One-step sucrose dilution of frozen-thawed sheep embryos

Sheep embryos of the late morula to early blastocyst stage were frozen, thawed and cultured to test several sucrose solutions for post-thaw dilution of the cryoprotective agent glycerol. Ewes of mixed breeding were superovulated and embryos were flushed from the uterus either surgically or at slaughter 5 d after estrus. Fifty-eight embryos were pooled in microdrops of modified Dulbecco's phosphate buffered saline (MDPBS) then randomly divided into four treatments. A 2 x 2 factorial design was used to compare 0.25 M sucrose in MDPBS as an in-straw cryoprotectant dilution with a standard step-wise dilution procedure within standard fast and slow freeze-thaw systems. After storage in liquid nitrogen for 6 to 8 d, the embryos were thawed and the cryoprotectant (1.4 M glycerol) removed before culture in microdrops of modified synthetic oviduct fluid under paraffin oil in water-saturated 5% CO(2) in air atmosphere at 37 C. No significant interaction was found between the freeze-thaw procedure and cryoprotectant + dilution procedures. Embryos in the fast freeze-thaw procedure had a mean development score of 1.3 +/- 0.3 and those in the slow freeze-thaw procedure had a mean score of 1.2 +/- 0.3. The mean development score 2.0 +/- 0.3 for the standard dilution procedure was superior (P<0.001) to the score of 0.6 +/- 0.2 for the 0.25 M sucrose dilution procedure. In a separate trial, 18 sheep morulae were collected and equilibrated with 1.4 M glycerol in MDPBS. A standard fast freeze-thaw procedure was used and, after 18 d of storage at -196 C, the glycerol was diluted from the embryo with 1.0 M sucrose. Culture was conducted in a similar manner and a mean development score of 1.0 +/- 0.3 was obtained. These results indicate standard cryoprotectant dilution procedures for sheep embryos are superior to dilution with 0.25 M sucrose. In a limited study, dilution with 1.0 M sucrose was also not as effective as standard dilution procedures.