2 ml of Brinster's medium. Then, the straws were immersed into an ice bath (0℃) for 5 minutes. Next, an equal volume of 2 M dimethyl sulfoxide was added to the sample straw. The medium with ova was then seeded at -6℃ and cooled to -100℃ at a rate of 0.4 to 0.5℃ per minute using an inexpensive freeze-thaw apparatus. After reaching -100℃, the sample straws were directly immersed in liquid nitrogen at -196℃ and stored for 1-9 days. Following the storage period, the sample straw were thawed at 12-15℃ per minute from -196℃ to 0℃. The samples were then diluted with fresh medium and placed inside a petri dish at room temperature (20-25℃). The ova were moved immediately to a fresh medium 3 times, then incubated at 37℃ for 72 hours. Sixty out of the 70 ova were recovered from the straws after the freezing experiment, and 41 out of the 60 ova (68.3%) developed in vitro to the blastocyst stage within 72 hours.

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