Rat one-cell embryo vitrification in F344, Long-Evans, and SD strain via small-volume vitrification and rapid warming in cryotubes

Abstract

Genome-edited animals can be created by introducing CRISPR/Cas9 systems into one-cell stage embryos, even in non-mice embryos. We developed a vitrification method for rat one-cell embryos of the F344 inbred, Long-Evans, and SD strains. Successful cryopreservation requires the avoidance of intracellular ice formation (IIF). We hypothesized that a new cryopreservation method could be developed by rapid warming to avoid IIF during warming, via cryopreservation in conventional cryotubes using small-volume vitrification and rapid warming. When cryopreserved one-cell embryos in a cryotube with 15 μL cryopreservation solution (mixture of 5 μL 5 % propylene glycol and 10 μL PEPeS) were warmed by adding 1 ml of 0.3 M sucrose solution at 50 °C, they developed to term at 67.2 % (F344), 56.3 % (Long-Evans), and 65.0 % (SD), which were comparable with those (60.3 %, 58.3 %, 67.0 %, respectively) of non-cryopreserved control embryos. Thus, rat one-cell embryos from several strains can be cryopreserved even with cryotubes via rapid warming.