Survival of frozen or vitrified bovine blastocysts produced in vitro in synthetic oviduct fluid

Abstract

Simplification of in vitro culture conditions offers the advantage of limiting problems due to variation in composition of biological fluids and co-culture with epithelial cells. The aim of this work was to study cryosurvival of bovine embryos produced under partially defined conditions. A conventional freezing technique and a vitrification method that allow for direct transfer of embryos after thawing were compared. Following IVM and IVF, zygotes were cultured in modified synthetic oviduct fluid (SOF), with 10% fetal calf serum (FCS) added 48 h post insemination, or, in the absence of FCS, in a humidified atmosphere of 5%O 2, 5%CO 2, 90%N 2. Morphologically normal Day 8 and 9 blaslocysts were classified into 3 groups and were cryopreserved. Group A were blastocysts (< 150 μm); Group B consisted of expanded blastocysts (> 150 μm); while Group C were hatching or hatched blastocysts. The freezing solution consisted of 1.36 M glycerol and 0.25 M sucrose in PBS. The vitrification solution consisted of 6.5 M glycerol and 6% bovine serum albumin in PBS (VS3a). Thawed embryos were cultured on granulosa cells and survival was defined as the re-expansion and maintenance of the blastocoel over 24 and 72 h, respectively. Survival rates following vitrification were similar or better than after freezing (Day 8 embryo survival rate at 72 h was 72 vs 57%). More advanced embryos of the same group seemed to survive better. Embryos cultured without serum survived cryopreservation significantly better than those produced with addition of serum at 48 h post insemination (86 vs 61% at 72 h; P < 0.01). Hatched blastocysts survived freezing or vitrification well (80 to 100%). One calf was born following the transfer of 6 vitrified embryos. The results indicate that both freezing and vitrification methods are suitable for cryopreservation of bovine embryos produced in SOF.