PREGNANCIES FOLLOWING TRANSFER OF EQUINE EMBRYOS CRYOPRESERVED BY VITRIFICATION

The objective of this study was to investigate the in vitro and in viv o developmental abilities of equine embryos cryopreserved by vitrifica tion. Twenty-eight embryos were recovered from Native pony and Thoroug hbred mares at Days 5 to 7 by nonsurgical uterine flushing (detection of ovulation=Day 0). The vitrification solution contained 40% ethylene glycol, 18% Ficoll, and 0.3 M sucrose in PBS. The embryos were placed for 1 to 2 min in vitrification solution (Group 1) or following expos ure to 20% ethylene glycol in PBS for 10 to 20 min (Groups 2 and 3). S ingle embryos were loaded in 0.25-ml straws, cooled for 1 min in liqui d nitrogen vapor and immersed in liquid nitrogen. Straws were warmed i n water (20 degrees C, 20 sec), and the contents were expelled with 0. 5 M sucrose in PBS. Then the sucrose was diluted in 1-step (Groups 1 a nd 2) or 4-steps (Group 3). Embryos (n=21) were cultured for 120 h in TCM199 supplemented with 10% fetal bovine serum at 37 degrees C in 5% CO2 in air and evaluated morphologically. Development to the hatching or hatched blastocyst stage was obtained in 0/7, 4/7 and 4/7 embryos i n Groups 1, 2 and 3, respectively. An additional 7 embryos were vitrif ied-warmed according to the treatment of Group 2 (4 embryos) and Group 3 (3 embryos). Five embryos were selected after in vitro culture for 4 h and were transferred nonsurgically into the uterine horn of Day-4 recipient mares. Transfer of 2 embryos (both Day-6 blastocysts: Group- 2 treatment) resulted in pregnancies with a viable fetus at Day-60 of the gestation period.