123 VITRIFICATION OF IN VITRO-PRODUCED BOVINE BLASTOCYSTS BY ADDITION OF CRYOPROTECTANT IN ONE STEP

Abstract

Vitrification combined with in-straw dilution may replace conventional cryopreservation of bovine embryos, but this requires further study for practicality. Our objectives were to compare three ethylene glycol concentrations (6, 7, and 8 M) and two equilibration times (2.5 and 3.5 min) for one-step addition of cryoprotectant. In vitro-matured oocytes from slaughterhouse ovaries, fertilized using sperm of 3 bulls, were cultured in chemically defined medium (CDM-1/CDM-2) plus FAF-BSA to produce 420 blastocysts. Day 7.5 embryos were placed into HCDM-2 (HEPES-buffered medium) and then transferred to a 6 μL drop of vitrification solution (V) (6, 7, or 8 M ethylene glycol, 0.5 M galactose, and 18% w/v Ficoll 70 in HCDM-2). Immediately thereafter, 1 cm column of DHCDM (0.5 M galactose in HCDM-2) was drawn into a 0.25 mL straw, followed by a 0.5 cm column of air and another 7 cm of DHCDM. Another 0.5 cm column of air was aspirated before the 6 μL of V (0.5 cm) containing the embryos were aspirated; then 0.5 cm of air followed. Finally, DHCDM was drawn until the first column came into contact with the cotton plug. Straws were then heat-sealed and plunged into liquid nitrogen slightly above the embryos after 2.5 or 3.5 min equilibration. The rest of the straw was then submerged slowly. Straws were thawed in air for 10 s and then in 37°C water for 20 s. Straws were held at room temperature (24°C) for 4 min before being expelled into HCDM-2. They were then placed into CDM-2 + 5% FCS for culture. Quality score (1 = excellent, 2 = fair, 3 = poor), survival (S) as determined by expansion of blastocysts, and hatching (H) were assessed at 24 and 48 h post-thaw. Data from 6 replicates (2/bull) were analyzed by ANOVA after arc sin transformation of percentage data. S and H responses were calculated as a percentage of non-frozen controls in the same replicate. Control survival and hatching rates were: 24S: 90%, 24H: 50%, 48S: 90%, 48H: 72%. Quality scores at both 24 and 48 h were higher (P < 0.05) for 8 M than 6 M ethylene glycol (2.68 and 3.24 for 24 h; 2.55 and 3.17 for 48 h); values for 7 M ethylene glycol were intermediate. Equilibration time had no effect on embryo quality (P > 0.1). Neither ethylene glycol concentration nor exposure time affected survival or hatching at 24 or 48 h (P > 0.1). Survival rates (as a % of control) at 48 h were: 8 M: 57%, 7 M: 55%, 6 M: 36% and hatching: 8 M: 39%, 7 M: 30%, and 6 M: 21%; 2.5 min tended to be better than 3.5 min for survival at 24 h, hatching at 24 h, survival at 48 h, but not hatching at 48 h (56% and 43%, 30% and 26%, 55% and 44%, 28% and 32% respectively). Higher concentrations of ethylene glycol proved beneficial in terms of embryo quality, with the same trend for survival and hatching rates. One-step addition of cryoprotectant for vitrification shows potential for simplifying embryo cryopreservation. However, further research is needed to produce more acceptable survival rates and to study vitrification of in vivo-produced embryos.