Cryopreservation of equine embryos by open pulled straw, cryoloop, or conventional slow cooling methods

Cryopreservation of equine embryos with conventional slow-cooling procedure s has proven challenging. An alternative approach is vitrification, which c an minimize chilling injuries by increasing the rates of cooling and warmin g. The open pulled straw (OPS) and cryoloop have been used for very rapid c ooling and warming rates. The objective of this experiment was to compare e fficacy of vitrification of embryos in OPS and the cryoloop to conventional slow cool procedures using 0.25 mL straws. Grade 1 or 2 morulae and early blastocysts (less than or equal to 300 mum i n diameter) were recovered from mares on Day 6 or 7 post ovulation. Twenty- seven embryos were assigned to three cryopreservation treatments: (1) conve ntional slow cooling (0.5 degrees /min) with 1.8 M ethylne glycol (EG) and 0.1 M sucrose, (4) vitrification in OPS in 16.5% EG, 16.5% DMSO and 0.5 M s ucrose, or (3) vitrification with a cryoloop in 17.5% EG, 17.5% DMSO, 1 M s ucrose and 0.25 muM ficoll. Embryos were evaluated for size and morphologic al quality (Grade I to 4) before freezing, after thawing, and after culture for 20 h. In addition, propidium iodide (PI) and Hoechst 33342 staining we re used to assess percent live cells after culture. There were no differenc es (P > 0.1) in morphological grade or percent live cells among methods. Me an grades for embryos after culture were 2.9 +/- 0.2, 3.1 +/- 0.1, and 3.3 +/- 0.2 for conventional slow cooling, OPS and cryolop methods, respectivel y. Embryo grade and percent live cells were correlated, r = 0.66 (P < 0.004 ). Thus OPS and the cryoloop were similarly effective to conventional slow- cooling procedures for cryopreserving small equine embryos. (C) 2001 by Els evier Science Inc.