Researcher 2010 Viability And DNA Fragmentation Assessment Of Bovine Embryos Vitrified By Different Methods

Abstract: This study was conducted to compare the efficiency of four vitrification methods: straws in goblet, straws in Hasler device, cryloop and cryotop methods on blastocyst viability and the extent of apoptosis using the TUNEL technique in bovine embryos. Blastocysts were produced in vitro by standard procedures. The base medium for vitrification was Syngro holding medium. Embryos were vitrified by the four methods following a two step addition of cryoprotectant. Embryos were first exposed to 5 M EG (28%) for 3 min at 20-22 °C, and then transferred into a vitrification solution composed of 6.5 M EG (36%), 0.5 M galactose (9% w/v), and 18% (w/v) Ficoll 70 in Syngro plus 1% PVA for 45 s. Embryos were loaded in the four devices and were placed in liquid nitrogen. The postwarm survival of the blastocysts was assessed by in vitro culture for 24 h. The proportions of embryos developing into expanded/hatched blastocysts were assessed after 48h. Embryos were stained and the number of blastomeres was determined by counting the number of nuclei. DNA fragmentation was assessed by TUNEL assay. No significant difference was found in survival rates directly after warming and hatching rates among the four methods. There was a significant difference (P<0.05) in embryo viability among the four methods at 24 h post warming. Embryos vitrified-thawed using cryotop exhibited higher proportions of development into hatched blastocysts but without any significant difference between the other methods. A significant decrease in total cell number was observed in vitrified blastocysts than fresh embryos. In vitrified expanded and hatched blastocysts, the cell number was significantly increased and mitotic cells were decreased compared to fresh embryos. All vitrification methods induce DNA damage to blastocystes. Damage was minimal in cryotop and straw in goblet and maximal with cryloop and straws in the Hasler device. In conclusion, bovine blastocyst can be successfully cryopreserved by vitrification using the four devices. Relatively low survival rates of blastocysts and increase in DNA damage were obtained using the cryoloop device compared to other methods