Abdelhady, A. W.; Mittan-Moreau, D. W.; Crane, P. L.; McLeod, M. J.; Cheong, S.-H.; Thorne, R. E.

Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction using cryopreserved oocytes and embryos. We use synchrotronbased time-resolved x-ray diffraction and tools from protein cryocrystallography to characterize ice formation within bovine oocytes after cooling at rates between ~1000 {degrees} C/min and ~600,000 {degrees}C/min and during warming at rates between 20,000 and 150,000 {degrees}C/min. Maximum crystalline icediffraction intensity, maximum ice volume, and maximum ice grain size are always observed during warming. All decrease with increasing CPA concentration, consistent with the decreasing free water fraction. With the cooling rates, warming rates and CPA concentrations of current practice, oocytes may show no ice after cooling but always develop substantial ice fractions on warming, and modestly reducing CPA concentrations causes substantial ice to form during cooling. With much larger cooling and warming rates achieved using cryocrystallography tools, oocytes soaked as in current practice remain essentially ice free during both cooling and warming, and when soaked in half-strength CPA solution oocytes remain ice free after cooling and develop small grain ice during warming. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes, establish the character of ice formed, and suggest that substantial further improvements in warming rates are feasible. Ice formation can be eliminated as a factor affecting post-thaw oocyte viability and development, allowing other deleterious effects of the cryopreservation cycle to be studied, and osmotic stress and CPA toxicity reduced.