ULTRARAPID FREEZING OF MOUSE EMBRYOS
SHIVANI VASUTHEVAN
SHIVANI VASUTHEVAN
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Abstract
The "ultrarapid" technique for murine embryo cryopreservation involves a brief exposure (2.5-3 minutes) of embryos to a high concentration (3.0-4.5 M) of dimethyl sulfoxide (DMSO) and 0.25 M of sucrose at room temperature before direct plunging into liquid nitrogen. The objective of this study was to determine the efficacy of this technique for the cryopreservation of two-cell mouse embryos under various conditions. Three bicarbonate-based culture media namely T6, Medicult and Human Tubal Fluid (HTF) medium were found to result in high rates of survival (96.2%-98.4%) and in vitro development (77.9%-87.8%) when employed to make up the 3.5 M DMSO freezing solution. The elevated pH of these solutions in the absence of a 5 % CO2 atmosphere did not have a detrimental effect on the cryosurvival and developmental capacity of the frozen-thawed embryos. Upon comparing the use of phosphate-, Hepes- and bicarbonate-buffered T6 medium containing 3.5 M or 4.5 M DMSO and 0.25 M sucrose for cryopreservation, phosphate-buffered T6 containing 3.5 M DMSO was found to result in a reduced in vitro blastocyst formation rate (72 % ) compared to bicarbonate- and Hepes-buffered T6 (84.5% and 86.5% respectively). Upon differential staining of the developing blastocysts, it was followed that blastocysts derived from the phosphate-buffered T6 group possessed a significantly smaller inner cell mass (mean cell number of 21. 6) and a reduced mean total cell number (86.6) compared to blastocysts of tile other media groups. With 4.5 M of DMSO, although all three media yielded consistently low blastocyst formation rates (69.2%-75.6%), freeze-thaw in Hepes-T6 yielded blastocysts with a significantly higher mean ICM (26.0) and total blastocyst (97.3) cell number compared to freeze-thaw in the other two media. On transferring the developing blastocysts from the bicarbonate-, Hepes- and phosphate-buffered T6 groups frozen with 3.5 M DMSO into pseudopregnant females, similar implantation (64.0%-76.5%) and fetal formation (60.0%-69.6%) rates were obtained with all three media and the non-frozen group. However, a significantly higher resorption rate (10.6%) was obtained with the phosphate-buffered group in comparison to the nonfrozen group (2.6%). Lastly. the efficacy and safety of the "ultrarapid two-step" technique was determined. It was found that the survival (97.0%-97.4%) and in vitro developmental capacity (85.3%-86.4%) of two-cell embryos frozen-thawed by this technique was comparable to that of embryos frozen-thawed by the classical ultrarapid technique (98.5%-100% and 90.1 %-90.6% respectively) regardless of whether a single or multi-step method of DMSO removal was employed. The fetal developmental rates oi the blastocysts derived from the two-step frozen-thawed group (60.0%) was also comparable to that of the one step frozen-thawed group (71.8%). It was therefore concluded that the additional pre-freeze 5-minute equilibration step in 1.5 M DMSO may be an unnecessary addition to the existing protocol. In conclusion, the classical one step ultrarapid freezing technique is a simple, inexpensive and yet convenient method for cryopreserving two-cell mouse embryos. The added advantage of being able to utilize three bicarbonate media commonly employed for embryo culture to make up the cryoprotectant solutions as well, should promote the application of this technique for the routine cryopreservation of mouse embryo on a wider scale.
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1993
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