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.

The objective of the study was to vitrify mouse embryos with the cryoloop technology using a new combination of vitrification mediums. Embryos were exposed to a 2- step loading of CPA, ethylene glycol and propylene glycol, before being placed on the surface of a thin filmy layer formed from the vitrification solution in a small nylon loop. After warming, the CPA was diluted out from the embryos by a 3-step procedure. 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 developing normally (89.1%) in vitro after thawing.