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 concent ration of cryoprotectant (7.0 mol/l ethylene glycol with 0.5 mol/l sucrose) and freezing rates (1200-10300 degreesC/min) during rapid freezing of mous e 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 degreesC) before stora ge in liquid nitrogen. METHODS: Morphologically intact mouse zygotes (n = 8 91) pre-equilibrated in 1.5 mol/l ethylene glycol for 5 min were either loa ded in 0.25 nil straws containing cryoprotectant or loaded in OPS with 2 mu l cryoprotectant. After 20 or 45 s of loading the straws or mixing in cryop rotectant and loading in OPS, they were plunged either directly in to liqui d nitrogen or were plunged first in to liquid nitrogen in a super cooled ch amber and then stored in liquid nitrogen. Zygotes were thawed and intact em bryos cultured in vitro. RESULTS: The rate of survival was higher (91%, P < 0.01) when zygotes were frozen with rapid freezing compared with super rap id, 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 t hose frozen with super rapid and super OPS freezing rates (81 and 75%). A h igher rate of survival was observed when zygotes were exposed to cryoprotec tant for 45 s and frozen with super OPS than with OPS freezing (75 versus 6 2%; P < 0.05). The rate of cleavage and development of intact zygotes to bl astocysts was not different among the different groups. CONCLUSION: Exposur e 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 survi val and development and increasing the freezing rate from 1200-10300 degree sC/min was of no advantage when using a rapid freezing procedure for freezi ng mouse pronuclear stage embryos.