Title: Open-pulled Straw (OPS) Vitrification of Mouse Hatched Blastocysts
Abstract: Abstract This study was first employed to investigate the developmental potential of mouse hatched blastocyts (HBs) vitrified by a two-step open-pulled straw (OPS) method. HBs were obtained by culture of morulae in vitro. First, the embryos were placed in four cryprotectant solutions—that is, 10% ethylene glycol (EG), 10%E + 10%D (10% EG and 10% dimethyl sulphoxide (DMSO) in mPBS), EFS30 (30% EG, Ficoll, and sucrose) and EDFS30 (15% EG, 15% DMSO, Ficoll, and sucrose)—at 25°C for 0.5 to 10 min, respectively, to determine their optimal survival after rapid dilution in 0.5 M sucrose. Secondly, based on the above best survival, the embryos were plunged into liquid nitrogen after first pretreatment in 10%E for 0.5 min and then 0.5 min equilibration in EFS30 (Group 1), or 10%E + 10%D and EDFS30 for 0.5 min, respectively (Group 2). When warming, three methods were used to dilute the cryoprotectants from the vitrified embryos. The embryos were assessed by the re-expansion of the blastocoel or development to term. The result showed that all the vitrified-warmed HBs got high in vitro survival rates (83.7% to 98.9%). The highest in vitro survival rates (87.8% in Group 1, 98.9% in Group 2) were obtained when the vitrified embryos were diluted first in 0.3 M sucrose for 5 min, then in 0.15 M sucrose for 2 min (method C). When the vitrified embryos diluted with method C were transferred, their survival rate in vivo (35.5% to 42.2% of the total) were similar to (P > 0.05) that of control (45.7%). These results demonstrate OPS method was highly efficient for the cryopreservation of mouse HBs. Keywords: Hatched blastocystKunming mouseOPSVitrification C. Zhou and G.-B. Zhou contributed equally to this work. This work was supported by Beijing key agricultural project (H022020060420) and state “863” planning (Project 2004AA213071). Notes 1EG = ethylene glycol, DMSO = dimethyl sulfoxide. 2FS = mPBS medium containing 300 g/L Ficoll, 171.2 g/L sucrose, and 3 mg/ml BSA. 3A day earlier before the 10%E and 10%E + 10%D were used, they were supplemented with 3 mg/ml BSA. 1Survival embryos are defined as the ones that re-expanded after 24 h of culture. 2Number of survival embryos/Number of embryos recovered. 3Hatched blastocysts were exposed mPBS for 30 min before culture. ∗P < 0.05 ∗∗P < 0.01, significantly different from the control. 1The mouse hatched blastocysts were vitrified after first pretreatment with 10%E for 0.5 min then exposure to EFS30 for 0.5 min in Group 1, while in Group 2, 10%E + 10%D for 0.5 min then exposure to EDFS30 for 0.5 min. 2A: The vitrified embryos were warmed in 0.5 M sucrose for 5 min. B: The vitrified embryos were warmed first in 0.5 M sucrose for 2 min, then in 0.3 M sucrose for 5 min. C: The vitrified embryos were diluted first in 0.3 M sucrose for 5 min, then in 0.15 M sucrose for 2 min. 3Embryos that re-expanded after 24 h of culture. a-bvalues with different superscripts in the same column differ significantly (P < 0.05); ∗∗P < 0.01, significantly different from the control. 1The mouse hatched blastocysts were vitrified after first pretreatment with 10%E for 0.5 min then exposure to EFS30 for 0.5 min in Group 1, while in Group 2, 10%E + 10%D for 0.5 min then exposure to EDFS30 for 0.5 min. 2Number of recipients delivered/number of recipients transferred. aValues with same superscripts within each column did not differ significantly (P > 0.05).
Publication Year: 2007
Publication Date: 2007-01-24
Language: en
Type: article
Indexed In: ['crossref', 'pubmed']
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Cited By Count: 11
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