Slow-cooling techniques are widely used in cryopreservation of bovine embryos. We have previously developed a simple, two-step vitrification technique for direct transfer in the field; however, simplification to one-step vitrification would be attractive. Therefore, factorial combinations of two techniques (one-step and two-step) 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. Briefly, oocytes were aspirated from 2–8-mm follicles of ovaries obtained at a slaughterhouse, matured, fertilized and cultured in vitro with standard procedures using chemically defined media (CDM1/2 or G1/2). Two-step embryos were transferred in 1μL into 1mL of V1-CDM (5M ethylene glycol (EG) in HEPES-buffered holding medium (HCDM2)), and one-step embryos into a 7-μL droplet of V2-CDM (7M EG, 0.5M galactose and 18% w/v Ficoll 70 in HCDM2) for 3min at 24°C. Next, embryos for the two-step method were moved in 1μL into a 7μL droplet of V2-CDM at 24°C. Droplets containing embryos (one or two-step) were loaded into 0.25-mL straws preloaded with a 1-cm column of D-HCDM (0.5M galactose in HCDM2), then 0.5cm air, and then 7cm of D-HCDM followed by 0.5cm air. The column containing the embryos (0.5cm (7μL)) was followed with 0.5cm air and 1cm 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 was then slowly immersed. The time from loading to plunging was 40–50s. Straws were thawed in air (24°C) for 10s and then in water horizontally at 37°C until ice disappeared. Straws were gently shaken to mix the columns; then, after 5min 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 (Table 1) 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 (P>0.1), indicating that, after thawing, embryos can be kept at room or body temperature. Also, main effect means for re-expansion and hatching for one-step or two-step addition of cryoprotectant were similar (P>0.1), but 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.
Table 1
Main effect means (least-square means±SEM) of vitrified embryos (% of non-vitrified controls)
A Discussion of Weighting Procedures for Unit Nonresponse