Physiological stress levels predict survival probabilities in wild rabbits
Sonia Cabezas a,⁎ , Julio Blas b , Tracy A. Marchant b , Sacramento Moreno a
a
Department of Applied Biology, Estación Biológica de Doñana, Spanish Council for Scientific Research–CSIC, Avenida María Luisa s/n. E-41013, Sevilla, Spain
b
Department of Biology, University of Saskatchewan, 112 Science Pl., Saskatoon, SK, Canada S7N 5E2
Abstract
Among vertebrates, short-term elevations of glucocorticoid hormones (corticosterone or cortisol) facilitate a suite of physiological and
behavioral changes aimed at overcoming environmental perturbations or other stressful events. However, chronically elevated glucocorticoids can
have deleterious physiological consequences, and it is still unclear as to what constitutes an adaptive physiological response to long-term stress. In
this study, we experimentally exposed European wild rabbits Oryctolagus cuniculus to a source of long-term stress (simulated through a 2- to 4week period of captivity) and tested whether glucocorticoid physiology predicted two major components of rabbit fitness: body condition and
survival probability. Following exposure to long-term stress, moderately elevated serum corticosterone and fecal glucocorticoid metabolites levels
in the wild rabbits were negatively associated with body condition, but positively associated with subsequent survival upon release. Our results
suggest that the cost of maintaining elevated corticosterone levels in terms of decreased body condition is balanced by the increased chance of
survival upon release.
.
Keywords: Body condition; Corticosterone; European wild rabbit; Fecal glucocorticoid metabolites; Non-invasive monitoring; Oryctolagus cuniculus; Stress;
Survival; Translocation
Introduction
Animals respond physiologically to the perception of an
array of external noxious or stressful stimuli, including
predation attempts, harsh weather, habitat change and anthropogenic disturbances, through a rapid cascade of endocrine
secretions within the hypothalamic–pituitary–adrenal (HPA)
axis (Axelrod and Reisine, 1984; Sapolsky et al., 2000;
Wingfield et al., 1997, 1998). This response is widely conserved
across vertebrates and ultimately involves the secretion of
glucocorticoids (GC, cortisol or corticosterone) from the
adrenals. The presence of elevated GC allows individuals to
cope with short-term stressors through a complex set of
physiological and behavioral changes aimed at halting nonessential activities while the stressful perturbation persists.
Mobilization of body energy stores, increased gluconeogenesis,
⁎ Corresponding author. Fax: +34 954 621 125.
E-mail addresses: scabezas@ebd.csic.es, sonia.cabezas@usask.ca
(S. Cabezas).
and the promotion of food-searching and dispersal behaviors are
some of the adaptive changes activated by GC. Wingfield et al.
(1998) proposed that the activation of the HPA axis under
stressful situations constitutes a distinct “emergency” life history
stage in vertebrates.
The degree of stress response activation often correlates with
the overall health of an individual, and the quantification of GC
levels is often used to infer the health status of animal
populations (Creel et al., 1996; Morton et al., 1995; Romero,
2004; Wasser et al., 2000; Wingfield et al., 1997). In particular,
the utilization of a capture and restraint protocol (Wingfield,
1994) has become a popular approach whereby the adrenocortical response of wild individuals to a short period of capture
and human handling is measured, and inferences are drawn
about how the population would cope with other sources of
stress. Although this approach is a valuable tool to evaluate the
response to stress over the course of a few minutes to hours (i.e.,
acute stressors), it is not clear whether short-term responsiveness can predict adaptation to longer term stress (i.e., chronic
stressors). This is important because many anthropogenic
disturbances (e.g., habitat degradation, exposure to tourism,
contamination episodes) and a number of wildlife management
practices (e.g., habitat restoration, reintroductions and reinforcements of animal populations) involve perturbations that last
for considerable periods of time, and a prolonged activation of
the HPA axis may reverse earlier adaptive responses to the
stressful event. Some studies have shown that chronic GC
elevation may produce potentially detrimental effects, especially reduced survival and fitness through infertility, impaired
resistance to disease, inhibition of growth and atrophy of body
tissues (Rogovin et al., 2003; Romero and Wikelski, 2001;
Sapolsky et al., 2000; Suorsa et al., 2003). Others, however,
suggest that chronic but moderate GC elevation provides
adaptive advantages to the individual (Cote et al., 2006;
Pravosudov, 2003). Therefore, the interpretation of interindividual differences in the HPA axis responsiveness may
yield opposite conclusions depending whether the stressor is
acute or chronic and also depending on the actual level of
activation of the GC release (Romero, 2004).
Despite differences between short- and long-term responsiveness to stressors, few studies have experimentally evaluated
what constitutes an adequate response to long-term perturbations or whether differences in the associated stress response
can actually predict the fitness of an individual. In this context,
the translocation of animals, used to improve and recover
natural populations of wildlife, might provide useful information about adaptive responses to chronic stress. The establishment of quarantine periods prior to release is a highly
recommended veterinary practice in translocation programs
because the potential transmission of new disease agents into
the release area, may have a major impact on native populations
and on the overall success of the translocation program
(Woodford and Rossiter, 1994). Quarantine periods imply
handling and captivity of animals over several days or weeks,
can be performed in a standardized manner, and often involve
subsequent monitoring of the introduced animals. Consequently, translocation programs constitute a unique opportunity to
study how individuals cope with a long-term source of stress
and whether assessment of GC secretion can provide predictive
information on major components of fitness such as body
condition and survival. In this sense, the study of HPA
responsiveness during a quarantine period constitutes an
experimental capture and restraint protocol, but modified so
that the exposure to the source of perturbation lasts for weeks
rather than minutes or hours. This information could be then
used to evaluate and predict a population-level response to
chronic stress exposure as occurs with many of the anthropogenic disturbances threatening wildlife populations.
We tested these possibilities in European wild rabbits
(Oryctolagus cuniculus) during a translocation program within
the Doñana Natural Park in Huelva province of south-west
Spain. In particular, we studied whether GC secretion and
metabolism during the quarantine period could explain two
major components of individual rabbit fitness: body condition
and survival probability. Native populations of European wild
rabbits have suffered a considerable decline since the early
1950s in the Iberian Peninsula. This is a major conservation
concern because the viability of several endangered predators
endemic to this region (the Spanish imperial eagle Aquila
adalberti and Iberian lynx Lynx pardina in particular) relies
almost exclusively on rabbits as a prey source (Ferrer and
Negro, 2004). As a consequence, translocations of wild rabbits
are one of the most frequently used management tools to boost
the density of natural populations in Spain (Cabezas, 2005;
Calvete and Estrada, 2004; Moreno et al., 2004). In our study,
wild individuals were captured, maintained in captivity during a
quarantine period, and then released and monitored for survival
through radio-tracking. The physiological response to stress
during the quarantine period was assessed by measuring
circulating corticosterone (CORT) and GC metabolites (GCM)
in feces. Finally, we analyzed the association between the GC
measurements and various components of individual fitness of
the rabbits, including body condition and survival in the wild.
Methods
Experimental design
Between February and March 2002 we captured 44 adult wild rabbits (24
males and 20 females) in the province of Cadiz (SW Spain). After capture,
rabbits were transported to experimental facilities, sexed, weighed (mean ± SE:
1062 ± 14.3 g) and identified with numbered metallic ear-tags. All rabbits were
adults and sexually mature as determined by body mass and the period of year
in which they were captured (Soriguer, 1981). Captures were carried out
several weeks prior to the start of the seasonal peak of reproduction, which
normally occurs in late spring in nearby populations (Villafuerte et al., 1997).
All females were checked for signs of pregnancy and lactation by means of
palpation of the abdomen and mammary glands. Only one of the 20 females
was pregnant, but the fetuses were aborted few days after capture. We allowed
a 3-week recovery period before release and this animal showed body mass
and corticosterone values well within the recorded range for other rabbits at
the end of the quarantine; consequently, we decided not to eliminate it from
our experimental sample. Standardized sanitary protocols have been
established for the rabbit translocation program at Doñana National Park in
concert with Spanish laws (Calvete et al., 2005). We followed these protocols
in consultation with the veterinary staff of the Junta de Andalucia, and
prioritized ethical considerations over scientific goals. At the beginning of the
quarantine period all individuals were treated for external and internal
parasites and subcutaneously vaccinated against myxomatosis and rabbit
hemorrhagic disease (RHD) using doses of commercial vaccines recommended for domestic rabbits. During the captive period, all animals were
housed individually in flat-deck cages commonly used for domestic rabbits,
and provided ad libitum access to water and food (commercial pelleted food
and hay). Rabbits were sheltered indoors but exposed to natural photoperiod
and temperature. The captive period lasted between 2 and 4 weeks to conform
to a release schedule planned as part of the translocation program. Two weeks
was the minimum time the rabbits spent in quarantine; this was based on the
myxomatosis incubation period and the time following vaccination needed to
develop immunity against myxomatosis, and RHD in domestic rabbits
(Argüello, 1991).
Two days before being released, rabbits were weighed (mean ± SE: 940 ±
15.1 g) and the cubit length was measured for subsequent calculation of body
mass index by means of residuals of the reduced major axis regression
between log10-weight and log10-cubit length (Green, 2001). In addition, blood
and fecal samples were collected to determine CORT and GCM levels,
respectively. The collected fresh fecal samples corresponded to the night
previous to blood extraction (i.e., between 11:00 pm and 9:00 am), and blood
samples were drawn through puncture in the ear vein between 9:30 am and
2:00 pm. Serum CORT shows daily variations in rabbits (Szeto et al., 2004)
and we recorded the exact time of day of blood collection for each rabbit in
order to control for this potential source of variation in the statistical analyses.
We also recorded the time elapsed between catching each rabbit from its cage
and taking the blood sample (mean ± SE: 4.2 ± 0.2 min); this was also
considered in the statistical analyses. Serum (obtained after blood centrifugation) and fecal samples were stored frozen at − 20 °C until GC
quantification.
After the captive period, 28 rabbits (14 males and 14 females) were equipped
with a radio-collar, weighing approximately 20 g and containing an activity
sensor (Biotrack, Wareham, UK), and released in the Doñana Natural Park.
Survival was monitored by performing daily localization of each individual over
the 30 days following release. When the activity sensor indicated lack of
movement, we searched the field grounds until the animal remains were located.
Other studies have previously used the ICN antiserum to measure GCM in a
wide range of mammalian and avian species (Wasser et al., 2000) and we
expected that the ICN RIA would also prove suitable for the measurement of
GCM in rabbit feces. However, we found that many of the rabbit fecal samples
measured were below or very near the detection limit (ED80 of 19.4 pg) of the
ICN RIA, and generated GCM values about 10-fold lower than those determined
from the Sigma RIA. The sample dilutions used in ICN RIA were similar to
those used in the Sigma RIA; lower sample dilutions were not used in the ICN
RIA due to the non-specific interference observed in the Sigma RIA.
Serum corticosterone measurement
Data analysis
Corticosterone is the major adrenal glucocorticoid secreted by the European
rabbit (Szeto et al., 2004). Serum levels of CORT were analyzed following
extraction with diethyl ether and utilizing techniques described fully elsewhere
(Wayland et al., 2002). Extraction efficiency, measured in samples spiked with
3
H-corticosterone, was 96%. Dried ether extracts were reconstituted in a small
volume of assay buffer and frozen at − 20 °C until CORT was measured by
radioimmunoassay (RIA). This RIA employs an antiserum purchased from
Sigma Chemicals (Oakville, Ontario) and 3H-corticosterone (Amersham
Biosciences, Baie d'Urfe, Quebec). Cross-reactivity of the antiserum with
other secreted steroids, including cortisol and progesterone, is low (Wayland et
al., 2002). The minimum detection limit of the RIA, defined as the dose of
CORT which produced a relative binding (%B/Bo) of 80% in the RIA (ED80),
was 14.5 pg. Serum extracts were measured over two assays. Assay variability
was determined as the %coefficient of variation (%CV) resulting from repeated
measurement of samples spiked with a known amount of CORT (n = 3) in each
assay. The within-assay variability in the two assays was 1.3% and 4.6%.
Between assays variability was determined to be 3.0%. Serial dilutions of the
rabbit serum extracts generated a displacement curve parallel to the
corticosterone standard curve. All serum extracts were diluted several fold
with assay buffer to correspond to a level near the midrange (ED50) of the assay.
To analyze the association between serum CORT and fecal GCM levels and
the body condition index prior to the release of individuals, we performed
Generalized Linear Models (GLM) using the Genmod procedure (SAS, 1997)
with normal distribution of errors and identity link function. In a first model
(Model 1) we analyzed serum CORT concentration (S-CORT) as a function of
sex (S), body mass index at the end of the captivity period (BMI), length of the
captivity period in weeks (LC), delay time between capture and bleeding in
minutes (DT); time of day at blood sampling (T); and square time (T2) to allow
non-linear fitting. In a second model (Model 2) the fecal GCM concentration (FGCM) was analyzed as a function of sex (S), body mass index (BMI) and length
of the captivity period (LC). GLM models were constructed through a backward
stepwise procedure. From initial saturated models comprising all the dependent
variables, we sequentially eliminated the least significant terms until obtaining a
minimum adequate model where all the retained variables had a significant
effect on CORT or GCM concentrations at a > 5% rejection probability.
The risk of dying during the first month following release was analyzed
through a Cox's proportional hazard regression model for censured data
(Therneau and Grambsch, 2001) that considered the following independent
Fecal glucocorticoid metabolites measurement
Glucocorticoid metabolites in fecal samples were extracted utilizing
procedures modified from Wasser et al. (2000). Dried feces (approximately
200 mg) were placed in a glass vial with approximately 5 ml of 90% HPLC
graded methanol (VWR International, Missisauga, Ontario) and homogenized at
maximum speed (approximately 25,000 RPM) with a hand-held motorized
homogenizer (Omni 2000, Pro Scientific, Connecticut). The homogenized
samples were then placed in a shaking water bath at room temperature for
30 min, followed by centrifugation at 4000×g. The supernatant was air dried
overnight, followed by reconstitution in 1.0 ml methanol. An aliquot of the
reconstituted methanolic supernatant was then diluted 1:5 in assay buffer and
stored at − 20 °C. Samples were further diluted in assay buffer prior to
measurement of the GCM.
The GCM content of the diluted fecal extracts was determined with the same
RIA used to measure serum CORT levels as described above (Sigma RIA) as
well as a second RIA (ICN RIA) developed using an anti-corticosterone
antiserum purchased from MP Biomedicals (Solon, Ohio). Serial dilutions of the
rabbit fecal extracts were assessed for parallelism to the CORT standards in the
Sigma RIA. Lower dilutions of the methanol extracts (ranging from 1:5 to 1:40)
displayed significant interference and non-parallelism in the Sigma RIA. Further
analysis revealed that this was due to non-specific (i.e., non-displaceable)
binding between the fecal extracts and the 3H-corticosterone. The constituent(s)
in the methanol extract that contributed to the interference in the Sigma RIA at
these lower dilutions is not clear. However, this interference in the RIA
disappeared with further dilution of the extracts. To minimize the possibility that
any given sample might display a non-specific interference (i.e., non-parallelism
to the standard curve) in the Sigma RIA, all fecal samples were analyzed at
multiple dilutions (from 1:80 to 1:320) and were found to be parallel to the
standard curve over this dilution range. Samples were analyzed in a total of three
separate assays. The within-assay variability was determined to be 2.8, 5.0 and
2.2%CV in the three assays; the between assay variability was determined to be
9.3%CV.
The ICN RIA for fecal GCM was developed using 3H-corticosterone as the
tracer and other assay procedures identical to those used in the Sigma RIA.
Fig. 1. Relationship between the serum corticosterone concentration (a) and
fecal GCM concentration (b) and the body mass index of wild rabbits at the end
of the captivity period.
variables: serum corticosterone (S-CORT) and fecal GCM (F-GCM) concentrations, sex (S), body mass index (BMI), and length of the captivity period (LC).
The final model was obtained by means of a backward selection procedure
where non-significant terms were sequentially removed until obtaining a
minimum adequate model where all the retained variables had a significant
effect at a > 5% rejection probability.
Results
GLM analysis showed a negative association between both
serum CORT and fecal GCM levels and the BMI of the
individuals at the end of the captivity period (Model 1:
F1,38 = 4.72; P = 0.030; Model 2: F1,38 = 5.56; P = 0.018; Figs. 1a
and b respectively, Table 1). We found no significant differences
in serum CORT or fecal GCM levels between males and females
(Table 1). The duration of the captivity period (LC) did not show
any significant effect on either serum CORT or fecal GCM levels
(Table 1). The CORT concentration was also not affected by the
time of the day at blood sampling (T and T2 variables) nor by the
delay time between capture and blood collection (DT) (see
Table 1). The correlation between CORT levels in serum and
GCM levels in feces was not significant (R = 0.081; P = 0.640).
During the 30-day monitoring period following release, 67%
rabbits (42% males and 58% females) survived. The Cox's
regression model showed that the risk of dying during the 30
first days after release was independent of sex, body mass index
or duration of the captivity period, but negatively related to both
serum CORT and fecal GCM concentrations (Table 2, Figs. 2
and 3).
Table 2
Results from the Cox's regression model assessing the risk of death in wild
rabbits as a function of the recorded variables
Effect
χ12
P
Coef
SE
Length of the captivity period (LC)
Sex (S)
Body mass index (BMI)
Serum CORT concentration (S-CORT)
Fecal GCM concentration (F-GCM)
0.2
2.2
2.3
7.6
7.5
0.655
0.138
0.129
0.006
0.006
–
–
–
− 0.203
− 6.224
–
–
–
0.105
2.679
For each effect, it is shown the Chi-squared value and associated probability
obtained during the backwards procedure. Predicted coefficients and standard
errors (SE) are shown for the variables retained in the final model (i.e.,
α ≤ 0.05).
(2004). During captivity, wild rabbits showed typical fear and
escape reactions to human presence and handling, as well as an
overall body mass decline along the quarantine period,
averaging 9% below initial values at capture. Reference values
of serum CORT and fecal GCM titers for wild rabbits under
non-stress conditions are lacking in the literature. However, we
measured serum CORT and fecal GCM levels after 2–4 weeks
of continuous exposure to stress. Consequently, we interpret
variability in these measures as part of the individual
physiological response to a moderately long-lasting stressor,
and therefore reflect variation in chronic stress levels rather than
baseline or acute levels.
Wild rabbits showed a considerable variability both in
circulating CORT and excreted fecal metabolites, each of which
ranged over an 8-fold difference among individuals. This
Discussion
Our experimental quarantine provided a challenging and
unpredictable environment to wild rabbits, which was prolonged over a moderately long period of several weeks.
Although all animals had access to basic needs such as food,
water and shelter, the confinement in a reduced artificial space
together with the social isolation from conspecifics and the
repeated and unpredictable human presence, represented
exposure to a chronic source of stress as defined by Romero
Table 1
Results from Generalized Linear Models explaining the association between
serum corticosterone concentration (Model 1, ng/ml), and fecal GCM
concentration (Model 2, ng/mg) of wild rabbits, and several variables considered
χ2
df
P
%Deviance
explained
Model 1 (S-CORT)
Body mass index (BMI)
Sex (S)
Time (T)
Square time (T2)
Length of the captivity period (LC)
Delay time capture and bleeding (DT)
4.72
1.65
0.99
0.22
0.07
0.02
1,38
1,37
1,36
1,35
1,34
1,33
0.030
0.199
0.319
0.639
0.788
0.878
11
Model 2 (F-GCM)
Body mass index (BMI)
Sex (S)
L ength of the captivity period (LC)
5.56
1.87
1.41
1,38
1,37
1,36
0.018
0.171
0.234
13
Fig. 2. Survival probability as a function of serum corticosterone concentration
(ng/ml). Upper and lower error bars show recorded corticosterone values
(mean ± 1 standard error) for rabbits that respectively survived (triangle) and
did not survive (square) the 30-day period following release. The central panel
represents predicted survival (mean ± 1 standard error) according to the Cox
regression model for a set of individuals showing average fecal GCM
concentration (i.e., 0.712 ng/mg).
Fig. 3. Survival probability as a function of fecal GCM concentration (ng/mg).
Upper and lower error bars show recorded GCM values (mean ± 1 standard
error) for rabbits that respectively survived (triangle) and did not survive
(square) the 30-day period following release. The central panel represents
predicted survival (mean ± 1 standard error) according to the Cox regression
model for a set of individuals showing average serum corticosterone
concentration (i.e., 8.78 ng/ml).
suggests that within our population, individuals naturally
differed in their physiological responses and ability to cope
with a standardized source of stress. Importantly, the variability
in GC endocrine parameters was associated with an individual's
body condition and was able to predict subsequent survival.
Individuals with higher serum CORT and fecal GCM levels
showed lower scores of body condition. The body mass index
explained 11% of the variance in serum CORT levels and 13%
of the variance in fecal GCM levels. These results were
consistent with previous evidence found in other mammalian
and avian taxa (Cote et al., 2006; Johnson et al., 2004; Robin et
al., 1998; Romero and Wikelski, 2001; Suorsa et al., 2003;
Wingfield et al., 1997).
The association between body condition and GC levels
found in our study can be explained by two hypotheses that are
not mutually exclusive. First, it is possible that the perception of
stressful stimuli associated to captivity triggered elevations in
GC secretion, which in turn modulated changes in body
condition through its effects on carbohydrate, lipid and protein
metabolism (Sapolsky et al., 2000). Second, it is also possible
that the captive environment caused changes in the condition of
wild rabbits not directly related to GC levels, and CORT was
subsequently elevated as a consequence of decreased body
weight (Pérez-Rodríguez et al., 2006). Unfortunately, the
correlational nature of our results does not allow us to conclude
whether elevated GC secretion was the cause or the consequence of the significant association with body condition.
Although the association between long-term HPA activation
and reduced body condition is often presented as a deleterious
side-effect of chronic exposure to stress, the body mass index
had no effect on the subsequent survival of rabbits in our
experiment. This suggests that the range of variation in body
condition found in our study was still within normal “healthy”
values for rabbits. Therefore, the condition of those individuals
with higher GC levels was still adequate to allow recovery and
adaptation following release. In fact, our results indicate that
both serum CORT and fecal GCM levels were good predictors
of individuals' ability to cope with an unpredictable novel
environment, and possibly to adjust their physiology and
behavior to maximize survival.
Several studies of translocation programs have shown that
rabbits suffer high mortality during the first weeks after release
(Calvete and Estrada, 2004; Letty et al., 2000, 2002). This
mortality has been attributed to several factors, but deterioration
of physiological status as a consequence of stress induced by
handling and the novelty of the release environment are often
cited (Letty et al., 2002, 2003). Our results are in agreement
with the observation that physiological state upon release is a
major component explaining the success of translocation.
However, they also indicate that HPA activation under
conditions of long-term exposure to stress does not necessarily
imply deleterious consequences to an individual. In fact, we
found that CORT levels in serum and GCM in fecal samples
were positively correlated with the short-term survival of
translocated wild rabbits. Moderately elevated CORT secretion
in wild rabbits seems to be beneficial and allow for an adequate
adaptation during captivity and subsequent survival upon
release (see also Cote et al., 2006; Meylan and Clobert, 2005;
Pravosudov, 2003).
Elevated serum CORT during a period of stress may trigger a
suite of behavioral and physiological responses, including a rise
in locomotive activity and adequate mobilization of energy
stores (Wingfield et al., 1998). Thus, long-term but moderate
elevations of CORT may enable individuals to avoid potentially
deadly circumstances, thereby explaining the higher survival
probabilities found in our study. Elevated GC levels, rather than
suggesting impaired negative feedback as a consequence of the
prolonged quarantine period (Romero, 2004), possibly allowed
rabbits to display better escape reactions during predation
attempts or other emergency situations. Conversely, reduced
GC levels under the long-term stressful environment simulated
by the quarantine may indicate that some individuals showed an
acclimation to our experimental settings. With acclimation,
animals fail to respond to repeated or chronic stressors, and
reduce overall GC secretion (Romero, 2004). Such an
acclimation to stress in our study, may have allowed acclimated
individuals to maintain a better body condition during the
quarantine. However, the acclimation process alters the HPA
axis physiology such that GC responses to novel stressors are
enhanced compared to responses of non-acclimated animals,
through a mechanism known as facilitation (Bhatnagar and
Vining, 2003; Romero, 2004). Therefore, it is possible that the
variability in GC levels shown by wild rabbits during our
experimental quarantine reflected different degrees of acclimation, with the more acclimated individuals gaining the
advantage of a better body condition at the expense of an
inadequate adaptation upon release. Overall, our data suggest
that an increase in GC secretion under long-term stressful
conditions is a basic adaptive mechanism allowing animals to
face a wide range of critical and energy demanding situations.
The relationship between HPA activation and survival is still
largely unexplored, and the few studies testing this association
in vertebrates have yielded contradictory results. While some
studies report a negative association between GC levels and
survival (Brown et al., 2005; Rogovin et al., 2003; Romero and
Wikelski, 2001; Suorsa et al., 2003), other works report opposite
results (Cote et al., 2006; Meylan and Clobert, 2005), or even
curvilinear associations (Brown et al., 2005). In order to
compare our results to previous studies, we have to consider
that our GC measures were recorded under conditions of longterm exposure to a moderate stressor. To our knowledge, only
one study has previously tested the effect of GC exposure on
survival probabilities in vertebrates under conditions of chronic
stress (Romero and Wikelski, 2001). In their study, Romero and
Wikelski (2001) found that chronic GC levels in fasting
Galapagos Marine iguanas were negatively associated with
body condition and survival probabilities after the “El Niño”
famine event of 1998. This is opposite to the pattern we found
for translocated wild rabbits. The apparent contradiction could
be explained by a parabolic association between chronic GC
levels and survival, such as the one we propose in Fig. 4. Under
conditions of long-term chronic stress, animals are expected to
elevate their circulating GC above normal “baseline” values
(Wingfield et al., 1998), and the degree of GC elevation would
explain body condition and survival (top and lower graphs in
Fig. 4). Reduced GC levels may indicate acclimation, a process
that may allow individuals to maintain a good body condition
but at the expense of inadequate HPA responsiveness in a novel
environment, which would imply reduced survival. Intermediate
GC levels may indicate that animals are coping with the source
Fig. 4. Proposed association between glucocorticosteroid levels of wild rabbits
under conditions of long-term stress (i.e., chronic glucocorticosteroid, X-axis)
and both body condition (top graph, Y-axis) and survival (lower graph, Y-axis).
of stress, showing a moderate body condition score as a
consequence of mobilized energy resources, but also showing
adequate responsiveness to the challenging environment, and
therefore optimal survival. Finally, extremely elevated GC
levels would promote skeletal muscle catabolism as an extreme
emergency source of energy, leading to critically reduced levels
of body condition that decrease the probability of survival. The
latter situation was likely to have occurred in the study of marine
iguanas (Romero and Wikelski, 2001), because the environmental perturbation removed access to food resources for
several months and led to mass mortality in the iguana
population. In our study, however, chronic perturbation (i.e.,
the quarantine period) did not deprive animals from access to
food, water or refuge, and therefore we would not expect
extreme HPA responses among healthy individuals.
The primary purpose of our study was to establish the
association between physiological (HPA) responses to stress
and individual fitness of rabbits. We assessed the HPA
response by quantifying both circulating CORT levels and
the GCM concentration in feces of the rabbits. To our
knowledge this is the first time that the Sigma RIA system
has been used to measure fecal GCM levels. Various authors
have articulated the need for careful validation of assays when
measuring GCM in feces (Buchanan and Goldsmith, 2004;
Palme, 2005; Touma and Palme, 2005). A number of findings
suggest that our results for the fecal GCM are a valid
measurement of HPA activation in the rabbits. We carefully
assessed each fecal sample for parallelism in the RIA and
included internal control samples to measure the sensitivity
and variability of each assay. The levels of GCM found in the
rabbit fecal samples are within the range detected in other
mammalian species and with a variety of other GCM assay
systems (Monclús et al., 2006; Teskey-Gerstl et al., 2000;
Wasser et al., 2000). Finally, the overall conclusions from our
study are based on both serum CORT and fecal GCM
measurements. An identical relationship between the physiological stress response, BMI and survival in rabbits was
obtained, regardless of whether stress was assessed utilizing
serum CORT or fecal GCM values. Nevertheless, further
validation of glucocorticoid measurements in fecal samples
should be carried out for the European wild rabbit.
Interestingly, the ICN RIA displayed poor cross-reactivity
with rabbit fecal GCM in our study. This was somewhat
surprising as the ICN antiserum has been used to detect GCM in
a variety of mammalian species (Wasser et al., 2000). However,
Touma et al. (2003) also found that the ICN antibody does not
cross-react with fecal or urine GCM in laboratory mice. Species
differences in glucocorticoid metabolism are well-known, and
our findings clearly indicate that the ICN antiserum is also
unsuitable for detecting GCM in rabbits. In preliminary
experiments in birds, we found that the Sigma RIA does not
detect sulphonated or glucuronidated GCM although it does
appear to recognize some other form of a polar GCM in various
avian species (T.A. Marchant and S. Poland, unpublished
results). Further studies will be needed to determine the nature
of the polar GCM detected by the Sigma RIA in rabbit feces.
However, our finding of a strong relationship between, BMI,
survival and either serum CORT or fecal GCM suggests that the
Sigma RIA may be more broadly suited to future studies of the
impact of stress on wild rabbit populations.
The lack of a significant correlation between serum CORT
and fecal GCM in our study is not totally unexpected, given
that the blood samples were obtained at a single time point
distinct from the fecal sample collection period, and that fecal
GCM levels represent the integration of a number of variables,
including fecal elimination rates, gut transit times and the daily
pattern in CORT secretion, over a fairly long period of time
(Monclús et al., 2006; Teskey-Gerstl et al., 2000; Palme, 2005;
Wasser et al., 2000). In our study, blood samples were collected
at a time when circulating CORT levels have been found to
reach their daily maximum in rabbits (Szeto et al., 2004). In
contrast, the fecal GCM concentrations likely represent blood
CORT levels 1 or more days earlier and over an uncertain
period within the daily cycle of CORT secretion. It is entirely
likely that a significant correlation between serum CORT and
fecal GCM would be found with a more intensive blood
sampling protocol over the time frame represented by the fecal
samples.
Acknowledgments
We thank A. Melero, R. Moreno and D. Doblas for their help
during the data collection, and Drs. E. Angulo and C. Calvete
for their useful comments on previous drafts. Very special
thanks to Dr. G.R. Bortolotti and M. Delibes for their comments
and support and to T. German for helping with the laboratory
analyses. Two anonymous referees provided useful comments
on previous manuscript drafts. The staff of Doñana Natural Park
provided technical and logistical help. S.C. and the research
project were supported by funds from the European Union
(FEDER 1FD1997-0789) and the Spanish Ministerio de Ciencia
y Tecnología (MCYT, project BOS2001-2391-C02-01). J.B.
was supported by a postdoctoral grant (from MCYT) and the
Isabel Maria Lopez Martinez Memorial Scholarship from the
University of Saskatchewan.
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