title: Feasibility of a nylon-mesh holder for vitrification of bovine germinal vesicle oocytes in subsequent production of viable blastocysts.
abstract: To improve the feasibility of nylon-mesh holder for vitrification of bovine cumulus-oocytes complexes (GV-COCs) having germinal vesicle, this study was conducted to demonstrate effects of sugars and protocol of exposure in vitrification on subsequent in vitro maturation, ultrastructural changes, and in vitro development in bovine immature oocytes after cryopreservation using nylon mesh. Before vitrification, GV-COCs were exposed to the cryoprotectant, which was composed of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll-70, and 0.3 M sucrose (EFS40) or 0.3 M trehalose (EFT40), either by single step or in a stepwise way. The maturation rates in the stepwise exposure with EFS40 or EFT40 were significantly higher (P < 0.05) compared with the corresponding rates in the single step. In the stepwise exposure, few abnormalities were observed compared with the single-step exposure, where most oocytes showed a highly vacuolated cytoplasm with many ruptured mitochondria. Cleavage rates in fertilized oocytes previously exposed stepwise to EFS40 or EFT40 were significantly higher than those exposed by the single-step procedure. The cleaved embryos derived from the stepwise exposure to EFS40 developed to blastocysts. After transfer of blastocysts derived from vitrified GV oocytes, a female calf was born. These results indicate that vitrification of large numbers of bovine GV-COCs using a nylon-mesh holder accompanied with stepwise exposure minimizes structural damage in organelles, resulting in yield of viable blastocysts following in vitro embryo production.
authors.affiliation: Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiyamachi, Sendai 981-8555, Japan. abe-y@bios.tohoku.ac.jp
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Abe
authors.forename: Kenshiro
authors.initials: K
authors.lastname: Hara
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Matsumoto
authors.forename: Jin
authors.initials: J
authors.lastname: Kobayashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sasada
authors.forename: Hans
authors.initials: H
authors.lastname: Ekwall
authors.forename: Heriberto
authors.initials: H
authors.lastname: Rodriguez-Martinez
authors.forename: Eimei
authors.initials: E
authors.lastname: Sato
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D002417:Cattle; D003451:Cryoprotective Agents / Q000494:pharmacology; D019855:Ethylene Glycol / Q000494:pharmacology; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods; D008677:Metaphase / Q000502:physiology; D009865:Oocytes / Q000502:physiology*; D013395:Sucrose / Q000494:pharmacology; D014021:Tissue Preservation / Q000295:instrumentation* / Q000379:methods*; D014199:Trehalose / Q000494:pharmacology
chemical_list: D003451:Cryoprotective Agents; D013395:Sucrose; D014199:Trehalose; D019855:Ethylene Glycol