Effect of dehydration prior to cryopreservation of large equine embryos

Cryopreservation of equine embryos>300microm in diameter results in low survival rates using protocols that work well for smaller equine embryos. These experiments tested the potential benefit of incorporating a dehydration step prior to standard cryopreservation procedures. Forty-six, day 7-8, grade 1, equine embryos 300-1350microm in diameter were subjected to one of the following treatments: (A) 2 min in 0.6M galactose, 10min in 1.5M glycerol, slow freeze (n=21); (B) 10min in 1.5M glycerol, slow freeze (n=15); (C) 2min in 0.6M galactose, 10min in 1.5M glycerol, followed by exposure to thaw solutions, then culture medium (n=5); (D) transferred directly to culture medium (n=5). Frozen embryos were thawed and subjected to a three-step cryoprotectant removal. Five embryos from each treatment were evaluated morphologically after 24 and 48h culture (1=excellent, 5=degenerate/dead). All treatments had at least 4/5 embryos with a quality score >or=3 at these time points except treatment B (2/5 at 24h, 1/5 at 48h). Subsequent embryos from treatment A (n=16) or B (n=10) were matched in sets of two for size and treatment, thawed, and immediately transferred in pairs to 13 recipients. Only two recipient mares were pregnant; one received two 400microm embryos from treatment A, and the other one 400 and one 415microm embryo from treatment B. There was no advantage of incorporating a 2min dehydration step into the cryopreservation protocol for large equine embryos.