title: Equilibrium vitrification of mouse embryos at various developmental stages.
abstract: Previously, we developed a new method by which 2-cell mouse embryos can be vitrified in liquid nitrogen in a near-equilibrium state, and then kept at -80°C for several days. In the present study, we examined whether or not the method was effective for mouse embryos at other developmental stages. Eight-cell embryos, morulae, and expanded blastocysts of ICR mice were vitrified with ethylene glycol-based solutions, named EFSc because of their composition of ethylene glycol (30-40%, v/v) and FSc solution. The FSc solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 1.5 M sucrose. The extent of equilibrium was assessed by examining how well vitrified embryos survived after being kept at -80°C. When 8-cell embryos and morulae were vitrified with EFS35c or EFS40c and then kept at -80°C, the survival rate was high even after 4 days in storage and remained high after re-cooling in liquid nitrogen. On the other hand, the survival of vitrified-expanded blastocysts kept at -80°C was low. Therefore, 8-cell embryos and morulae can be vitrified in a near-equilibrium state using the same method as for 2-cell embryos. A high proportion of C57BL/6J embryos at the 2-cell, 8-cell, and morula stages vitrified with EFS35c developed to term after transportation on dry ice, re-cooling in liquid nitrogen, and transfer to recipients. In conclusion, the near-equilibrium vitrification method, which is effective for 2-cell mouse embryos, is also effective for embryos at the 8-cell and morula stages. The method would enable handy transportation of vitrified embryos using dry ice.
authors.affiliation: Laboratory of Animal Science, College of Agriculture, Kochi University, Nankoku, Kochi, Japan.
authors.forename: Bo
authors.initials: B
authors.lastname: Jin
authors.forename: Keiji
authors.initials: K
authors.lastname: Mochida
authors.forename: Atsuo
authors.initials: A
authors.lastname: Ogura
authors.forename: Chihiro
authors.initials: C
authors.lastname: Koshimoto
authors.forename: Kazutsugu
authors.initials: K
authors.lastname: Matsukawa
authors.forename: Magosaburo
authors.initials: M
authors.lastname: Kasai
authors.forename: Keisuke
authors.initials: K
authors.lastname: Edashige
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000502:physiology*; D019855:Ethylene Glycol / Q000494:pharmacology; D005260:Female; D005362:Ficoll; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D009028:Morula / Q000502:physiology*; D013534:Survival; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents; D005362:Ficoll; D019855:Ethylene Glycol