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adrenocorticotrophic hormone and dexamethasone challenge
test’). The bird was placed in a cloth bag after the first sample, was used as a measure of body size. Factor one was plotted
and after 30 min a second blood sample was taken to measure
stress-induced CORT levels. Handling and placing in a cloth
bag is a standardized and well-established method to induce
stress responses in birds.
Experiment 2: population stress response
In this experiment, we collected a blood sample consecutively
in nine birds nesting in the same area (2 m diameter) at 5 min
intervals, i.e. at 0, 5, 10, 15, 20, 25, 30, 35 and 40 min after
the onset of the stressor, which was our entrance into the
chamber. This reflects the stress response of birds to human
disturbance and not to handling as in experiment 1. The pro-
cedure was repeated on different days until four groups of the
inner sub-colony and three groups of the outer sub-colony
were sampled. Immediately after blood sampling the birds
were measured and put back into their nests.
Experiment 3: adrenocorticotrophic hormone
and dexamethasone challenge test
In 46 individuals (23 of the inner colony and 23 of the outer
colony) a baseline sample was obtained within 1 min after
capture. Immediately after taking the first blood sample, birds
were injected intramuscularly with ACTH (Sigma; n = 6 inner
colony and n = 6 outer colony), dexamethasone (DEX,
Soldesam; n = 6 inner colony and n = 6 outer olony) or saline
(SAL; n = 3 inner colony and n = 3 outer colony). A further
eight individuals were not injected and were used as control
birds (CTL; n = 8 inner colony and n = 8 outer colony). Birds
were then placed in cloth bags, and a second blood sample
was taken after 30 min. In this experimental test, we used bio-
metrics in order to discriminate gender (Albores-Barajas et al.,
2010) and balance sexes between treatments.
Radioimmunoassay procedure
and biochemical validations
We measured plasma CORT concentration using a commer-
cial available CORT 125I radioimmunoassay kit (catalogue no.
07-120102; ICN Biomedicals/MP Biomedicals, Solon, OH,
USA). We followed the protocol of the company with the fol-
lowing modifications as described by Washburn et al. (2002):
the volume of all reagents was halved; the dilution of the sam-
ples was 1:50 instead of 1:200; and the standard curve was
extended by two points, resulting in eight standard points to
increase the sensitivity. Even though this protocol had been
validated for different bird species, we repeated the validation
for our species and could confirm the parallelisms. All samples
were analysed in duplicate, with an interassay coefficient of
variation <15% and intra-assay coefficient of variation
between 12 and 20%. The detection limit was 3 ng/ml plasma.
Statistics
We calculated body condition by entering bird biometrics
(wing length, tarsal length, bill depth and width and head
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Conservation Physiology • Volume 3 2015
size) into a principal components analysis, and factor one Downloaded from http://conphys.oxfordjournals.org/ by guest on March 12, 2016
against the weight of the individual, and the residuals were
used as an indicator of body condition (Catry and Furness,
1999).
We used generalized linear models (GLM) and two-way
repeated-measures analysis of variance to determine
d­ ifferences between disturbed and non-disturbed sub-­
colonies and serial samples (baseline and 30 min after cap-
ture). We used Spearman’s test in order to identify
correlations between variables. Data were analysed using
SPSS (SPSS, 2004) and R version 2.15.1 (R Development
Core Team, 2014), with significance defined as P < 0.05
for all analyses.
Results
We first tested for difference in body condition between indi-
viduals of the inside and outside sub-colonies, finding no sig-
nificant differences (sub-colony, F1,23 = 1.028, P = 0.321; and
sub-colony + year, F2,22 = 0.985, P = 0.390).
Experiment 1: individual stress response,
0–30 min (n = 8)
Storm petrels had a flat stress response in 2011 (time,
F1,6 = 0.004, P = 0.949; Fig. 1a) that did not differ between dis-
turbed (n = 4) and non-disturbed birds (n = 4; sub-colony,
F1,6 = 3.185, P = 0.125; and time × sub-colony, F1,6 = 0.17,
P = 0.899). Similar results were obtained in 2012 with the
birds of the control group of experiment 3 (time, F1,19 = 0.025,
P = 0.877; Fig. 1b). In fact, to increase the sample size, we also
analysed the individual stress response of control birds of
experiment 3 (see below in Experiment 3 paragraph), which,
like the birds of experiment 1, were handled to induce a stress
response but were not treated with any hormone or drug.
However, baseline values in 2012 were, although not signifi-
cantly, lower than in 2011 (F1,27 = 3.812, P = 0.061). There
was no significant difference between disturbed and non-dis-
turbed birds (sub-colony, F1,19 = 0.007, P = 0.934; and
time × sub-colony, F1,19 = 0.281, P = 0.602).
Considering all individuals sampled at 0 and at 30 min in
2011 (n = 8, experiment 1) and 2012 (n = 21, control group of
experiment 3), we found no correlation between body condi-
tion and both baseline CORT levels (S = 2152, ρ = 0.172,
P = 0.408) and stress response, namely change between 0 and
30 min (S = 259, ρ = 0.002, P = 0.995). We then fitted a linear
regression analysis to establish whether body condition alone
or additively with year or population had an effect on the
individual stress response. In all cases, we observed no signifi-
cant effects (body condition, F1,23 = 0.450, P = 0.125; body
condition + year, F2,22 = 1.422, P = 0.262; and body condi-
tion + sub-colony, F2,22 = 0.250, P = 0.782). We then tested for
the effect of the sampling year on individual stress response
and observed a tendency for year effects (year, F1,27 = 3.401,
P = 0.076).