Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA.
Division of Animal Sciences, University of Missouri, Columbia, MO, USA.
Sci Rep. 2024 Aug 13;14(1):18809. doi: 10.1038/s41598-024-69528-8.
Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction of humans, domestic and research animals, and endangered species using cryopreserved oocytes and embryos. The nature of ice formed in bovine oocytes (similar in size to oocytes of humans and most other mammals) after rapid cooling and during rapid warming was examined using synchrotron-based time-resolved x-ray diffraction. Using cooling rates, warming rates and CPA concentrations of current practice, oocytes show no ice after cooling but always develop large ice fractions-consistent with crystallization of most free water-during warming, so most ice-related damage must occur during warming. The detailed behavior of ice at warming depended on the nature of ice formed during cooling. Increasing cooling rates allows oocytes soaked as in current practice to remain essentially ice free during both cooling and warming. Much larger convective warming rates are demonstrated and will allow routine ice-free cryopreservation with smaller CPA concentrations. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes and establish the structure and grain size of ice formed. Ice formation can be eliminated as a factor affecting post-warming oocyte viability and development in many species, improving outcomes and allowing other deleterious effects of the cryopreservation cycle to be independently studied.
冰的损伤和冰抑制冷冻保护剂（CPAs）的潜在毒性是人类、家畜和研究动物以及濒危物种使用冷冻卵子和胚胎进行辅助繁殖的关键问题。使用基于同步加速器的时间分辨 X 射线衍射研究了快速冷却和快速升温过程中牛卵母细胞中形成的冰的性质（大小与人类和大多数其他哺乳动物的卵母细胞相似）。使用当前实践中的冷却速率、升温速率和 CPA 浓度，卵母细胞在冷却后没有形成冰，但在升温过程中总是会形成大的冰相——这与大多数游离水的结晶一致，因此大多数与冰相关的损伤必须发生在升温过程中。升温过程中冰的详细行为取决于冷却过程中形成的冰的性质。增加冷却速率可以使卵母细胞在冷却和升温过程中保持基本无冰状态，如当前实践中浸泡的方式。更大的对流升温速率也得到了证明，并且可以在使用更小 CPA 浓度的情况下实现常规的无冰冷冻保存。这些结果阐明了冷却、升温以及 CPA 浓度在卵母细胞中形成冰方面的作用，并确定了形成的冰的结构和晶粒尺寸。在许多物种中，冰的形成可以消除对升温后卵母细胞活力和发育的影响因素，提高结果，并允许独立研究冷冻保存周期的其他有害影响。
