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Peer-reviewedCC BYScientific Reports

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

  • Satoshi Ohara1
  • Shiori Nakamura1
  • Kyogo Hagino2
  • Yuu Yoshimi2
  • Naoya Matsumoto3
  • Masami Yamanaka4
  • Masanao Nakanishi4
  • Sei Watanabe4
  • Hideyuki Ito5
  • Miho Inoue-Murayama6
  • Toshio Tsubota1
  • Jumpei Yamazaki1
1 more authors
  • Michito Shimozuru1
6 affiliations
  1. 1Faculty of Veterinary Medicine, Hokkaido University
  2. 2Noboribetsu Bear Park
  3. 3Azabu University
  4. 4Shiretoko Nature Foundation
  5. 5Kyoto City Zoo
  6. 6Wildlife Research Center, Kyoto University
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Abstract

Age is important for understanding the life history of wildlife. In this study, we established a non-invasive age estimation method based on DNA methylation levels using fecal DNA collected from 43 brown bears of known age (0–28 years; 27 females and 16 males) living in both captivity (18 bears) and the wild (25 bears). Bisulfite pyrosequencing was performed to determine the methylation levels of fecal DNA, and the best model was constructed based on four cytosine-phosphate-guanine (CpG) sites: one adjacent to DLX5 and three adjacent to SLC12A5. The mean absolute error after leave-one-out cross-validation was 2.08 years, and the median absolute error was 0.99 years; these results demonstrate high accuracy. Furthermore, a method was implemented for quantifying host DNA copy numbers, demonstrating that analyses tend to fail or exhibit large estimation errors when the amount of host DNA is insufficient. This is a significant advancement compared to previous techniques that relied on fecal DNA methylation levels. Applying this method to field surveys will greatly contribute to ecological research and the development of appropriate conservation and management strategies for bears, while facilitating future epigenetic clock studies on other animals.

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Cite

BibTeXRIS
BibTeX
@article{ohara2026establishment,
  title = {{Establishment of a non-invasive age estimation method based on fecal DNA methylation levels in brown bears}},
  author = {Satoshi Ohara and Shiori Nakamura and Kyogo Hagino and Yuu Yoshimi and Naoya Matsumoto and Masami Yamanaka and Masanao Nakanishi and Sei Watanabe and Hideyuki Ito and Miho Inoue-Murayama and Toshio Tsubota and Jumpei Yamazaki and Michito Shimozuru},
  journal = {Scientific Reports},
  year = {2026},
  month = jul,
  doi = {10.1038/s41598-026-63215-6},
  url = {https://doi.org/10.1038/s41598-026-63215-6},
  note = {CC BY},
}
RIS
TY  - JOUR
TI  - Establishment of a non-invasive age estimation method based on fecal DNA methylation levels in brown bears
AU  - Satoshi Ohara
AU  - Shiori Nakamura
AU  - Kyogo Hagino
AU  - Yuu Yoshimi
AU  - Naoya Matsumoto
AU  - Masami Yamanaka
AU  - Masanao Nakanishi
AU  - Sei Watanabe
AU  - Hideyuki Ito
AU  - Miho Inoue-Murayama
AU  - Toshio Tsubota
AU  - Jumpei Yamazaki
AU  - Michito Shimozuru
PY  - 2026
DA  - 2026/07/01
JO  - Scientific Reports
DO  - 10.1038/s41598-026-63215-6
UR  - https://doi.org/10.1038/s41598-026-63215-6
AB  - Age is important for understanding the life history of wildlife. In this study, we established a non-invasive age estimation method based on DNA methylation levels using fecal DNA collected from 43 brown bears of known age (0–28 years; 27 females and 16 males) living in both captivity (18 bears) and the wild (25 bears). Bisulfite pyrosequencing was performed to determine the methylation levels of fecal DNA, and the best model was constructed based on four cytosine-phosphate-guanine (CpG) sites: one adjacent to DLX5 and three adjacent to SLC12A5. The mean absolute error after leave-one-out cross-validation was 2.08 years, and the median absolute error was 0.99 years; these results demonstrate high accuracy. Furthermore, a method was implemented for quantifying host DNA copy numbers, demonstrating that analyses tend to fail or exhibit large estimation errors when the amount of host DNA is insufficient. This is a significant advancement compared to previous techniques that relied on fecal DNA methylation levels. Applying this method to field surveys will greatly contribute to ecological research and the development of appropriate conservation and management strategies for bears, while facilitating future epigenetic clock studies on other animals.
SN  - 2045-2322
ER  -