Cryopreservation Competence of Chicken Oocytes as a Model of Endangered Wild Birds: Effects of Storage Time and Temperature on the Ovarian Follicle Survival

SIMPLE SUMMARY: Many avian species are classified as endangered, and the development of the gamete preservation technique is essential to preserve their genetic diversity. Since cryopreservation and transplantation of ovarian tissues are currently the only methods of female fertility preservation in birds after birth, transporting the ovary to the laboratory in good condition is key for successful female fertility preservation. Here, we report using chickens as a model of wild birds for which storage at low temperature (4 °C), but not room temperature, can maintain the ovary, ensuring oocyte survival, morphology and protein expression of germ cell markers for at least 3 days. Furthermore, a vitrification study confirmed that oocytes after ovary storage at a low temperature can be preserved by ovarian tissue cryopreservation. On the other hand, ovaries stored at room temperature decreased oocyte survival, while increasing gene expression of apoptosis and oxidation stress markers. Our study not only verifies the fact that the storage of hen ovaries at low temperatures protects oocytes, which can be preserved by vitrification, but it also contributes to understanding the effect of ovary storage on the integrity and cryotolerance of immature follicles within the hen ovary. ABSTRACT: For the conservation of endangered avian species, developing gamete preservation technologies is essential. However, studies in oocytes have not been widely conducted. In this study, assuming that the ovaries are transported to a research facility after death, we investigated the effect of ovary storage on oocytes for the purpose of cryopreserving avian female gametes by using a chicken as a model of endangered avian species. After excision, the ovaries were stored at either a low temperature (4 °C) or room temperature for 1–3 days. Ovarian follicles stored under different conditions for each period were examined by neutral red staining, histology, and gene and protein expression analysis. In addition, the pH of the storage medium after preserving the ovaries was measured. Then, ovarian tissues were vitrified to determine the cryopreservation competence. Storing the ovarian tissues at 4 °C kept the follicles viable and morphologically normal for 3 days with slow decline. In contrast, although different storage temperature did not influence follicle viability and morphology after only 1 day of storage, ovarian tissues stored at room temperature rapidly declined in structurally normal follicles, and viable follicles were rarely seen after 3 days of storage. Gene and protein expression analysis showed that apoptosis had already started on the first day, as shown by the higher expression of CASP9 under room temperature conditions. Furthermore, high expression of SOD1 and a rapid decline of pH in the storage medium under room temperature storage suggested the influence of oxidative stress associated with low pH in this condition on the follicle survivability in hen ovarian tissues. Our cryopreservation study also showed that ovarian tissues stored at 4 °C could recover after cryopreservation even after 3 days of storage. The described storage conditions and cryopreservation methods, which preserve chicken follicle survival, will lay the foundation of ovarian tissue preservation to preserve the fertility of wild female birds.