Microwave-assisted dehydration, long-term storage at non-freezing temperatures, and rehydration of cat germinal vesicles†.

ABSTRACT

Germinal vesicles are alternative targets for female fertility preservation due to their availability and high resilience against non-physiological conditions. Preserved germinal vesicles can then be transferred to fresh cytoplasts to reconstitute viable oocytes. Here, we describe a germinal vesicle preservation method that employs non-ionizing microwave radiations imparting energy to water molecules, which results in rapid and homogeneous drying of the sample. Trehalose is added as a xero-protectant before the radiations, enabling isothermal vitrification of the disaccharide sugar during drying. While the technique is still considered experimental, studies have shown that DNA and structural integrity can be effectively maintained in dried/rehydrated germinal vesicles. Importantly, the dry-preservation approach allows supra-zero temperature storage of the samples, offering a cost-effective and energy-saving alternative to traditional methods relying on ultra-low freezing temperatures. The protocol outlines a comprehensive procedure involving germinal vesicle oocyte collection, trehalose loading, microwave drying, storage, and rehydration. The simplicity of the protocol facilitates the ease of manipulation, making it an accessible method for researchers. While initially developed for domestic cats, the protocol can be adapted for other species with necessary modifications, considering potential species-specific responses to dehydration stress.