Slow-cooling techniques are widely used for cryopreserving bovine embryos. We have developed a simple, two step vitrification technique for direct transfer of embryos in the field; however, simplification to one step addition of cryoprotectants would be attractive. Therefore, factorial combinations of one step vs two step addition of cryoprotectant and two post-thaw temperatures until culture (24 and 37°C) were studied. Blastocysts (n=220) sired by two bulls were obtained in vitro in four replicates. Oocytes were aspirated from 2-8 mm follicles of ovaries obtained at a slaughterhouse, and matured, fertilized and cultured in vitro with standard procedures using chemically defined media (CDM1/2 or G1/2). Two step embryos were transferred in 1 ml into 1 ml of V1-CDM (5 M ethylene glycol (EG) in Hepes-buffered holding medium (HCDM2)) for 3 min at 24°C and then were moved in 1 ml into a 7 ml droplet of V2-CDM (7 M EG, 0.5 M galactose and 18% w/v Ficoll 70 in HCDM2) at 24°C. Droplets containing embryos then were loaded into 0.25-ml straws preloaded with a 1 cm column of D-HCDM (0.5 M galactose in HCDM2), then 0.5 cm air, and then 7 cm of D-HCDM followed by 0.5 cm air. The column containing the embryos (~0.5 cm (7 ml)) was followed with 0.5 cm air and 1 cm of D-HCDM. Straws were heat-sealed and plunged vertically, sealed end first, into liquid nitrogen just covering the embryo, and the rest of the straw then was slowly immersed. The time from placing embryos in V2-CDM, including loading to plunging was 40-50 sec. One step embryos were treated similarly except that they were placed in V2-CDM for 3 min before plunging. Straws were thawed in air (24°C) for 10 sec and then in water at 37°C until ice disappeared. Straws were gently shaken to mix the columns; then, after 5 min at 24 or 37°C, embryos were expelled and cultured in CDM2 + 5% FCS. Re-expansion and hatching rates were evaluated 48 h post thaw. Data (Table1) were calculated as a percentage of non-vitrified controls for respective replicates (control means: re-expansion 87%; hatching 74%) and analyzed by ANOVA.

There were no main effects of post-thawing temperature or vitrification technique (P>0.1) for either re-expansion or hatching, although there was a tendency for higher survival for the two step procedure. Further refinements of the one step technique including EG concentrations, embryological stages and equilibration times should be studied.