Establishment of pregnancies after serial dilution or direct transfer by vitrified equine embryos

Experiments were conducted to determine viability of equine embryos in vivo after vitrification. In a preliminary study (Experiment 1), embryos were exposed in three steps to vitrification solutions containing increasing concentrations of ethylene glycol and glycerol (EG/G); the final vitrification solution was 3.4 M glycerol + 4.6 M ethylene glycol in a base medium of phosphate-buffered saline. Embryos were warmed in a two-step dilution and transferred into uteri of recipients. No pregnancies were observed after transfer of blastocysts >300 μm (n = 3). Transfer of morulae or blastocysts ≤300 μm resulted in four embryonic vesicles (4/6, 67%). In a second experiment, embryo recovery per ovulation was similar for collections on Day 6 (28/36, 78%) versus Days 7 and 8 (30/48, 62%). Embryos ≤300 and >300 μm were vitrified, thawed and transferred as in Experiment 1. Some embryos ≤300 μm were also transferred using a direct-transfer procedure (DT). Embryo development rates to Day 16 were not different for embryos ≤300 μm that were treated as in Experiment 1 (10/22, 46%) or transferred by DT (16/26, 62%). Embryos >300 μm (n = 19) did not produce embryonic vesicles.