The effect of media, serum and temperature on in vitro survival of bovine blastocysts after open pulled straw (OPS) vitrification

摘要

In 5 experiments, 862 in vitro produced cattle embryos were vitrified and warmed using open pulled straw (OPS) techniques, then cultured in vitro for 3 days. The culture medium in experiments 1 to 4 was synthetic oviduct fluid supplemented with amino acids (SOFaa) and 5% calf serum. In experiment 1, replacement of TCM-199 + 20% calf serum with PBS + 20% calf serum in the holding medium during vitrification and warming did not result in significant differences in re-expansion (92 vs. 95%) and hatching rates (79 vs. 72%). In experiment 2, the phosphate buffered saline (PBS) holding medium was supplemented with either 20% calf serum, 5 mg/ml bovine serum albumin (BSA) or 3 mg/ml polyvinyl-alcohol. Although the re-expansion rates did not differ (98, 95 and 93% respectively), there was a decrease in the hatching rate after vitrification with polyvinyl-alcohol (77 and 78 vs. 51% respectively). In experiment 3, the influence of temperature of equilibration media prior to- and rehydration media after vitrification was investigated. When the temperature of these media was adjusted to 20deg C instead of the standard 35degree C, the re-expansion and hatching rates decreased markedly. Increasing the time of equilibration with diluted cryoprotectant solution at 20degree C eliminated these differences. In experiment 4, the ethylene-glycol and DMSO cryoprotectant mixture was replaced with ethylene glycol-ficoll-trehalose solution. No difference in the re-expansion (89 vs. 96% respectively) or hatching rate (79 vs. 84% respectively) was detected. In experiment 5, the vitrified-warmed blastocysts were cultured in SOFaa medium supplemented with 5% calf serum or with 5 mg/ml BSA. Although the re-expansion rates were identical in the 2 groups (95%), the hatching rates were lowerwhen embryos were cultured in BSA (71 and 47% respectively). It is concluded that this technique, with its advantages of rapid cooling and warming, may be used with media of different chemical composition according to the requirements of the biological structure and that serum supplementation of the medium is needed for hatching to occur after OPS vitrification.