85 VITRIFICATION IN VIVO OF LARGE EQUINE EMBRYOS AFTER VITRIFICATION OR CULTURE

Cryopreservation of large (>300 μm) equine embryos has been unsatisfactory using slow-cooling or vitrification techniques. The objective of the present experiments was to compare three methods for vitrification of large embryos using modified vitrification protocols for equine (Eldridge-Panuska et al. 2005 Theriogenology 63, 1308–1319) and bovine (2003 J. Anim. Sci. 81 Suppl. 1, 143) embryos. For Method 1, embryos (n = 14) 350–550 or 550–750 µm were exposed, respectively to, VS1 [1.4 M glycerol (G) in mPBS] for 5 min or 7 min, moved to VS2 [1.4 M G, 3.6 M ethylene glycol (EG) in mPBS] for 5 min or 7 min, and then transferred to VS3 (3.4 M G, 4.6 M EG in mPBS) for 40 or 60 s. In Method 2, embryos (n = 13) 350–550 or 550–750 μm were exposed, respectively, to VS1 for 10 or 14 min, VS2 for 10 or 14 min and VS3 for 60 or 90 s. Straws (0.25 mL) for Methods 1 and 2 were loaded with two columns of DS (0.5 M galactose in mPBS) and a small column of VS3 containing the embryo. Straws were heat-sealed and deposited in a goblet suspended in liquid N2 and containing vapor for 1 min and then plunged. Straws were warmed in air (24°C) for 10 sec and then in water at 20°C for 10 sec. Straws were shaken to mix the columns; after 3 min, embryos were expelled and re-hydrated in two additional solutions containing 0.3 and 0.15 M galactose for 3 min each. For Method 3, embryos (n = 17) 300–750 μm were placed in 1.5 M EG in mPBS for 5 min; 3 M EG in mPBS for 10 min; 5 M EG in mPBS for 5 min and 7 M EG, 0.5 M galactose, 18% w/v Ficoll 70 in mPBS for less than a minute. The droplet containing an embryo was loaded into a 0.25 mL straw that was preloaded with two columns of DS and followed by a small column of DS. Straws were heat-sealed and plunged vertically, sealed end fist, into liquid nitrogen covering the embryo, then the remainder of the straw was slowly immersed. Straws were warmed in air (24°C) for 10 s and then in water at 37°C for 10 s. Straws were shaken to mix the columns; after 3 min at 37°C, embryos were rehydrated as in Methods 1 and 2. Embryos were transferred nonsurgically to recipients 5 d after ovulation, and pregnancy diagnoses were performed 5 to 9 d after transfers. No embryonic vesicles were observed for embryos vitrified with Methods 1 and 2. The pregnancy rate for embryos vitrified with Method 3 was 35% (6/17) overall, and 55% (6/11) for embryos between 300–400 μm. No pregnancies resulted from embryos >400 μm. More studies are needed to optimize methodologies for dehydration, equilibration, and warming of large equine embryos.