Fecal corticosterone metabolite (FCM) measurement
FCM levels reflect the level of circulating corticosterone that occurred
10–12 h previously in root voles (He et al., 2013), and FCM is derived
primarily from plasma-free corticosterone in rodents (Sheriff et al.,
2010). Faecal samples for the FCM analysis were collected during the
first 2 h of trapping (09:00–11:00 AM), and each captured animal was
collected once within a 3-day trapping session; thus, all animals
provided only a single sample in each trapping session. Meanwhile, each
trap was cleaned with water before collecting the faecal sample,
ensuring that the samples were not influenced by the previous trapping
or time of day. Traps used to sample faeces only had a few carrots.
Faecal samples from pregnant
females were not collected to avoid confounding effects of reproduction
states on FCM levels (McDonald, 1998; Edwards & Boonstra, 2018). The
total number of faecal samples was 546 and 832 in 2012 and 2015,
respectively, throughout each experiment, and they accounted for 59%
and 67% of the sum of minimum number known alive in each trapping
session throughout the duration of experiments in both years (excluding
reproductive females). The collected samples were, then, frozen in ice,
transported to the laboratory, and stored in a −20°C freezer until
analysis. FCM was measured following the methods outlined by Yang et al.
(2018), previously validated for root voles. First, the collected faecal
samples were lyophilised (Labconco, Kansas City, MO, USA) for 14–18 h,
ground into particles, and homogenised in 0.5 mL NaOH solution (0.04 M).
The extraction of FCM was performed by adding 5 mL of
CH2Cl2 to the sample (0.1 g), followed
by sonication for 15 min (Pihl & Hau, 2003) and centrifugation for 15
min at 3,000 g . After centrifugation, 1 mL of the solution was
taken from the organic layer, diluted with 3 mL
CH2Cl2, and then mixed with 4 mL of a
mixed solution of sulfuric acid and ethanol (7:3, v:v). The samples
were, then, shaken for 2 min and rested for 30 min before separation of
the sulfuric acid layer for fluorescence detection. The fluorescence
density in each sample was measured using an RF-540 IPC Fluorometer
(Shimadzu, Japan) at excitation and emission wavelengths of 470 and 520
nm, respectively, and the FCM concentration in each sample was
calculated based on the fluorescence densities produced by varying
concentrations of the standard (Chen et al., 2012).