
%0 Journal Article
%A Campos-Chillon, L. F. 
%A Cox, T. J. 
%A Seidel Jr, G. E. 
%A Carnevale, E. M. 
%T 85 VITRIFICATION <i>IN VIVO</i> OF LARGE EQUINE EMBRYOS AFTER VITRIFICATION OR CULTURE
%J Reproduction, Fertility and Development
%D 2005/12/14
%V 18
%N 2
%P 151-151
%K 
%R https://doi.org/10.1071/RDV18N2AB85
%U https://www.publish.csiro.au/paper/RDV18N2AB85 
%X Cryopreservation of large (>300 &mu;m) 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&ndash;1319) and bovine (2003 J. Anim. Sci. 81 Suppl. 1, 143) embryos. For Method 1, embryos (n &equals; 14) 350&ndash;550 or 550&ndash;750 &micro;m 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 &equals; 13) 350&ndash;550 or 550&ndash;750 &mu;m 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&deg;C) for 10 sec and then in water at 20&deg;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 &equals; 17) 300&ndash;750 &mu;m 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&percnt; 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&deg;C) for 10 s and then in water at 37&deg;C for 10 s. Straws were shaken to mix the columns; after 3 min at 37&deg;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&percnt; (6/17) overall, and 55&percnt; (6/11) for embryos between 300&ndash;400 &mu;m. No pregnancies resulted from embryos >400 &mu;m. More studies are needed to optimize methodologies for dehydration, equilibration, and warming of large equine embryos. 
