Effect of dehydration prior to cryopreservation of large equine embryos

Abstract

Cryopreservation of equine embryos > 300 μm 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 300–1350 μm in diameter were subjected to one of the following treatments: (A) 2 min in 0.6 M galactose, 10 min in 1.5 M glycerol, slow freeze ( n = 21); (B) 10 min in 1.5 M glycerol, slow freeze ( n = 15); (C) 2 min in 0.6 M galactose, 10 min in 1.5 M 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 48 h culture (1 = excellent, 5 = degenerate/dead). All treatments had at least 4/5 embryos with a quality score ⩾ 3 at these time points except treatment B (2/5 at 24 h, 1/5 at 48 h). 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 400 μm embryos from treatment A, and the other one 400 and one 415 μm embryo from treatment B. There was no advantage of incorporating a 2 min dehydration step into the cryopreservation protocol for large equine embryos.