Introduction

Maintaining a stable dry state is critical for long-term preservation of live biomaterials at suprazero temperatures. The objective of the study was to characterize the effect of moisture content on DNA integrity within the germinal vesicle (GV) of feline oocytes following dehydration and storage at 22–24 °C.

Methods

Using microwave-assisted drying, conditions that led to a predictable and stable moisture content in trehalose solutions were determined. To explore moisture content stability during storage, trehalose samples were dried for 15 min and stored in glass vials at 11 or 43% RH for 8 weeks. To examine whether this condition allowed proper storage of GVs, permeabilized cat oocytes were incubated in trehalose for 10 min and dried for 15 or 30 min. Oocytes then were rehydrated to assess DNA integrity either directly after drying or after 8 weeks of storage in an 11% RH environment. Raman spectroscopy was used to identify the states of dried samples during storage.

Results

Moisture content was stable during the storage period. There was no significant difference in DNA integrity between fresh and dried samples without storage. After 8 weeks of storage, DNA integrity was maintained in GVs dried for 30 min. Samples dried for 15 min and stored were compromised, suggesting crystallization of the preservation matrix during storage. Biostabilization was optimal when samples were directly processed to moisture contents consistent with storage in the glassy state.

Conclusion

Microwave-assisted drying processing and storage conditions were optimized to ensure stable long-term storage of structural and functional properties of genetic resources.

中文翻译：

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在超零温度下保存海藻糖玻璃中的雌性基因组：猫胚泡中水分含量与 DNA 损伤之间的关系

介绍

保持稳定的干燥状态对于在超零温度下长期保存活的生物材料至关重要。该研究的目的是描述水分含量对猫卵母细胞在 22-24°C 下脱水和储存后生泡 (GV) 内 DNA 完整性的影响。

方法

使用微波辅助干燥，确定了导致海藻糖溶液中水分含量可预测且稳定的条件。为了探索储存过程中的水分含量稳定性，将海藻糖样品干燥 15 分钟并在 11 或 43% RH 的玻璃小瓶中储存 8 周。为了检查这种条件是否允许正确储存 GV，将透化的猫卵母细胞在海藻糖中孵育 10 分钟并干燥 15 或 30 分钟。然后，在干燥后或在 11% RH 环境中储存 8 周后，将卵母细胞再水化以评估 DNA 完整性。拉曼光谱用于识别干燥样品在储存过程中的状态。

结果

贮藏期间水分含量稳定。未经储存的新鲜和干燥样品之间的 DNA 完整性没有显着差异。储存 8 周后，在干燥 30 分钟的 GV 中保持 DNA 完整性。干燥 15 分钟并储存的样品受到损害，表明保存基质在储存过程中结晶。当样品直接处理到与玻璃态储存一致的水分含量时，生物稳定性是最佳的。

结论

优化微波辅助干燥处理和储存条件，确保遗传资源结构和功能特性的长期稳定储存。