Satoshi Ohara, Jumpei Yamazaki, Kyogo Hagino, Yuu Yoshimi, Naoya Matsumoto, Masami Yamanaka, Masanao Nakanishi, Sei Watanabe, Hideyuki Ito, Miho Murayama, Toshio Tsubota, Michito Shimozuru

Age information is essential for understanding ecology and developing effective management strategies for bears. While traditional tooth-based age estimation methods have long been used, alternative approaches based on DNA methylation levels have recently been developed in bear species using blood samples. Although blood sampling is less invasive than tooth extraction, it still requires procedures such as capture and anesthesia. In this study, we aimed to establish a non-invasive age estimation method based on DNA methylation using fecal DNA. Fecal samples were collected from 57 brown bears of known age living in both captivity and the wild. Bisulfite pyrosequencing was performed to measure the DNA methylation levels in two genomic regions, revealing positive correlations between age and methylation levels. The best age estimation model was constructed using four DNA methylation sites (cytosine-phosphate-guanine [CpG] sites): one adjacent to DLX5 gene and three adjacent to SLC12A5 gene. This model demonstrated high accuracy, with a mean absolute error of 2.08 years and a median absolute error of 0.99 years after leave-one-out cross-validation. Furthermore, our approach incorporated host (i.e., bear-derived) DNA copy number quantification, demonstrating that analyses tend to fail or produce large estimation errors when the amount of host DNA is insufficient. This represents a significant advancement over previous techniques based on fecal DNA methylation. Applying this novel age estimation method in field studies is expected to enhance ecological research and facilitate the development of effective monitoring and management strategies for brown bears. In addition, the method has the potential to be extended to other animal species in future studies.