Vitrification of cleavage stage mouse embryos by the cryoloop procedure

By decreasing the volume of the cryoprotective solution it is possible to increase dramatically the freezing speed and - at the same time - reduce the toxicity and osmotic side effects of cryoprotectants (CPA). The objective of our study was to vitrify Day-3 cleavage stage mouse embryos (n = 229) with the cryoloop technology using a new composition of vitrification media. Embryos were exposed to a 2-step loading of CPA, ethylene glycol (EG) and propylene glycol (PG), before being placed on the surface of a thin filmy layer formed from the vitrification solution in a small nylon loop, then they were rapidly submerged into liquid nitrogen. After warming, the CPA was diluted out from the embryos by a 3-step procedure. Survival of embryos was based on morphological appearance after thawing and continued development to expanded blastocysts upon subsequent 48-hour culture. Embryos of the two control groups were either treated likewise except that they were not vitrified, or cultured in vitro without any treatment. Our data show that a high percentage of embryos survived (92.7%) vitrification in the mixture of EG and PG combined with cryoloop carrier and developed normally (89.1%) in vitro after thawing. To our knowledge this is the first report of the successful vitrification of cleavage stage mouse embryos using VitroLoop vitrification procedure.