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Mouse morulae were recovered from ICR females mated with males of the same strain after in-ducing superovulation by injections of PMSG and hCG. The morulae were frozen in a glass test tube containing 0.2m<I>l</I> PBS with 1.5M-DMSO and thawed slowly (25C/min; -75 to -10C). After stepwise dilution of DMSO with phosphate buffered saline, the morulae were washed twice and cul-tured for 2-3 days in a modified Krebs-Ringer-bicarbonate solution to determine their viability, which <BR>In Exp. 1, embryos were frozen to various temperatures at different cooling rates and were kept for 5min before thawing. When embryo were frozen at a rate of 5 or 10C/min, their survival rates de-creased as the freezing temperature declined, while 84-98% of embryos survived after freezing to -20, -50 or -75C at 0.25 or 1C/min. In Exp. 2, higher survival rates were obtained in the morulae thawed after rapid cooling to -75C from -50--60C (83-86%) than from -20--40C (2-38%) to which the embryos were precooled at 1C/min. In Exp. 3, embryos were frozen to -50C at 5C/min and then plunged into -75C ethanol after holding for different periods at -50C. Higher survival rates were obtained in the embryos held at -50C for 10-180min (59-73%) than in those held for 0-5min (4-14%) before plunging into -75C. In Exp. 4, embryos cooled at 1C/min were stored at -25, -50 or 75C for different periods before thawing. When embryos were stored at -25C, their survival rates sharply decreased as the storage period was increased, reaching 0% after storage for 24h. Two normal young were obtained from 8 embryos transferred into a recipient after storage for 90 days at -75C before thawing was assessed by the ability to develop to expanded blastocysts.<BR>From these results it is suggested that dehydration of the embryos is almost completed during the cooling down to -50C at a slow rate. But when the embryos were cooled rather rapidly (5C/min), dehydration of embryos is mostly achieved by holding them at -50C for a given period. The damage of the embryos stored at -25C seems to be due to "solution effects", because many of the damaged embryos looked morphologically normal under dissecting microscope after thawing. Such solution effects on the embryos, however, were moderated as the storage temperature was lowered.
- 日本繁殖生物学会の論文
日本繁殖生物学会 | 論文
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