Study area, animals, and blood sampling
Captive bears
Blood samples were obtained from 34 brown bears (17 males and 17
females) kept at Noboribetsu Bear Park (Noboribetsu, Hokkaido, Japan;
Figure 1) during 2020–2022. Their ages ranged from 2 to 34 years old
(Supplementary Table S_M1). Four individuals were rescued in the wild
at 0 years of age and were moved to the facility, while all others were
born in the facility. All individuals were fed bear pellets (ZOOFOOD
bear; Nosan Co., Kanagawa, Japan), concentrated feed formulated for cows
(Soyokazenokaori MG; Nippon Formula Food Manufacturing Co., Ltd.,
Kanagawa, Japan), dog food (Maibitto; PETLINE Inc., Gifu, Japan),
vegetables, and fruits once or twice daily. Water was provided ad
libitum . Bears fed in winter and did not hibernate. Prior to blood
sampling, bears were anesthetized via intramuscular administration of
xylazine HCl (1 mg/kg; Selactar; Bayer, Tokyo, Japan) and a 1:1 mixture
of zolazepam HCl and tiletamine HCl (2.0–4.0 mg/kg; Zoletil 100;
Virbac, Carros, France) using a blow dart. After blood sampling was
completed, atipamezole HCl (1 mg/kg; Atipame; Kyoritsu Co., Ltd., Tokyo,
Japan) was injected intramuscularly to aid recovery from anesthesia.
Blood samples were collected via the medial saphenous vein into vacuum
tubes containing ethylenediaminetetraacetic acid disodium (EDTA-2Na) as
an anticoagulant. The collected blood samples were stored at −80°C as
whole blood or buffy coat until genomic DNA extraction. Buffy coat
samples were obtained by centrifuging blood samples at 1,880 × g for 10
minutes.
Wild bears
We used 31 blood samples from 13 wild brown bears (1 male and 12
females) living in the Rusha area (44°11′N, 145°11′E) of the Shiretoko
Peninsula, eastern Hokkaido, Japan (Figure 1). Seven bears were sampled
multiple times at different ages. Their ages ranged from 1 to 26 years
old (Supplementary Table S_M1). In this area, long-term bear monitoring
surveys have been conducted since 1997 until the present, enabling age
determination based on visual and DNA-based identification (Shimozuru et
al. 2017).
Bears were anesthetized via intramuscular administration of a 1:1
mixture of zolazepam HCl and tiletamine HCl (5.5 mg/kg; Zoletil 100;
Virbac) and 75 µg/kg medetomidine HCL (Dorbene Vet; Kyoritsu Co., Ltd.)
based on estimated body weight using an air rifle. After immobilization,
bears were weighed, and blood was collected. When blood sampling was
completed, atipamezole HCl (375 µg/kg; Atipame; Kyoritsu Co., Ltd.) was
injected intramuscularly to aid recovery from anesthesia. Blood samples
were collected via the jugular vein into vacuum tubes containing
EDTA-2Na as an anticoagulant. The collected blood samples were stored at
−80°C as whole blood or buffy coat until genomic DNA extraction. Buffy
coat samples were obtained by centrifuging blood samples at 1,880 × g
for 10 minutes.
Additionally, two female cubs-of-the-year that were captured with their
mother in a barrel trap were sampled in Shibetsu Town, located in the
southeastern part of the Shiretoko Peninsula. Bears were anesthetized
using a blow dart for intramuscular administration of a 1:1 mixture of
zolazepam HCl and tiletamine HCl (3 mg/kg; Zoletil 100; Virbac) and 40
µg/kg medetomidine HCL (Dorbene Vet; Kyoritsu Co., Ltd.), and then were
awakened using atipamezole HCl (200 µg/kg; Atipame; Kyoritsu Co., Ltd.).
All other procedures were conducted as described above.
Information about the samples collected for this study is summarized in
Supplementary Table S_M1. Ages were determined at the time of blood
sampling based on the assumption that all bears were born on February 1
(Friebe et al. 2014).