Refinements in the cryopreservation of mouse ovaries

Here we describe a new technique for cryopreserving mouse ovaries by using 0.5-mL straws. One advantage of this method is that it uses the same controlled-rate freezer and programming routinely used for the cryopreservation of mouse embryos. Using a 0.5-mL French straw loaded in the same way as for embryo freezing (for example, the one-step dilution method) with 1 M sucrose as an osmotic buffer and 2 M propylene glycol as the cryoprotectant containing the ovary sample, we further standardized the 2 methodologies. Applying this technique, 11 ovarian halves were cryopreserved in straws and stored under liquid nitrogen. Straws containing the frozen ovarian halves were thawed in a water bath at room temperature and the recovered ovaries orthotopically implanted into 11 recipient female mice; 8 of the 11 frozen ovarian halves resulted in functional ovaries. The 73% pregnancy rate resulted in a total of 53 pups born, of which 38 (72%) were generated from cryopreserved ovaries. Ovarian cryopreservation has been demonstrated to be a valid option for banking mouse genetic resources. Unlike frozen embryos, cryopreservation of ovarian tissue preserves haploid gametes. Despite this limitation, ovarian cryopreservation is the only technique that can be used to preserve oocytes from aged or problematic breeders. This advantage is especially important in situations where the only males available in the line are infertile, aged, or problematic breeders.