Establishment of a non-invasive age estimation method based on fecal DNA methylation levels in brown bears

We would like to express our profound gratitude and appreciation to all the people who cooperated with the sampling, especially to all the staff of the Shiretoko Nature Foundation, Noboribetsu bear park, and Sapporo Maruyama Zoo (especially to Mr. Wataru Goshima and Mr. Michiaki Shimizu) for providing bear fecal samples. We wish to thank Hatsusaburo Ose and all the members of the Shiretoko Fishery Productive Association for their kind support. Finally, we thank Editage (www.editage.jp) for English language editing.

This study was supported by funding from the Japan Society for the Promotion of Science (JSPS) (https://www.jsps.go.jp/english/e-grants/index.html) KAKENHI (grant numbers: JP19K06833, JP22K14910, JP23K05312, JP24KJ0304, JP25K22873 and JP25H01002), the Collaborative Research Program of Wildlife Research Center, Kyoto University (grant no. 2021-A-16), and Grant for Basic Science Research Projects from The Sumitomo Foundation (grant no. 200561). This research was performed by the Environment Research and Technology Development Fund (JPMEERF20254002) of the Environmental Restoration and Conservation Agency provided by Ministry of the Environment of Japan.

1. Satoshi Ohara and Shiori Nakamura, contributed equally to this work. Michito Shimozuru and Jumpei Yamazaki contributed equally co-corresponding authors.

Authors and Affiliations

1. Faculty of Veterinary Medicine, Hokkaido University, Kita-18, Nishi-9, Kita-ku, Sapporo, 060-0818, Hokkaido, Japan

   Satoshi Ohara, Shiori Nakamura, Toshio Tsubota, Jumpei Yamazaki & Michito Shimozuru

2. Noboribetsu Bear Park, 224 Noboribetsuonsencho, Noboribetsu, 059- 0551, Hokkaido, Japan

   Kyogo Hagino & Yuu Yoshimi

3. Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, 252-5201, Kanagawa, Japan

   Naoya Matsumoto

4. Shiretoko Nature Foundation, 531 Iwaobetsu, Shari, 099-4356, Hokkaido, Japan

   Masami Yamanaka, Masanao Nakanishi & Sei Watanabe

5. Kyoto City Zoo, Okazaki Hosshojicho, Kyoto, 606-8333, Japan

   Hideyuki Ito

6. Wildlife Research Center, Kyoto University, 2-24 Tanakasekidencho, Sakyo-ku, Kyoto, 606-8203, Japan

   Miho Inoue-Murayama

7. One Health Research Center, Hokkaido University, Kita-18, Nishi-9, Kita-ku, Sapporo, 060-0818, Hokkaido, Japan

   Jumpei Yamazaki & Michito Shimozuru

Corresponding authors

Correspondence to
Jumpei Yamazaki or Michito Shimozuru.

Competing interests

The authors declare no competing interests.

Ethical approval

All procedures involved in sample collection from live animals in the Shiretoko National Park (incl. aSpecial Wildlife Protection Area) were conducted in accordance with the Guidelines for Animal Care andUse, Hokkaido University, and were approved by the Animal Care and Use Committee of the GraduateSchool of Veterinary Medicine, Hokkaido University (permit nos: 1106, 1151, 1152, 15009, 17005, 18-0083, 19-0021, 20-0146, and 23-0014) and by the Hokkaido Regional Environment Office and KushiroNature Conservation Office of the Ministry of the Environment, Japan (Permit nos: 2105071, 2205061,2305161, 2405291, and 2504221). In addition, all methods were carried out in compliance with ARRIVEguidelines.

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