Zona fracture damage and its avoidance during the cryopreservation of mammalian embryos

Although fracture damage to the zonae pellucidae and blastomeres is frequently observed after the cryopreservation of mammalian embryos, little is known of the mechanism by which this occurs. The incidence of damage to zonae was measured when bovine ova with normal zonae were frozen in straws or glass test tubes by standard embryo cryopreservation procedures that yield high rates of survival. Ova were examined for zona damage after warming by procedures that ought to produce little or no thermal stress (slow warming in 20 degrees C air) or high levels of stress (rapid warming in liquid baths). Ova frozen in straws exhibited no zona damage after slow warming at 150 degrees C/min in air (n = 206). However, the incidence of zona damage increased when the straws were warmed rapidly in 20 degrees C (n = 157) or 36 degrees C (n = 159) water (17 and 24%, respectively). Ova in straws warmed rapidly in nonaqueous liquids (ethylene glycol, or silicone oil) exhibited lower rates of zona damage (2 to 5%). Ova frozen in glass tubes exhibited a much higher incidence of zona damage than those frozen in straws, regardless of the warming conditions. Thus, 30% of 114 ova exhibited damage when tubes were warmed slowly at 25 degrees C/min in air, while 54% of 98 ova showed zona damage when tubes were warmed rapidly at 500 degrees C/min in 36 degrees C water. These results are consistent with the view that zona damage is associated with thermally-induced fracturing of the suspension during rapid changes of temperature.