Kyoto University is going ahead with a project to extract ovarian tissue from endangered mammal species after they die, and preserving the material by freezing it. The university has collected reproductive material from around 30 species including the Siberian tiger and Tsushima leopard cat.

It's hoped that the project could lead to the artificial breeding of rare wild animals through research into rearing immature eggs cells in the collected tissue, and the establishment of a technology or means to carry out in-vitro fertilization.

So far, prevailing techniques in artificial breeding at zoos and other institutions have largely been done using sperm, which is easier to obtain in large amounts, to inseminate females. The barriers are greater for obtaining eggs ready to fertilize, however, as some animals such as pandas, only ovulate once a year. Plus, even if the mature eggs are successfully extracted, their high water content reportedly makes them difficult to keep in frozen preservation.

Nevertheless, Mayako Fujihara, a Japan Society for the Promotion of Science Research fellow who specializes in conservation ecology, has been using the Wildlife Research Center of Kyoto University as a base to focus on issues around preserving ovarian tissue.

An ovary contains a vast amount of primordial follicles, which serve as the basis for eggs. In the case of a dog, for example, around 140,000 primordial follicles are estimated to be in its ovaries when it's born. It's possible that the follicles could be artificially matured to create new possibilities for breeding animals.

Aoba, a female Tsushima leopard cat, is seen at Fukuoka Zoo & Botanical Garden, in Fukuoka's Chuo Ward on Oct. 7, 2019. (Mainichi/Mayu Suenaga)

From 2016, Kyoto University has been asking zoos and other facilities in Japan for animals' ovaries after they die. So far it has received material from around 40 animals and some 30 species, including chimpanzees and koalas. It keeps the contributions stored in special liquid nitrogen receptacles.

The issue that presents itself now is the establishment of a technology or system to rear fertilizable eggs from the primordial follicles in the ovaries. In experiments using material from cats and dogs, researchers could only get as far as the secondary follicle stage of development -- two stages before the matured egg -- and a breakthrough with a more detailed mechanism is required.

Dog cells were successfully cultivated on the back of a laboratory rat, and it would be possible to do the same on a gel in the case of cat cells, which led researchers to conclude that the necessary time, nutrients and methods to bring the follicles to maturation may vary depending on the animal species.

Fujihara said, "The preservation of ovaries is effective to preserve the diversity of genes of rare animals. I also want to look at methods to freeze them at zoos."

Mayako Fujihara, a Japan Society for the Promotion of Science Research fellow, is seen at Kyoto University in Sakyo Ward, Kyoto, on Feb. 13, 2020. (Mainichi/Sachiko Miyakawa)

National institutions and others are engaging in efforts for the preservation of animals' genetic resources, which is hoped to lead to contributions in preserving species themselves. But under the present circumstances, the focus tends to be on resources such as somatic cells and sperm that are relatively easier to collect and store, and this too is presenting its own problems.

At the National Institute for Environmental Studies (NIES) in eastern Japan's Ibaraki Prefecture, reproductive cells and the cells from the hides of around 100 species of rare wild animals are kept in storage. However, the only animal that it keeps eggs for is the Tsushima leopard cat. Similarly, the Preservation and Research Center in Yokohama, which specializes in the rearing and breeding of rare animals, has stocks of sperm from around 50 animal species, but eggs from only three. The disparities are reportedly down to difficulties keeping eggs preserved in a healthy state. Tama Zoological Park in Tokyo only keeps sperm for around 20 species.

Manabu Onuma, a senior researcher at the NIES' Center for Environmental Biology and Ecosystem Studies, said, "It's rare for an institution to have the technology to preserve ovarian tissue, and there are also issues around different storage requirements for different species. But if the technology was put in place, perhaps we would see more efficient breeding of endangered species."

Institutions including the Ibaraki-based National Agriculture and Food Research Organization, a national research and development agency, are working on establishing preservation technology for the unfertilized and undeveloped eggs from cows and pigs, among other farm animals. But moves to do so are reportedly not widespread.

The organization's Embryo Production Research Unit leader Yuji Hirao expressed his hopes for increased uptake, saying, "At present, the major research theme is to improve breeding practices using sperm. If technology to preserve and cultivate immature eggs can become widespread, then the possibility of it being paired with other technology and work widens. It will be useful as an agricultural method to produce better quality meat, and other such improvements."

(Japanese original by Sachiko Miyakawa, Osaka City News Department)