title: Cryopreservation method affects DNA fragmentation in trophectoderm and the speed of re-expansion in bovine blastocysts.
abstract: This study investigated re-expansion dynamics during culture of bovine blastocysts cryopreserved either by slow-freezing or vitrification. Also, the extent and localization of membrane damage and DNA fragmentation in re-expanded embryos were studied. Frozen-thawed embryos showed a significantly lower re-expansion rate during 24 h of post-thawing culture compared to vitrified embryos. Vitrified embryos reached the maximum level of re-expansion rate by 12 h of culture whereas frozen embryos showed a gradual increase in re-expansion rate by 24 h of culture. When assayed by Hoechst/propidium iodide staining there was no difference in the numbers and ratio of membrane damaged cells between re-expanded frozen and vitrified embryos; however, the extent of membrane damage in blastomeres was significantly higher in both groups compared with non-cryopreserved embryos (control). TUNEL assay combined with differential ICM and TE staining revealed a significantly higher number and ratio of TE cells showing DNA-fragmentation in frozen-thawed re-expanded blastocysts compared to vitrified ones; however, vitrification also resulted in an increased extent of DNA fragmentation in TE cells compared with control blastocysts. In frozen-thawed blastocysts increased extent of DNA fragmentation was associated with reduced numbers and proportion of TE cells compared with vitrified and control embryos. The number and ratio of ICM cells and the extent of DNA fragmentation in ICM did not differ among control, frozen and vitrified groups. In conclusion, compared with vitrified embryos, blastocysts preserved by slow-freezing showed a delayed timing of re-expansion which was associated with an increased frequency of DNA fragmentation in TE cells.
authors.affiliation: NARO Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Inaba
authors.affiliation: NARO Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan; Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Miyashita
authors.affiliation: NARO Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan. Electronic address: somfai@affrc.go.jp.
authors.forename: Tamás
authors.initials: T
authors.lastname: Somfai
authors.affiliation: NARO Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan.
authors.forename: Masaya
authors.initials: M
authors.lastname: Geshi
authors.affiliation: NARO Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan.
authors.forename: Satoko
authors.initials: S
authors.lastname: Matoba
authors.affiliation: Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Dochi
authors.affiliation: NARO Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan; College of Agriculture and Life Sciences, Seoul National University, Seoul 151-742, Republic of Korea; Food and Fertilizer Technology Center, Taipei 10648, Taiwan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nagai
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000378:metabolism*; D001757:Blastomeres / Q000378:metabolism; D002417:Cattle; D018929:Cell Culture Techniques; D015925:Cryopreservation / Q000379:methods*; D053938:DNA Fragmentation; D004622:Embryo, Mammalian / Q000166:cytology; D005615:Freezing; D058989:Vitrification
keywords: Blastocyst; Bovine; DNA fragmentation; Freezing; Trophectoderm; Viability; Vitrification