(376) Development of a Yeast Bioassay for the Assessment of Phyto-Oestrogen Interactions in kākāpō

Mayako Fujihara¹, Takehito Kaneko², Takashi Nagamine³, Yumiko Nakaya³, Chinatsu Nishimoto⁴, Manabu Onuma⁵, Keisuke Saito⁶, Yukiko Watanabe⁶, Miho Inoue-Murayama¹

1            Wildlife Research Center, Kyoto University, Kyoto, Japan

2            Graduate School of Veterinary Science, Osaka Metropolitan University, Osaka, Japan

3            Okinawa Wildlife Federation, Okinawa, Japan

4       Graduate School of Science, Kyoto University, Kyoto, Japan

5            National Institute for Environmental Studies, Ibaraki, Japan

6       Institute for Raptor Biomedicine Japan, Kushiro, Japan

Abstract Text:

Japan is abundant in biodiversity with different climates in each region. Okinawa, the southern island of Japan, has endemic bird species, the Okinawa rail (Hypotaenidia okinawae), and Hokkaido, the most northern island, is a habitat for large raptors, the Steller’s Sea-Eagle (Haliaeetus pelagicus) and the White-tailed Eagle (H. albicilla). These are listed as national endangered species of wild fauna and flora in Japan (Law for the Conservation of Endangered Species of Wild Fauna and Flora), and are classified as endangered species in the Japanese Red Data Book. The Japanese Ministry of Environment has been leading breeding projects for these species to restore the population in their habitat, and sustainable reproduction with stable genetic diversity is the major issue for their ex-situ conservation. Therefore, developing reproductive techniques preserving and utilizing the germplasm are all essential to promote their captive breeding. Our team has worked together on a project to address these needs in Okinawa rail and large raptors by the Environment Research and Technology Development Fund (JPMEERF20244M01) of the Environmental Restoration and Conservation Agency of Japan. To develop the assisted reproductive techniques using the germplasm in wild birds, the optimal transportation and cryopreservation protocol for ovarian tissue were investigated by comparing different temperatures and cryoprotectants in chickens as model animals. In the meantime, we have built a transportation system of ovaries between their habitats and a laboratory. Then, the ovarian tissues of wild endangered birds were vitrified with the protocol developed in chicken, and the viability of oocytes before and after vitrification were examined by neutral red staining and histological observation.

The ovaries of the Okinawa rail (n = 7), the Steller’s Sea-Eagle (n = 1), and the White-tailed Eagle (n = 1) were collected from the animals that recently died in their habitat in Okinawa or Hokkaido. The ovaries from their wild habitat were transported at 4°C and arrived within 1 to 2 days at the laboratory in Kyoto. The viable oocytes were observed in all ovaries by neutral red staining regardless of species. The structurally normal oocytes were observed before and after ovarian tissue vitrification, although normality decreased after vitrification. Our ovarian tissue cryopreservation strategy in collaboration between habitat and laboratories, which increase oocyte survival from carcasses, will lay the foundation of germplasm preservation to preserve the fertility of wild female birds. Thus, we believe this project will provide both fundamental information and genetic resources for their reproduction and accelerate the ex-situ conservation of the Okinawa rail, the Steller’s Sea-Eagle, the White-tailed Eagle and other endangered avian species.