Abstract
This study evaluated the effects of cryopreservation by slow freezing on the mitochondrial function, DNA integrity, and developmental ability of bovine embryos and examined whether resveratrol treatment of the frozen-thawed blastocysts improved embryonic viability. In vitro produced bovine embryos were subjected to slow freezing. After thawing, the ATP content and mitochondrial DNA integrity (mtDNA), determined by real-time PCR targeting short and long mitochondrial sequences, was found to be lower in frozen-thawed embryos than in fresh embryos, and mtDNA copy number was significantly reduced during the 24-hr incubation post warming. Furthermore, immunostaining against double-strand DNA revealed DNA damage in frozen-thawed embryos. When frozen-thawed embryos were incubated in the medium containing 0.5µM resveratrol, SIRT1 expression, and survival rate of the embryos significantly improved compared with the vehicle-treated embryos. In addition, cell-free mtDNA content in medium was higher in case of resveratrol-treated embryos than of vehicle-treated embryos. In conclusion, slow freezing affects mitochondrial integrity and function in the blastocysts. In the frozen-thawed embryos, mitochondria were removed during post-thawing incubation and resveratrol enhanced the process, resulting in improved survivability of the embryos.
Published Version
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