Commercial cryopreservation of bovine embryos has relied mainly on slow-cooling techniques. We have developed a simple, two step vitrification technique that permits in-straw dilution so that embryos can be transferred in the field. The purpose of this experiment was to study factorial combinations of two equilibration concentrations of ethylene glycol (EG) (3.5 and 5 M), two thawing temperatures (20 and 37°C) and two post-thawing waiting times until culture (5 and 15 min). A total of 486 blastocysts sired by three bulls were obtained in vitro in six replicates. Briefly, oocytes were aspirated from 2-8 mm follicles from slaughterhouse ovaries, matured and in vitro fertilized and cultured with standard procedures. The vitrification procedure used 0.25-ml straws that were preloaded with a 1 cm column of D-HCDM (0.5 M galactose in Hepes-buffered holding medium (HCDM2)), then 0.5 cm air, and then 7 cm of D-HCDM. Embryos were transferred to 1 ml of V1-CDM (3.5 M or 5M ethylene glycol in HCDM2) for 3 min at room temperature (24°C). Next, embryos were moved in 1 ml into a 7 ml droplet of V2-CDM (7 M ethylene glycol, 0.5 M galactose, 18% w/v Ficoll 70 in HCDM2) at 24°C. In less than 1 min, the droplet containing the embryo was loaded, followed by 0.5 cm air and 1 cm of D-HCDM. The straw was sealed with heat and plunged with the sealed end vertically into liquid nitrogen covering the embryo, and the rest of the straw was slowly immersed. Straws were thawed in air (24°C) for 10 sec and then in water horizontally at 20 or 37°C until ice disappeared. Straws were gently shaken to mix the columns; then, after 5 or 15 min at 24 or 37°C, embryos were expelled and put in culture in CDM2 + 5% FCS. Re-expansion and hatching rates were evaluated 48 h post thawing. Data (Table 1) were calculated as a percentage of non-vitrified controls for respective replicates (control means: re-expansion 86 and hatching 54%) and analyzed by ANOVA.

Main effect means for re-expansion were higher (P=0.08) for 5 M (87 %) than 3.5 M (78%) of EG for equilibration; also, re-expansion (88 and 76% of controls) and hatching (86 and 54 % of controls) were higher for 37 than 20°C (P<0.05). There was no difference between post-thawing waiting times for either response (P>0.05), but there was an interaction between time and temperature for re-expansion (P<0.05), indicating poor survival when embryos were thawed at 20°C and kept for 15 min. Data (Table 1) indicate that 5 M and 37 °C are optimal procedures, and embryos can be kept up to 15 min. Further studies including transfer of embryos to recipients are necessary to corroborate these results.

Key words: embryo cryopreservation, vitrification, direct transfer.