Successful vitrification of pronuclear-stage rabbit zygotes by minimum volume cooling procedure.
Rabbit zygotes at the pronuclear-stage were cryopreserved by vitrification using a gel-loading tip (GL-tip), Cryoloop or Cryotop. In GL-tip and Cryoloop methods, zygotes were first exposed to 10% ethylene glycol (EG)+10% DMSO in TCM199+20% fetal bovine serum (FBS) for 2 min, and then equilibrated for 30 s in a vitrification solution composed of 20% EG+20% DMSO+0.6 M sucrose in TCM199+20% FBS. In Cryotop method, zygotes were first exposed to 7.5% EG+7.5% DMSO+20% FBS in TCM199 for 3 min, and then equilibrated for 1 min in a vitrification solution composed of 15% EG+15% DMSO+0.5 M sucrose+20% FBS in TCM199. In vitro culture of vitrified-warmed zygotes using GL-tip and Cryoloop resulted in low cleavage rates (2 and 5%, respectively) and no development into blastocysts. In contrast, zygotes vitrified-warmed using Cryotop exhibited higher proportions of cleavage (58%) and development into blastocysts (24%). When compacted morulae or early blastocysts were vitrified by these three procedures, 80-93% of them exhibited blastocoele expansion or zona hatching during the subsequent 48 h of culture. Use of Cryotop instead of GL-tip or Cryoloop for zygote vitrification, without changing conditions of solutions and periods for exposure, equilibration and post-warm dilution, resulted in cleavage and blastocyst development rates of 88 and 45%, respectively. A longer exposure time (10 min) of zygotes to 7.5% EG+7.5% DMSO+20% FBS in TCM199 resulted in higher proportions of zygotes cleaving (94%) and developing into blastocysts (51%) after Cryotop vitrification. Proportions of post-warm zygotes (10-min exposure group) and fresh control zygotes developing into newborn offspring were 36 and 53%, respectively. Pronuclear-stage rabbit zygotes were successfully cryopreserved by vitrification using the Cryotop method.
Hochi
S
S
Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano 386-8567, Japan. shochi@giptc.shinshu-u.ac.jp
Terao
T
T
Kamei
M
M
Kato
M
M
Hirabayashi
M
M
Hirao
M
M
D000818:Animals; D001755:Blastocyst / Q000502:physiology; D002417:Cattle; D002970:Cleavage Stage, Ovum; D015925:Cryopreservation / Q000295:instrumentation / Q000379:methods / Q000662:veterinary*; D046508:Culture Techniques; D004121:Dimethyl Sulfoxide; D019855:Ethylene Glycol; D005312:Fetal Blood; D006358:Hot Temperature; D009028:Morula / Q000502:physiology; D011817:Rabbits / Q000196:embryology*; D015053:Zygote / Q000502:physiology*
D019855:Ethylene Glycol; D004121:Dimethyl Sulfoxide