title: Open-pulled straw vitrification differentiates cryotolerance of in vitro cultured rabbit embryos at the eight-cell stage.
abstract: The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5-17.7%, 1.5-4.3% and 2.8-3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4-25.7 vs 13.7-26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8-3.7%) than in fresh controls (28.6-35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.
authors.affiliation: Department of Animal Science, National Chung Hsing University, Taichung, Taiwan, ROC.
authors.forename: T A
authors.initials: TA
authors.lastname: Lin
authors.forename: C H
authors.initials: CH
authors.lastname: Chen
authors.forename: L Y
authors.initials: LY
authors.lastname: Sung
authors.forename: M G
authors.initials: MG
authors.lastname: Carter
authors.forename: Y E
authors.initials: YE
authors.lastname: Chen
authors.forename: F
authors.initials: F
authors.lastname: Du
authors.forename: J C
authors.initials: JC
authors.lastname: Ju
authors.forename: J
authors.initials: J
authors.lastname: Xu
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian; D047108:Embryonic Development; D005260:Female; D011817:Rabbits / Q000196:embryology*; D027724:Reproductive Techniques, Assisted / Q000662:veterinary