Survival and viability of vitrified in vitro and in vivo produced ovine blastocysts

Ovine blastocysts were produced by maturation, fertilization and in vitro c ulture (IVM/IVF/IVC) of oocytes from slaughtered adult and prepubertal ewes and collection from superovulated and inseminated adult animals. Dulbecco' s PBS supplemented with 0.3 mM Na Pyruvate and 20% FCS was used as the basi c cryopreservation solution. The embryos were exposed to the vitrification solution as follows: 10% glycerol (G) for 5 min, then 10% G +20% ethylene g lycol (EG) for 5 min. Embryos were placed into 25% G + 25% EG in the center of 0.25- mL, straws and plunged immediately into LN2. Warming was done by placing the straws into a water bath at 37 degrees C for 20 sec, and their contents were expelled into a 0.5 M sucrose solution for 3 min; the embryos were then transferred into 0.25 M and 0.125 M sucrose solution for 3 min e ach. Warmed blastocysts were transferred to the culture medium for 24 h. Su rvival was defined as the re-expansion of the blastocoele. All surviving bl astocysts were transferred to synchronized recipient ewes, and the pregnanc y was allowed to go to term. Of 68 vitrified in vitro produced blastocysts, 46 re-expanded (67.6%) and 10 lambs were born (14.7%). From the 62 in vivo derived and vitrified embryos, 52 re-expanded (83.8%) and 39 lambs were bo rn (62.9%). The lambing rate of in vitro produced fresh transfer embryos wa s 40% (20 lambs/50 blastocysts transferred), and of the 32 in vivo derived blastocysts and transferred fresh, 26 lambs were born (81.2%). The results indicate that in vitro produced embryos can be successfully cryopreserved b y vitrification. (C) 2000 by Elsevier Science Inc.