title: Double vitrification of rat embryos at different developmental stages using an identical protocol.
abstract: The aim of the present investigation was to test the effectiveness of a method of vitrifying rat embryos at different stages of development (from early morula to expanding blastocyst) in a double vitrification procedure. Wistar rat embryos were vitrified and warmed in super-fine open-pulled straws (SOPS). Before being plunged into liquid nitrogen, the embryos were exposed to 40% ethylene glycol+0.75 M sucrose in TCM-199+20% fetal calf serum (FCS) for 20s at 38 degrees C. Subsequent warming and direct rehydration of the embryos was conducted in culture medium (TCM-199+20% FCS) at 38 degrees C. Early morula stage (7-10 blastomeres) embryos (n=358) were vitrified, warmed and cultured in vitro (EM group). Batches of these embryos were then cryopreserved again (revitrified) at the early blastocyst (EB group, n=87), blastocyst (B group, n=93) or expanding blastocyst stage (ExpB group, n=73). After the first (EM group) and repeated (EB, B, and ExpB groups) vitrification procedures, developmental rates of 81, 83, 34 and 76%, respectively were achieved (for EM-EB-ExpB P>0.1; for EM, EB, ExpB-B P<0.005). Our data demonstrate the possibility of using the described identical protocol for the SOPS vitrification of rat early morulae, early blastocysts and expanding blastocysts. The low survival rate of blastocysts subjected to double vitrification requires further investigation.
authors.affiliation: Department of Animal Production, Center for Agricultural Researches, P.O. Box 727, Zaragoza 50080, Spain. vovaisachenko@yahoo.com
authors.forename: Vladimir
authors.initials: V
authors.lastname: Isachenko
authors.forename: José
authors.initials: J
authors.lastname: Folch
authors.forename: Eugenia
authors.initials: E
authors.lastname: Isachenko
authors.forename: Frank
authors.initials: F
authors.lastname: Nawroth
authors.forename: Alexander
authors.initials: A
authors.lastname: Krivokharchenko
authors.forename: Gábor
authors.initials: G
authors.lastname: Vajta
authors.forename: Maria
authors.initials: M
authors.lastname: Dattena
authors.forename: José Luis
authors.initials: JL
authors.lastname: Alabart
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000502:physiology; D002417:Cattle; D015925:Cryopreservation / Q000379:methods*; D003470:Culture Media; D046508:Culture Techniques; D004622:Embryo, Mammalian / Q000502:physiology*; D005314:Embryonic and Fetal Development; D019855:Ethylene Glycol; D005260:Female; D005312:Fetal Blood; D006358:Hot Temperature; D016015:Logistic Models; D009028:Morula / Q000502:physiology; D009584:Nitrogen; D051381:Rats; D017208:Rats, Wistar; D013395:Sucrose
chemical_list: D003470:Culture Media; D013395:Sucrose; D019855:Ethylene Glycol; D009584:Nitrogen