Abstract
The effect of various combinations of plunge temperature and thawing protocol on the survival and viability of mouse oocytes was examined. The oocytes were frozen either in a standard freezing medium (ETFM, embryo transfer freezing medium) or in a low-sodium, choline-based freezing medium (CJ2), with 1.5 M 1,2-propanediol and 0.1 M sucrose, and using a conventional slow cooling method. The criteria used to assess survival were morphological state after thawing (intact or lysed), ability to become fertilized, and ability to develop to the two-cell, morula, and blastocyst stage in vitro. Oocytes frozen in CJ2 and plunged into liquid nitrogen (LN2) from −10, −20, or −33°C remained intact and developed to the blastocyst stage at significantly higher rates than oocytes frozen in ETFM. For oocytes plunged into LN2 from −33°C, very rapid thawing (10 s in 30°C water) was more detrimental than rapid or slow thawing (holding in air at room temperature for 10 or 30 s, respectively, prior to submersion in water at 30°C for 10 s). By contrast, oocytes plunged into LN2 from −10 or −20°C survived better when thawing was very rapid or rapid than when thawing was slow. With the current protocol CJ2 was very effective over a wide range of plunge temperatures (−20 to −33°C), although the optimal thawing protocol depended on the particular plunge temperature. Over 90% of oocytes surviving after slow cooling in CJ2 to −33°C could be plunged to −196°C with little or no further damage.
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