Cryopreservation of rat embryos at all developmental stages by small-volume vitrification procedure and rapid warming in cryotubes

Intracellular ice formation during cryopreservation is lethal to the cell, including during warming. Here, we examined the effect of sample volume and warming rate on the cryopreservation success of 1-cell rat embryos based on successful development into blastocysts in vitro and to term in vivo following embryo transfer. Embryos were equilibrated in 5% propylene glycol solution for 10 min, held for 40 s at 0 degrees C in cryopreservation solution (5%PG + PEPeS), and cooled by immersion in liquid nitrogen. When 1-cell embryos were cryopreserved in a volume of 30-100 mu L at a cooling rate of 5830-7160 degrees C/min and warmed at 35,480-49,400 degrees C/min by adding 1 mL of 0.3 M sucrose solution at 50 degrees C, 17.3-28.8% developed into blastocysts, compared with 57.0% of untreated embryos. However, when 1-cell embryos were cryopreserved in a smaller volume of 15 mu l at 7950 degrees C/min and warmed at 68,850 degrees C/min, 58.8 +/- 10.6% developed into blastocysts and 50.0 +/- 7.4% developed to term, comparable to that of non-treated embryos (57.0 +/- 5.4% and 51.4 +/- 3.1%, respectively). Cryopreserved embryos at other developmental stages also showed high in vitro culture potential similar to that of the control. Using a conventional cryotube and a small-volume vitrification procedure with rapid warming, we achieved high levels of subsequent rat embryonic development at all developmental stages.