Michito Shimozuru, Hokkaido University

Age is an essential information for understanding the ecology and management of bears. Instead of traditional age estimation method by counting cementum annuli of the teeth, those based on DNA methylation levels have been developed for wildlife over the past two decades. Recently, we have established age estimation method based on blood DNA methylation levels in multiple bear species. However, it is sometimes difficult to obtain fresh blood samples from dead bears, which makes it desirable to develop a method using more accessible samples. This study aimed to construct age estimation models based on liver- and muscle-derived DNA in brown bears and Asian black bears. Liver and muscle samples were collected postmortem from both captive and wild bears with known ages. After bisulfite treatment of the extracted DNA, PCR amplification was performed on 12 DNA regions, and DNA methylation levels at multiple cytosine-phosphate-guanine (CpG) sites were obtained by a next generation sequencer. The results showed a significant positive correlation between age and methylation levels at 11 CpGs located in 6 DNA regions in liver DNA. However, no such correlations were observed in any targeted CpGs in muscle DNA. An age estimation model based on the obtained methylation levels is under development and its accuracy is being validated. Using liver DNA for age estimation would allow for simpler and more accurate age determination of dead bears (hunted or killed for management purposes), thereby contributing to bear population monitoring and management.