Effect of conventional controlled-rate freezing and vitrification on morphology and metabolism of bovine blastocysts produced in vitro

This study compares the effects of conventional controlled-rate freezing an d vitrification on the morphology and metabolism of in vitro-produced bovin e blastocysts. Day 7 expanded blastocysts cultured in synthetic oviduct flu id with 5% fetal calf serum were frozen in 1.36 M glycerol, 0.25 M sucrose or vitrified in 25 % glycerol, 25 % ethylene glycol. Cell alterations and i n vitro development were evaluated immediately after thawing or after 72 h. The effect of cryopreservation on inner cell mass and trophectoderm (TE) c ell number as well as glucose, pyruvate and oxygen uptakes, and lactate rel ease by blastocysts were evaluated. Immediately after thawing, blastocysts showed equivalent cell membrane permeabilization after both cryopreservatio n procedures, while alterations in nuclear staining were more frequent in v itrified embryos. After culture, similar survival and hatching rates were o bserved. Both procedures decreased cell number immediately after thawing an d after 72 h. However, the number of TE cells was lower in frozen embryos t han in vitrified ones. In relation to this, frozen blastocysts showed a dec rease in glucose, pyruvate, and oxygen uptake, although those parameters we re not altered in vitrified embryos. An increased glycolytic activity was a lso observed in frozen embryos, indicating a stress response to this proced ure.