SURVIVAL OF FROZEN OR VITRIFIED BOVINE BLASTOCYSTS PRODUCED IN-VITRO IN SYNTHETIC OVIDUCT FLUID

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 stu dy cryosurvival of bovine embryos produced under partially defined con ditions. 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 ov iduct fluid (SOF), with 10% fetal calf serum (FCS) added 48 h post ins emination, or, in the absence of FCS, in a humidified atmosphere of 5% O-2, 5%CO2, 90%N-2. Morphologically normal Day 8 and 9 blastocysts wer e classified into 3 groups and were cryopreserved. Group A were blasto cysts (<150 mu m); Group B consisted of expanded blastocysts (>150 mu m); while Group C were hatching or hatched blastocysts. The freezing s olution consisted of 1.36 M glycerol and 0.25 M sucrose in PBS. The vi trification solution consisted of 6.5 M glycerol and 6% bovine serum a lbumin in PBS (VS3a). Thawed embryos were cultured on granulosa cells and survival was defined as the re-expansion and maintenance of the bl astocoel over 24 and 72 h, respectively. Survival rates following vitr ification were similar or better than after freezing (Day 8 embryo sur vival rate at 72 h was 72 vs 57%). More advanced embryos of the same g roup seemed to survive better. Embryos cultured without serum survived cryopreservation significantly better than those produced with additi on of serum at 48 h post insemination (86 vs 61% at 72 h; P<0.01). Hat ched 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 suit able for cryopreservation of bovine embryos produced in SOF.