Viability of bovine preimplantation embryos following cryopreservation

Abstract

In programs for the preservation of animal genetic resources a great significance belongs to a long-term storage of biological material (gametes, embryos) at extra-low temperatures. The aim of this study was to determine quality and viability of bovine embryos of Holstein breed cryopreserved by a vitrification procedure (n = 88) in comparison to intact (fresh) bovine embryos (n = 117). The embryos were recovered on 7th day after the first insemination by a flushing out of the uterine horns of superovulated cows with a Bioniche complete flush solution using a silicone two-way Foley catheter. The embryos of morula or early blastocyst stage were subjected to the two-step vitrification procedure using EFS vitrification solution (ethylene glycol - 40 % v/v, Ficoll 70 - 18 % w/v; 0.3 M sucrose in D-PBS + 20 % fetal calf serum and 5 µg.ml-1 gentamycin), pulled into the open-pulled straws and slowly immersed into liquid nitrogen. Following thawing the embryos were cultured for 48 hours in order to reach advanced developmental stage (expanded blastocyst), afterwards these embryos were analyzed for the developmental rate, embryo cell number and incidence of the dead cells. About 83 % of all control embryos were developed to the expanded blastocyst stage, whilst in the frozen-thawed group only 60 % of embryos reached this stage (p < 0.05). Frozen-thawed embryos contained significantly less number of embryonal nuclei when compared with fresh embryos. Dead cell incidence (TUNEL-index) was more than twice higher in the frozen-thawed embryos (9.53 %) in comparison to the fresh embryos (4.32 %), but this value did not exceed 10 %, the critical value which may compromise the embryo viability. Our experiments confirm that cryopreservation may affect embryo viability. Therefore, monitoring of embryo quality following cryopreservation would provide a basis for understanding embryo sensitivity to freezing protocols and help to improve a cryopreservation technique.