Conventional freezing of in vitro-produced and biopsied bovine blastocysts in the presence of a low concentration of glycerol and sucrose.
The purpose of this study was to develop a practical cryopreservation method for in vitro-produced (IVP) and sex-predetermined bovine blastocysts that will be applicable to direct transfer of the post-thaw embryos. Blastocysts were harvested 7 days after IVF and allocated to either an intact or biopsy group. The cryoprotective solution contained 0.7 M glycerol and 0, 0.05 or 0.1 M sucrose. Slow cooling at a rate of -0.5 C/min was terminated at -25, -30, or -35 C, and rapid cooling in liquid nitrogen was followed. After one-step thawing and dilution, the IVP blastocysts were cultured for 3 days to assess their survival. The post-thaw survival rate of intact blastocysts after termination of slow cooling at -30 C in 0.7 M glycerol plus 0.1 M sucrose (96.2%) was significantly higher than that at -25 C in 0.7 M glycerol alone (44.4%). The post-thaw survival rate of biopsied bovine blastocysts after termination of slow cooling at -25 C in 0.7 M glycerol alone (53.8%) tended to be lower than that at -25 C in 0.7 M glycerol plus 0.05 M sucrose (91.3%) or -30 C in 0.7 M glycerol plus 0.1 M sucrose (92.3%). Thus, addition of a small amount of sucrose to 0.7 M glycerol cryoprotective solution shortened the process of slow cooling for both the intact and biopsied bovine embryos. Judged from the survival levels in vitro after thawing and one-step dilution of embryos (>80%), this is an improved method of cryopreservation for subsequent direct transfer of IVP and biopsied bovine blastocysts.
Tominaga
Keiichiro
K
Hyogo Prefectural Institute for Agriculture, Forestry and Fisheries, Kasai, Japan. ktomi-@nike.eonet.ne.jp
Iwaki
Fumiyuki
F
Hochi
Shinichi
S
D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D002417:Cattle; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000008:administration & dosage / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005307:Fertilization in Vitro; D005990:Glycerol / Q000008:administration & dosage / Q000494:pharmacology*; D009994:Osmolar Concentration; D013395:Sucrose / Q000008:administration & dosage / Q000494:pharmacology*; D013696:Temperature; D014021:Tissue Preservation / Q000379:methods
D003451:Cryoprotective Agents; D013395:Sucrose; D005990:Glycerol