Physiology & Behavior 149 (2015) 310–316
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Physiology & Behavior
journal homepage: www.elsevier.com/locate/phb
Does a short-term increase in testosterone affect the intensity or
persistence of territorial aggression? — An approach using an individual's
hormonal reactive scope to study hormonal effects on behavior
Wolfgang Goymann a,⁎, Camila P. Villavicencio a, Beate Apfelbeck a,b
a
b
Abteilung für Verhaltensneurobiologie, Max-Planck-Institut für Ornithologie, Eberhard-Gwinner-Straße 6a, D-82319 Seewiesen, Germany
Institute of Biodiversity, Animal Health and Comparative Medicine, University of Glasgow, Glasgow G12 8QQ, Scotland, UK
H I G H L I G H T S
• We use hormonal reactive scopes to see how surges of testosterone affect behavior
• Surges of testosterone did not increase territorial aggression in black redstarts
• Surges of testosterone did not convey a competitive advantage in black redstarts
a r t i c l e
i n f o
Article history:
Received 23 March 2015
Received in revised form 18 June 2015
Accepted 20 June 2015
Available online 27 June 2015
Keywords:
Gonadotropin releasing hormone
GnRH
Territorial behavior
Hormone manipulation
Simulated territorial intrusion
STI
Challenge hypothesis
Androgen responsiveness
Hormonal reaction norm
a b s t r a c t
In this study, we describe an approach based on an individual's hormonal reactive scope to study short-term
effects of hormones on behavior. The control of territorial aggression has been traditionally linked to testosterone. Males of some vertebrate species show an increase in testosterone during territorial interactions and
implantation studies suggest that such an increase in testosterone enhances the intensity and persistence of
aggression. Here, we tested whether a short-term maximum release of testosterone – based on an individual's
hormonal reactive scope – affects the intensity or persistence of territorial aggression in male black redstarts, a
bird species in which testosterone does not increase during territorial encounters. An injection with
gonadotropin-releasing-hormone (GnRH) induced a physiological peak in plasma testosterone that was specific
for each individual (=individual reactive scope). However, such short-term surges in an individual's testosterone concentration did not affect the intensity or persistence of aggression. In conclusion, this study demonstrated
(1) that a species that naturally does not increase testosterone during male–male encounters would not benefit
from such an increase in terms of being more aggressive, (2) that behavioral studies using GnRH-injections
represent a promising approach to study species differences in androgen responsiveness, and (3) that injections
of releasing or tropic hormones in general may be a suitable approach to study short-term influences of
hormones on behavior. These injections effectively mimic the potential short-term changes in hormones that
can occur in the real life of individuals and enable us to study the effects of hormonal changes on behavior or
other traits within an ecological and evolutionary framework.
© 2015 Elsevier Inc. All rights reserved.
1. Introduction
A classical approach to study the effect of hormones in ecophysiological studies of animals is to insert implants containing a
hormone and observe the effect of the elevated hormone concentrations
on the trait in question (see e.g. [1–4] for pioneering examples of early
studies in free-living birds). Typically, such implants elevate hormone
concentrations for longer periods than normal (e.g. [5–7]) and thus
⁎ Corresponding author.
E-mail address: [email protected] (W. Goymann).
http://dx.doi.org/10.1016/j.physbeh.2015.06.029
0031-9384/© 2015 Elsevier Inc. All rights reserved.
may be of limited value to study the effects of short-term elevations of
hormone concentrations on behavior.
Here, we exemplify an alternative approach to study short-term
effects of changes in hormone levels on behavior, that is based on an
individual's hormonal reactive scope, i.e. the range of concentrations
of a hormone (from ‘baseline’ to the potential maximum) that an
individual can express during a particular life-history stage in response
to external or internal cues. Following Williams [8] this could also be
considered a hormonal reaction norm. We use the hypothalamic–pituitary–gonadal axis (HPG) with testosterone (hormone) and territorial
aggression (behavior) as an example to illustrate the practicability of
W. Goymann et al. / Physiology & Behavior 149 (2015) 310–316
this approach, but it could be easily adapted to other behaviors or hormonal systems.
The control of territorial aggression in birds and other vertebrates
has been linked to testosterone (e.g. [9–11]). In a classic study on the
song sparrow (Melospiza melodia) Wingfield [12] observed that
implants containing testosterone (subsequently termed testosterone
implants) increased the intensity and persistence of territorial aggression towards a simulated territorial intruder (a caged song sparrow
and playback of song). This is particularly interesting in combination
with the observation that song sparrows belong to the minority of
bird species studied to date that show a short-term increase in
testosterone concentrations during territorial interactions [13]. Together these studies by Wingfield suggest that short-term increases in
testosterone observed during territorial encounters may affect a song
sparrow's motivation to defend a territory during the reproductive
period.
Unlike the song sparrow and a few other species, the majority of
birds tested so far do not show a testosterone surge after simulated territorial intrusions (see [14–23] for recent studies and a review [24] for
studies conducted before 2007). This includes the black redstart
(Phoenicurus ochruros), a socially monogamous and bi-parental songbird of the western Palaearctic, and our main study species regarding
territorial aggression (e.g. [15–17]). Although black redstarts do not
increase testosterone in response to simulated territorial intrusions,
they have the hormonal reactive scope to do so (and may increase
testosterone following other cues, i.e. when stimulated by receptive
females). We ask whether a short-term rise in testosterone could potentially mediate a higher intensity and persistence of aggression also in
males of species – such as the black redstart – that do not elevate testosterone during male–male challenges. This androgen response may just
not have (yet) evolved in such species, even though an increase in
testosterone could be beneficial, i.e. increase the likelihood to deter a
potential rival during territorial challenges (alternatively, the ancestors
of black redstarts may have lost the androgen response to territorial
challenges, because it did not have a selective advantage).
To answer this question we elicited a short-term increase in plasma
testosterone within an individual's hormonal reactive scope using injections of gonadotropin releasing hormone (GnRH). GnRH is the hypothalamic releasing hormone that causes the pituitary to secrete luteinizing
hormone into the circulation, which then induces the testes to produce
testosterone. Injections of GnRH are a well-established method to study
the functioning of the hypothalamic–pituitary–gonadal axis (HPG) and
have been used in mammals and domesticated birds for many years
(reviewed by [25]). The use of GnRH injections as an indicator of gonadal activity in wild birds has been pioneered by John Wingfield [26] and
since then this approach has been extensively used by many researchers
to study the gonadal potential to produce testosterone in free-living
wild birds (e.g.[15,17,19,20,27–46]). However, such GnRH injections
have been rarely used as a means to test the effect of brief elevations
in testosterone on the behavior of free-living animals (but see [47]).
Birds and other vertebrates show large individual differences in
baseline breeding concentrations of testosterone, and also the capacity
to maximize testosterone output differs largely between individuals
(and life-history stages). For example, baseline concentrations of testosterone during the mating stage range from less than 0.2 ng/ml up to
about 12 ng/ml between male black redstarts that hold a territory and
are paired with a female [48]. Upon injection of a sufficient amount of
GnRH the testes of male black redstarts presumably maximize testosterone production. This results in a wide range of short-term maxima in
plasma testosterone concentrations that reflect each individual's
capacity to produce this hormone. Thus, by injecting GnRH one can
induce a short-term rise in testosterone from an individual's postcapture baseline to the respective individual's maximum concentration.
This increase represents the individual's hormonal reactive scope of
testosterone during a particular life-history stage and is repeatable
within individuals [33,49]. Unlike testosterone implants GnRH
311
injections cannot produce a peak in testosterone that would go beyond
an individual's physiological maximum. Instead, GnRH injections lead to
short-term increases of testosterone within the individual physiological
range. This physiological increase of testosterone allows us to link the
change in hormone concentration with changes in territorial aggression. We consider GnRH-injections (and more generally releasing
hormone or tropic hormone injections) a useful approach to study
short-term behavioral effects of transient increases in testosterone
within the physiological reactive scope of an individual. This may be
particularly useful in studies within an eco-physiological framework.
In this study, we first present reanalyzed data from our previous
studies on male black redstarts to show that GnRH-injections lead to a
short-term surge in testosterone during the mating and parental phases
of the first brood and to demonstrate that there is large variation in the
potential maxima between individuals. These data show that in both
periods natural and GnRH-induced testosterone concentrations of
black redstarts were high indicating that the HPG axis is active [48].
Second, we present a new experiment, in which we passively caught
territorial male black redstarts during the parental phase of their first
brood. According to the challenge hypothesis testosterone could interfere with parental care. Therefore, in a socially monogamous and
bi-parental species, this should be the period with the largest benefit
of maintaining lower testosterone levels but a high potential to increase
testosterone (androgen responsiveness) in case of social instability [50].
Also, during this period black redstarts are easiest to catch without a
dummy or playback lure. After capture, we quickly injected an experimental group with GnRH and a control group with saline. After
measurement and banding we immediately released them back onto
their territory to conduct a simulated territorial intrusion 35 min after
the injection of GnRH (when testosterone concentrations were presumably maximal) or saline. If short-term surges of testosterone increase
the intensity and persistence of territorial aggression also in a bird
species that does not elevate testosterone during territorial encounters,
we predicted that GnRH-injected birds would respond more intensely
towards a simulated territorial intrusion and also would be more
persistent in their territorial response in the period after the simulated
territorial intrusion in comparison to control birds that were injected
with saline. GnRH injections have been used to study changes in behavior before (e.g. [51–55]), but to the best of our knowledge this is only the
second time the approach is used in free-living animals within an ecologically relevant setting. The first time it was used in free-living
European ground squirrels (Spermophilus citellus), in which GnRH
injections led to an increase in testosterone and agonistic behavior of
males during the premating phase [47].
2. Methods
The study was performed in small villages in the vicinity of the
Max-Planck-Institut für Ornithologie, Germany (N 47°, E 11°,
500–600 m above sea level). Using data from our previous studies [15,
17,33] we demonstrate that GnRH induced an increase in testosterone
within 30 min after injection during the mating and the first parental
phase in male black redstarts. 67 male black redstarts that were in the
mating phase before their first clutch (April–May) and 31 male black
redstarts that were feeding their first nestlings or fledglings (May–
June) were caught and an initial blood sample of about 100 μl was
taken within 5 min after capture. Then, birds were injected intramuscularly with 1.25 μg chicken GnRH-I (Bachem H 3106) in 50 μl isotonic
saline (see [15–17,44]), a dose that has been demonstrated to elicit a
maximal testosterone response in the dark-eyed junco (Junco hyemalis),
with about 20–22 g body mass an at least 25% larger bird than the black
redstart [33]. Thirty minutes after the injection, a second blood sample
of 50–100 μl was taken to measure the GnRH-induced increase in
testosterone.
Plasma was immediately separated by centrifugation with a Compur
minicentrifuge (Bayer Diagnostics). The amount of plasma was
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W. Goymann et al. / Physiology & Behavior 149 (2015) 310–316
measured with a Hamilton syringe and stored in 500 μl ethanol. After
returning from the field samples were stored at −80 °C. Testosterone
concentration was determined by direct radioimmunoassay (RIA,
following [56], for assay details see [16,17]).
The main study was performed in May and June 2009 and 2010 and
does not contain previously published data. Only males that were
feeding nestlings or fledglings of their first brood were included in the
experiment, because during this breeding sub-stage the HPG-axis of
males is highly responsive and they are relatively easy to catch without
a dummy and/or playback. Males were caught with mealworm-baited
ground traps, while searching for food. Traps were observed at all
times from a car in the vicinity and males were immediately removed
from the traps upon capture. Within 3 min of capture the birds were
injected with 1.25 μg chicken GnRH-I (Bachem H 3106) in 50 μl isotonic
saline (GnRH group, N = 11) or with saline alone (control group, N =
11) into the pectoralis major muscle. After measurement and banding
with a numbered aluminium ring (Vogelwarte Radolfzell) and a combination of three color rings the birds were released back onto their respective territories within less than 10 min after capture. 35 min after
the GnRH-injection – when testosterone levels can be expected to be
maximal (see [15]) – a simulated territorial intrusion experiment was
performed in the center of the focal male's territory. Control and experimental groups were similar in body mass (expressed as means and 95%
credible intervals in squared brackets: saline: 16.8 [16.3–17.2] g; GnRH:
16.6 [16.1–17.1] g), tarsus length (saline: 23.7 [23.3–24.0] mm; GnRH:
23.4 [23.0–23.7] mm), and volume of the cloacal protuberance (estimated by expressing it as a cylindrical shape (V = π ∗ (CP width / 2)2 ∗ CP
height); saline: 278 [230–326] mm3; GnRH: 319 [271–367] mm3).
Two birds (one of the GnRH group and one of the saline group) did
not respond to the STI and were excluded from all further analyses. Furthermore, the digital voice recorder did not work when recording the
behavior of one GnRH-injected bird, thus resulting in a final sample
size of 10 control and 9 GnRH-injected birds for the behavioral analysis.
The sample size should have been sufficient to detect the typical effect
size of testosterone manipulation studies (i.e. Cohen's d of 1.2 or larger).
All experimental procedures were approved by the governmental authorities of Oberbayern, Germany.
2.1. Simulated territorial intrusion
To elicit a territorial response a remote-controlled loudspeaker
(Foxpro Scorpion, digital game caller, FOXPRO Inc. Lewistown,
USA) was put underneath a stuffed decoy to play back the territorial
song of a potential rival at a sound pressure level of 65 dB SPL at 1 m
(as measured with a CEL 573.B1 Sound Level Analyser). Each individual was exposed to a different playback. Playbacks had been recorded in spring 2009 in the same population of redstarts [57] and we
only used playbacks that were recorded from males that were at
least 10 km away from the location of the focal bird. As decoys we
used three different stuffed males in full adult plumage that were
protected by an inconspicuous cage made of a wire frame and a
mist net. This procedure had been successfully used to elicit a territorial response in male black redstarts in previous studies [15,16,58].
Using a digital voice recorder, we recorded the following behaviors
of the territory owner for 20 min: (1) latency to respond to the STI
either by singing or approaching the decoy, (2) the first time the
male was in a 5 m radius around the decoy, (3) the time the male
spent in this 5 m radius, (4) the proportion of time the territory
owner was fluffed, (5) the number of head noddings, which are typical threat postures of male black redstarts [58], and (6) the number
of songs. Furthermore, we noted whether the male attacked the
decoy. After 20 min we stopped the playback with a remote control
and remotely hid the decoy in a plastic box below the wire cage
by pulling a string. Then, we recorded the behaviors as above for
another 10 min after the STI.
2.2. Statistical analysis
Statistical analyses were conducted using R version 3.03 [59] and a
Bayesian statistical approach using the packages ‘arm’ [60] and ‘lme4’
[61]. General linear models were used to relate the increase in testosterone after GnRH-injection and the concentration of testosterone after
GnRH-injections with baseline (post-capture) testosterone for the
mating and parental phase separately. To compare the behavior of the
control group with that of the GnRH-injected group general linear
models were used with the maximum likelihood (ML) method.
Model residuals were analyzed using graphical methods (i.e. qq
plots of residuals fitted values versus residuals) for homogeneity of
variance, violation of normality assumptions or other departures from
model assumptions and model fit. For inferences from the models we
obtained Bayesian posterior parameter estimates and their 95% credible
intervals, using the function sim (running 10,000 simulations) and an
uninformed prior distribution [62]. In frequentist statistics, the statistical test provides a p-value describing the probability that the null hypothesis is true given the data. Bayesian statistics does not provide
such p-values. Instead, meaningful differences between groups can be
assessed by comparing the ranges of the 95% credible intervals between
groups. The 95% credible interval provides an estimate for the group
mean with a probability of 0.95. If the credible interval of one group
does not overlap with the mean estimate of another group, the groups
can be assumed to differ from each other. In our view, Bayesian estimates and credible intervals enable a more intuitive and meaningful
comparison of groups than the frequentist statistics and its reliance on
an arbitrary α-value referring to the likelihood of the null hypothesis
being true. If not indicated otherwise, data are presented as individual
data points in combination with Bayesian posterior means and their
respective 95% credible intervals (reported in squared brackets).
3. Results
3.1. Effectiveness of GnRH-injections
GnRH-induced levels of testosterone showed a positive relationship
with baseline testosterone concentrations during the mating phase
(F1,65 = 65.11; adjusted R2 = 0.49; slope: 1.29 [0.97;1.61]; Fig. 1a)
and the parental phase (F1,29 = 22.54; adjusted R2 = 0.42; slope: 0.64
[0.36;0.92]; Fig. 1d), suggesting that animals that started with high
baseline levels of testosterone also expressed high levels 30 min after
the GnRH injection. However, the magnitude of the rise in testosterone
after the GnRH injection was unrelated to initial baseline levels during
the mating phase (log10-transformed testosterone concentrations:
F1,65 = 0.003, adjusted R2 = 0.015; slope: − 0.009 [− 0.327;0.299];
Fig. 1b) and slightly negatively related to initial baseline levels of testosterone during the parental phase (F1,29 = 7.02, adjusted R2 = 0.16;
slope: −0.36 [−0.64;0.08]; Fig. 1e). Thus, in the mating phase, individuals with low baseline concentrations of testosterone increased testosterone secretion in a similar manner as individuals with high baseline
concentrations. In contrast, during the parental phase the magnitude
of the change in testosterone was higher for individuals that started
with lower baseline concentrations of testosterone. This suggests that
during the parental phase males with low levels of baseline levels of testosterone could increase testosterone secretion more than males with
high levels of baseline testosterone (compare slopes in Fig. 1c, f). In
six individuals with high initial concentrations of testosterone these
levels slightly decreased after GnRH injections, suggesting that their
concentrations were already maximal.
3.2. Behavior of saline- and GnRH-injected black redstarts during and after
a simulated territorial intrusion
Both saline- and GnRH-injected male black redstarts responded to
the simulated territorial intrusion. However, the large overlap of the
W. Goymann et al. / Physiology & Behavior 149 (2015) 310–316
313
Fig. 1. Post-capture baseline and GnRH-induced testosterone in 67 male black redstarts during the mating phase (a, b, c) and 31 male black redstarts during the parental phase (d, e, f).
Baseline testosterone and GnRH-induced testosterone concentrations were positively related during the mating phase (a) and the parental phase (d). Baseline testosterone and the magnitude of the GnRH-induced increase in testosterone concentrations were not related in the mating phase (b), and negatively related in the parental phase (e). These results are illustrated
by the slopes of individual hormonal reactive scopes from baseline to GnRH-induced testosterone concentrations (please note logarithmic scale), indicating similar slopes during the mating phase (c), but steeper slopes for individuals that were starting with lower baseline concentrations during the parental phase than individuals that started with higher baseline concentrations during that phase (f).
95% credible intervals of the GnRH-injected birds with the mean
estimates of the saline-injected birds indicated that the two groups
did not differ in any of the parameters measured to estimate the degree
of territorial aggression. In particular, there were no differences in the
latency to respond to the STI, the latency to approach within 5 m of
the decoy, the time spent within 5 m of the decoy, the percent of time
they fluffed their body feathers, the rate of head noddings, and the
number of songs they sang during the STI (Fig. 2).
Also during the 10 min period after the STI, GnRH-injected birds did
not respond differently from saline-injected birds. They did not spend
more time within 5 m of the area of the hidden decoy, and also the
rates of head noddings and the number of songs did not differ between
the groups (Fig. 3).
4. Discussion
During the mating and parental phase, the large majority of black
redstarts showed large differences in baseline testosterone concentrations and responded to injections of GnRH with a surge of testosterone
production. Post-capture baseline and GnRH-induced concentrations of
testosterone were positively correlated, and the magnitude of the
increase in testosterone after the GnRH-injection was unrelated to
initial testosterone during the mating phase, but negatively related to
initial testosterone in the parental phase. Hence, similar to studies in
other species (e.g. [15,32,38,40,46]) GnRH led to a release of testosterone within an individual's physiological capacity. These GnRH-induced
short-term increases in testosterone are similar to the short-term
increases in testosterone that can occur in the real life of birds and
other vertebrates and mimic the dynamic nature of the HPG axis. The
GnRH injection is only a minor add-on to the handling stress to birds
and probably affects them less than, for example, taking a blood sample.
This is confirmed by our study in which all birds except two responded
to the STI experiment already 35 min after the injection.
During the mating phase, individuals started from different baseline
concentrations, but overall showed a similar magnitude in change after
GnRH-injections. This could indicate that the post-capture baseline of
testosterone during the mating phase was actually a good estimate of
the breeding baseline for each bird and that the GnRH-induced increase
represented a good estimate of the hormonal reactive scope of an individual. During the parental phase the situation was slightly different in
that individuals that started with lower baseline plasma testosterone
concentrations were capable of increasing testosterone secretion more
than individuals that started with higher baselines (some of which
could not increase testosterone). These differences could possibly
indicate that during the parental phase some individuals had already
elevated levels of testosterone to begin with and thus expressed a
limited capacity to further increase testosterone after GnRH-injection.
In most other species tested so far, increases in testosterone after
GnRH injection were lower during the parental phase than during
mating [32,33,63]. During the parental phase it might be beneficial for
males to keep testosterone levels within a narrower range than during
mating, because in some species high levels of testosterone may
suppress paternal behavior. In, male dark-eyed juncos, for example,
GnRH-induced testosterone levels are negatively correlated with feeding behavior [38]. However, in males of other species – including black
redstarts – no such relationship was found [30,44,64].
In black redstarts we found that the GnRH-induced short-term
increase in testosterone did not affect the intensity or persistence of
aggression during or after simulated territorial intrusions with a stuffed
decoy and song playback. We did not include a formal positive control of
naïve – i.e. previously uncaught – birds subjected to an STI, because we
have previously conducted extensive STI experiments with black
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W. Goymann et al. / Physiology & Behavior 149 (2015) 310–316
Fig. 2. Behavior of saline (N = 10) and GnRH-injected (N = 9) male black redstarts during the 20 min STI period; a) latency to respond to the STI, b) latency to approach within 5 m of the
decoy, c) time spent within 5 m of the decoy, d) percent of time with fluffed body feathers, e) rate of head nodding and, f) number of songs. Large black triangles and error bars indicate
posterior mean estimates and their respective 95% credible intervals; small open triangles indicate individual measurements.
redstarts of the same population. However, if we compare the data
presented here with our previously published data on STI experiments,
then the response during and after an STI did not differ between previously naïve males and males that were caught and injected with saline
or GnRH 35 min before the STI (see e.g. Table 1 in [15], Figs. 1, 2, and 3 in
[16], and Fig. 2 in [17]). This similarity suggests that the short-term
stressor of passively catching a male black redstart without playback
lure, measuring it, injecting it with saline or GnRH, and releasing it
back onto its territory did not affect the strength of its territorial
response 35 min after the incident. Hence, because the agonistic
response to an STI of naïve and uncaught birds was similar to the one
of previously caught and injected birds it is unlikely that the hypothalamic pituitary adrenal stress response influenced the behavior.
The absence of a GnRH-induced short-term increase in testosterone
on the intensity and persistence of territorial aggression in black
redstarts is in contrast with a previous study in song sparrows, in
which testosterone implants increased the intensity and persistence of
territorial aggression [12]. However, one major difference between
song sparrows and black redstarts is that song sparrows increase testosterone during simulated territorial intrusions [13]. In contrast, no such
increase is observed in black redstarts [15–17]. Furthermore, blocking
of testosterone and estrogen action within a breeding and outside a
breeding context did not alter territorial aggression in male black redstarts [48,57,65]. In this study, we show that a short-term rise in testosterone would be ineffective in increasing the intensity or persistence of
territorial aggression in a species that otherwise lacks a testosterone response during male–male interactions. Hence, a rise in testosterone
during territorial encounters may be selected for in song sparrows, because this short-term elevation in testosterone may help male song
sparrows to maintain their territory. In contrast, a rise in testosterone
in male black redstarts may not represent a selective advantage, because it has no effect on territorial behavior. Further studies on species
that differ in their androgen responsiveness to territorial intrusions
are required to test whether the patterns observed in song sparrows
W. Goymann et al. / Physiology & Behavior 149 (2015) 310–316
315
Fig. 3. Behavior of saline (N = 10) and GnRH-injected (N = 9) male black redstarts during the period 10 min after the STI; a) time spent within 5 m of the covered decoy, b) rate of head
nodding and, c) number of songs. Large black triangles and error bars indicate posterior mean estimates and their respective 95% credible intervals; small open triangles indicate individual
measurements.
and black redstarts hold as a general rule. Given the current gaps in understanding androgen responsiveness in birds and other vertebrates
[24,66] the use of hormonal reactive scopes would be a promising approach to advance our understanding of hormone–behavior relationships in natural contexts.
5. Conclusions
In this study, we presented an approach to study the effects of
short-term increases in hormones on behavior within the physiological
reactive scope of individuals. In our case, a short-term rise in testosterone to its presumable physiological maximum did not affect territorial
behavior in a species that does not modulate testosterone during
male–male challenges. Apart from seasonal variation in hormone levels,
increases in hormone levels during particular life-history stages can be
dynamic and both the HPG and the HPA axis are flexible systems that
are regulated by negative feedback loops. Hormone implants interfere
with these dynamic systems by “chronically” elevating hormone levels
usually above an individual's natural maximum. It is, therefore, often
unclear how results of implant studies should be interpreted within
an ecological and evolutionary context. Releasing or tropic hormone
injections (e.g. GnRH and adrenocorticotropic hormone) elevate target
hormones (e.g. sex steroids and glucocorticoids) to an individual's maximum level and do not interfere with the negative feedback loops of the
HPG or HPA axis. They, therefore, offer the potential to study short-term
effects of elevated hormone levels on behavior or other traits in
question. Injections of releasing or tropic hormones are particularly
useful to mimic and study the potential behavioral effects of shortterm changes in androgens (i.e. androgen responsiveness to social
challenges) or glucocorticoids (i.e. acute stressors).
Acknowledgments
The authors thank the members of the Research Group of Evolutionary Physiology and in particular Michaela Hau and Robert de Bruijn for
discussions and the Max-Planck-Gesellschaft, and especially Manfred
Gahr for funding. CPV acknowledges a stipend from becas Chile. We
also thank 4 anonymous referees for constructive comments that
helped to improve the manuscript. The authors declare that they have
no conflict of interest.
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