Effect of freezing rate and exposure time to cryoprotectant on the development of mouse pronuclear stage embryos.

BACKGROUND The effects of exposure time (20 versus 45 s) to a high concentration of cryoprotectant (7.0 mol/l ethylene glycol with 0.5 mol/l sucrose) and freezing rates (1200-10 300 degrees C/min) during rapid freezing of mouse pronuclear stage embryos on survival and development to blastocysts were investigated. Different freezing rates were achieved by directly plunging the straws (rapid freezing) and open pulled straws (OPS) in liquid nitrogen (OPS freezing) and by plunging the straws (super rapid) and OPS (super OPS) in a super cooled liquid nitrogen chamber (at -212 degrees C) before storage in liquid nitrogen. METHODS Morphologically intact mouse zygotes (n = 891) pre-equilibrated in 1.5 mol/l ethylene glycol for 5 min were either loaded in 0.25 ml straws containing cryoprotectant or loaded in OPS with 2 microl cryoprotectant. After 20 or 45 s of loading the straws or mixing in cryoprotectant and loading in OPS, they were plunged either directly in to liquid nitrogen or were plunged first in to liquid nitrogen in a super cooled chamber and then stored in liquid nitrogen. Zygotes were thawed and intact embryos cultured in vitro. RESULTS The rate of survival was higher (91%, P < 0.01) when zygotes were frozen with rapid freezing compared with super rapid, OPS and super OPS freezing rates with an exposure time of 20 s (70, 65, and 76% respectively). When zygotes were exposed to cryoprotectant for 45 s and frozen with rapid freezing rates, the survival was higher (86%, P < 0.01) compared with those frozen with OPS (62%) but was not different from those frozen with super rapid and super OPS freezing rates (81 and 75%). A higher rate of survival was observed when zygotes were exposed to cryoprotectant for 45 s and frozen with super OPS than with OPS freezing (75 versus 62%; P < 0.05). The rate of cleavage and development of intact zygotes to blastocysts was not different among the different groups. CONCLUSION Exposure of zygotes to a high concentration of cryoprotectant (7.0 mol/l ethylene glycol with 0.5 mol/l sucrose) for 20 or 45 s did not influence their survival and development and increasing the freezing rate from 1200-10 300 degrees C/min was of no advantage when using a rapid freezing procedure for freezing mouse pronuclear stage embryos.