RECIPIENT SYNCHRONIZATION AFFECTS VIABILITY OF VITRIFIED OVINE BLASTOCYSTS

We assessed the viability of vitrified ovine blastocysts in vitro and in recipient ewes at different degrees of synchronization. Expanded ovine blastocysts obtained from superovulated Sarda ewes were exposed to a vitrification solution at 20 degrees C according to the following procedures: the blastocysts were added into 200 mu l glycerol (1.4 mol) for 5 min, then into 200 mu l glycerol (1.4 mol) and ethylene glyco (13.6 mol) for 5 min. These embryos were transferred into 25 mu l glycerol (3.4 mol) and ethylene glycol (4.6 mol) and loaded into the center of 0.25-ml straws separated from air bubbles by 2 columns of 90 mu l sucrose solution (0.5 mol). The straws were plunged immediately into liquid nitrogen (LN(2) ). The basic vitrification and thawing solution was phosphate buffered saline (PBS) supplemented with 20% fetal calf serum (FCS). The embryos were thawed by agitating the straws in a water bath at 20 degrees C, and expelling them into Petri dishes and mixing them, and placing the embryos into 200 mu l sucrose solution (0.25) mol for 5 min. The percentage of hatched blasticysts, after exposed and vitrified-thawed embryos, was 96.8% (30/31) and 82.8% (53/64), respectively, after 4 d of co-culture in TCM 199 medium with 10% FCS in 5% CO2 humidified atmosphere in air at 39 degrees C. Vitrified-thawed blastocysts, which re-formed the blastocoelic cavity after 24 h of co-culture as in the in vitro experiment, were surgically transferred into the uterine horn of recipients (2 embryos per ewe) at 6, 7 and 8 d after estrus. The pregnancy and lambing rates in recipient ewes at 6, 7 and 8 d after estrus were 72.7%, 90%, 54.5% and 72.7%, 80%, 45.4%, respectively. Both pregnancy and lambing rates differed significantly (P < 0.05) between the Day 7 and Day 8 groups.