The effect of media, serum and temperature on in vitro survival of bovine blastocysts after Open Pulled Straw (OPS) vitrification

The recently introduced Open Pulled Straw (OPS) vitrification technique has successfully been used for cryopreserving porcine embryos as well as for b ovine embryos and oocytes. The aim of this work is to investigate several f actors on the in vitro survival of bovine blastocysts. In 5 experiments, a total of 862 in vitro produced blastocysts and expanded blastocysts was vit rified and warmed using the OPS technology, then cultured in vitro for an a dditional 3 days. The culture medium in Experiments 1 to 4 was SOFaa with s upplements and 5% calf serum (CS). In Experiment 1, the replacement of TCM- 199+20% CS with PBS+20% CS in the holding medium during vitrification and w arming did not result in significant differences in the re-expansion (92 vs 95%) and hatching rates (79 vs 72%). In Experiment 2, the PBS holding medi um was supplemented with either 20% CS, 5 mg/mL bovine serum albumin (BSA) or 3 mg/mL,polyvinylalcohol (PVA). Although the re-expansion rates did not differ (98, 95 and 93%, respectively), there was a decrease in the hatching rate after vitrification with PVA (77 and 78 vs 51%, respectively). In Exp eriment 3, the influence of temperature of equilibration media prior to and rehydration media after the vitrification was investigated. When the tempe rature of these media was adjusted to 20 degrees C instead of the standard 35 degrees C, both the re-expansion and the hatching rates decreased marked ly. However, increasing the time of equilibration with the diluted cryoprot ectant solution at 20 degrees C eliminated these differences. In Experiment 4, the ethylene-glycol and dimethyl sulfoxide cryoprotectant mixture was r eplaced with ethylene glycol-ficoll-trehalose solution. No difference in th e re-expansion (89 vs 96%, respectively) or hatching rate (79 vs 84%, respe ctively) was detected. In Experiment 5, the vitrified-warmed blastocysts we re cultured in SOFaa medium supplemented with 5% CS or 5 mg/mL BSA. Althoug h the re-expansion rates were identical in the 2 groups (95%), the hatching rates were lower when embryos were cultured in BSA (71 and 47%, respective ly). These findings indicated the possible broader application for OPS, as they demonstrated that the physical advantages of rapid cooling and warming may be accompanied by different chemical composition (holding media, cryop rotective additives) according to the requirements of the biological struct ure. Our study also shows the need for serum supplementation of the medium for hatching to occur after OPS vitrification. (C) 1999 by Elsevier Science Inc.