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First published online March 2, 2006
Journal of Experimental Biology 209, 1035-1043 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02112
How much stress do researchers inflict on their study animals? A case study using a scincid lizard, Eulamprus heatwolei
Biological Sciences A08, University of Sydney, NSW 2006, Australia
* Author for correspondence at present address: Forestry and Environmental Studies, Yale University, CT 06511 USA (e-mail: tracy.langkilde{at}yale.edu)
Accepted 17 January 2006
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Key words: corticosterone, ethics, lizard, Eulamprus heatwolei, methods, research
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;
Guillette et al., 1995).
Second, inflicting high levels of stress on our study organisms is ethically
indefensible (Association for the Study of
Animal Behaviour, 2003; Office
of Technology Assessment, 1986). This latter issue has grown to
become a major community concern over recent decades, stimulating the creation
of institutional ethics committees to regulate researchers and hence, prevent
excessively stressful procedures (Hagelin
et al., 2003; Jennings,
1994; National Health and
Medical Research Council, 2004;
National Research Council,
1996). For example, many ethics committees forbid toe-clipping as
a technique for individual identification, insisting instead on `less
stressful' measures such as microchip implantation
(Mellor et al., 2004).
Approval by such a committee and adherence to accepted ethical codes of
conduct are now prerequisites for conducting research in many parts of the
world: for example, wildlife authorities may require ethics approvals before
providing research permits, and journals may adhere to specific guidelines
that outlaw some previously popular research methods
(Association for the Study of Animal
Behaviour, 2003; Department of
Sustainability and Environment, 1975;
Fish and Wildlife Division,
2003).
Although judgments about stress thus dictate the kinds of research methods
that can be used, the term `stress' itself generally remains poorly defined
even in the regulations that guide the activities of ethics committees (e.g.
National Health and Medical Research
Council, 2004; University of
Canterbury Animal Ethics Committee, 2004). Such definitions range
from emotive descriptions [e.g. `fearful' or `frustrated'
(Petherick, 2005)], to very
general statements such as `the biological responses an animal exhibits in an
attempt to cope with a threat to its homeostasis'
(Carstens and Moberg, 2000).
The term `stress' is often used interchangeably with `distress', though the
latter is sometimes used to describe a more severe condition.
Despite broad consensus about the need for ethical treatment of research
animals, the difficulty in defining stress has discouraged attempts to
actually measure the effects of experimental procedures on animal welfare.
Therefore, ethics committees generally have to base their decisions on
intuition, a notoriously unreliable criterion when dealing with organisms that
are phylogenetically remote from ourselves. Ectothermic vertebrates (such as
reptiles and amphibians) are of special interest in this respect because they
are popular research subjects, but are physiologically and behaviourally so
divergent from endothermic vertebrates (such as humans) that it is difficult
for us to judge stress in a lizard (for example) by overt behavioural
symptoms. How, then, can we proceed to replace subjectivity with objective
information? The central problem involves developing a way to measure `stress'
and fortunately, existing literature provides a useful and well-understood
methodology in this respect. An increase in glucocorticoids is a classic
marker of physiological stress in vertebrates
(Belliure et al., 2004), with
corticosterone being the principal glucocorticoid in reptiles, amphibians,
birds, and many rodents (Romero,
2004). Although plasma corticosterone levels can be influenced by
a variety of factors including reproductive stage
(Woodley and Moore, 2002), and
thus elevated levels do not always indicate stress, they can be used reliably
for this purpose under controlled conditions when other factors are held
constant. Thus, our working definition of stress is `the physiological
response of an animal to an experimental stimulus, as measured by elevated
circulating glucocorticoid levels'. Changes in plasma corticosterone levels
have been used to assess the impact of capture, handling, restraint and
confinement on reptilian species (Moore et
al., 1991; Cree et al.,
2003; Cree et al.,
2000; Mathies et al.,
2001; Kreger and Mench,
1993; Cash et al.,
1997), but the amount of stress induced by alternative commonly
used research procedures beyond initial capture and housing has not been
previously assessed. The present paper describes a study that set out to
quantify the ways in which plasma corticosterone levels in lizards are
affected by a variety of methods that are commonly used in fields of research
such as behavioural ecology and microevolution. For comparison with the levels
of stress induced by `natural' events in a lizard's life, our design included
treatments such as tail autotomy, and encountering potentially threatening
stimuli such as the scent of a predator or the actual presence of a larger
competitor.
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), with
strong, highly aggressive competition among these species for the warmest
refuge sites (Langkilde and Shine,
2004). The current study focuses on one of these species,
Eulamprus heatwolei (Wells & Wellington, Scincidae). As one of
the smallest of the five taxa (approximately 85 mm snout–vent length
SVL) (Cogger, 2000),
E. heatwolei is often excluded from preferred refuges by more
dominant species (Langkilde and Shine,
2004). Eulamprus heatwolei is insectivorous, and females
give birth to a litter of one to eight offspring per year in late summer
(Greer, 1989). Tail autotomy
is common in natural populations (83.5% of adult E. heatwolei
captured from Kanangra-Boyd National Park as part of a separate study had
previously autotomised their tails, N=85; T.L. and R.S., unpublished
data). Previous research has shown that E. heatwolei exhibit elevated
plasma corticosterone levels in response to prolonged handling
(Langkilde et al., 2005).
Collection and husbandry of animals
To obtain natural baseline corticosterone levels, blood samples were
obtained from 10 male and 10 female E. heatwolei immediately upon
capture from the field at Kanangra-Boyd National Park, in the first 2 weeks of
November 2004. In this and all other cases, we took a 70 µl blood sample
from the post-orbital sinus using a 75 µl heparinized capillary tube. For
the experimental component of this study, we captured 250 adult E.
heatwolei (100 males and 150 females) by hand from Kanangra-Boyd National
Park in the first 2 weeks of November 2004. This species breeds synchronously,
and at this time of year all lizards are just starting to mate, and females
are commencing ovulation (T.L. and R.S., unpublished data). All lizards were
sexed upon capture and abdominally palpated to determine the number of
follicles, and whether or not they had ovulated. They were then placed into
individual canvas bags, transferred to the University of Sydney, and
individually housed in plastic containers (20x12x10 cm;
LxWxD) with a paper substrate, a water dish, and a shelter. The
room was maintained at 16°C, and heat for thermoregulation was provided by
strips of heating tape (reaching 40°C) running under one end of each
container on a 9 h on:15 h off cycle. The overhead fluorescent lights in the
room were set on a 12 h on:12 h off cycle, mimicking field conditions. Water
was provided ad libitum, and lizards were fed four crickets twice
weekly. Lizards were maintained under these conditions for 14 days before the
experiment started, as previous research suggests that lizards adapt to
captivity (with corticosterone levels returning to baseline levels) within
this time (Jones and Bell,
2004; Manzo et al.,
1994).
Experimental design
(1) How long after exposure to a stressor should a blood sample be obtained?
To obtain a reliable estimate of the physiological stress caused by a
particular treatment, a blood sample must be taken after plasma corticosterone
concentrations have increased, but before they decrease again. To assess the
timing of changes in plasma corticosterone after the application of a
stressor, we assigned 50 female E. heatwolei to one of five
treatments (N=10 females in each group). Forty females (from groups
1–4) were exposed to a potential stressor by obtaining a blood sample
and then chasing them around the inside of an empty container
(60x33x20 cm; LxWxD) for 30 s before returning them to
their enclosures. We obtained blood samples from the females assigned to the
first treatment group 5 min after the initiation of the stressor, after 30 min
for the second treatment group, after 1 h for the third group, and after 2 h
for females in the fourth group. Females assigned to the fifth treatment acted
as controls; they were not disturbed in any way, and a blood sample was
obtained immediately upon capture from the enclosure. Results from this
experiment determined the timing of sampling in the subsequent
experiments.
(2) How does treatment affect plasma corticosterone levels?
Lizards were randomly assigned to one of 10 treatments (N=10 males
and 10 females per treatment), designed to mimic common laboratory research
techniques and natural events, as well as appropriate controls. The treatments
were as follows.
),
for 1 h.
All lizards were returned to their home enclosures upon completion of the
treatments. Blood samples were obtained 1 h after the initiation of each
treatment. Reproductive stage can affect circulating corticosterone
(Woodley and Moore, 2002),
however there was no significant difference between treatments in the number
of follicles females possessed (F[1,98]=0.03,
P=0.87), or whether or not they had been ovulated
(
2=10.76, d.f.=9, P=0.29).
(3) How do a lizard's sex and size influence its response?
To enable us to determine whether a lizard's sex and/or size influences its
plasma corticosterone levels, or its response to treatments, we measured
SVL and mass of each animal. Sex was determined by hemipene eversion
upon capture.
(4) How long does a response last, and do lizards acclimate to the stressor?
Ethical treatment of research animals requires us to evaluate the duration
of any stress response as well as its peak intensity: all else being equal, a
treatment causing a transitory response is preferable to one that causes
corticosterone levels to remain elevated for days or weeks. We monitored the
plasma corticosterone concentrations of E. heatwolei exposed to three
treatments: tail autotomy (N=8 males and 8 females), toe-clipping
(N=5 males and 5 females), and microchip implantation (N=10
males and 10 females), as well as controls (N=8 males and 8 females),
by obtaining additional blood samples from these animals at two intervals
(after 2 h and after 14 days) following initiation of the treatment. The
number of animals differed among treatment groups for logistical reasons.
Animals may be able to acclimate to a repeated or prolonged stressor, such
that plasma corticosterone levels fall despite continued exposure to stress.
In such a case, plasma corticosterone levels may provide a poor indicator of
actual stress levels experienced by the study organism. However, such
acclimated animals may respond more strongly than naïve controls if they
are subsequently exposed to a new or additional stressor
(Romero, 2004). To test
whether any observed decrease in corticosterone levels after 14 days was due
to a `real' reduction in stress (vs acclimation to a persistent
stressor), we exposed these animals to an additional stressor and obtained a
final blood sample 1 h later to measure plasma corticosterone. The blood
sample obtained 14 days after the initiation of the original treatment, in
combination with chasing the individual around an empty container
(60x33x20 cm; LxWxD) for 30 s, acted as the `novel'
stressor for this part of the study. All lizards were placed back into their
familiar enclosures after we applied the stressor (i.e. before we obtained the
blood sample).
(5) Do time of day and/or handling duration affect the stress response?
Plasma corticosterone levels may vary significantly over a 24-h period
(Chan and Callard, 1972;
Dauphin-Villernant and Xavier,
1987; Jones and Bell,
2004) (but see Tyrrell and
Cree, 1998), and be confounded by the stress caused by handling
the animal and obtaining the blood sample. Thus, we recorded the time of day
that each sample was obtained, as well as the total duration of handling from
first disturbance until the completion of blood sampling.
(6) Is there a simpler way to measure the stress response?
Measuring plasma corticosterone levels is invasive, time-consuming, and
costly, so it would be useful to identify a simpler alternative. We assessed
the validity of one alternative potential measure of `stress': breathing rate.
Exposure to external stimuli typically elicits increased respiratory rates in
lizards, and these are easily quantified by counting lateral expansions and
contractions of the chest cavity (Avery,
1993; Langkilde and Shine,
2005). We recorded the number of breaths per 30 s for each lizard
immediately after the initiation (in cases where treatments were applied for 1
h, e.g. unfamiliar enclosure) or completion of each treatment. These data
allow us to assess the relationship (if any) between breathing rate and plasma
corticosterone level, and to compare the patterns of response revealed by
these two measures.
Obtaining and analyzing blood samples
A total of 456 blood samples were obtained during the course of this
experiment. Although standardizing the time of day that all these samples were
obtained would have minimized the potential effect of diel variations in
corticosterone levels, it would have dramatically lengthened the duration of
this study, thus introducing variation due to factors such as time of year and
reproductive stage [factors that can have a significant impact on
corticosterone levels (Woodley and Moore,
2002)]. Thus, all blood samples were taken (from the postorbital
sinus, as described above) during the animals' normal activity period
(0900–1700 h), when levels are least variable
(Jones and Bell, 2004), with
trials timed so that lizards within each treatment were evenly sampled over
this period. In addition, the time of day that blood samples were obtained was
recorded, and included as a factor in the analyses (see above).
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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(2) How does treatment affect plasma corticosterone levels?
Treatment significantly affected plasma corticosterone levels [two-factor
analysis of covariance (ANCOVA) with treatment and sex as the factors, SVL as
the covariate, and plasma corticosterone levels 1 h post stimulus as the
dependent variable: treatment F[9,188]=11.57,
P<0.0001, all interactions non-significant;
Fig. 2A]. Fisher's PLSD
post-hoc tests identified four significantly different levels of plasma
corticosterone among our test animals (i.e. no statistically significant
differences within any of these groups, but each group different
from the others). The first group (A; lowest plasma corticosterone levels)
contained the field animals. The second group (B; slightly above field levels)
comprised the laboratory control animals together with those that were handled
and measured, those that were exposed to predator scent, and those that were
toe-clipped. The third group (C; high levels of plasma corticosterone)
comprised animals that had been implanted with microchips, had blood samples
taken, had autotomised their tails, had been placed into an unfamiliar
enclosure, or had been tested for locomotor speed. The final group (D; very
high levels of plasma corticosterone) contained the lizards that had been
exposed to aggressive heterospecific animals.
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(3) How do a lizard's sex and size influence its response?
Plasma corticosterone levels consistently were at least threefold higher in
female lizards than in males (two-factor ANCOVA with treatment and sex as the
factors, SVL as the covariate, and plasma corticosterone levels 1 h post
stimulus as the dependent variable: sex F[1,188]=139.46,
P<0.0001, all interactions non-significant;
Fig. 2A). In contrast, a
lizard's body size did not influence its plasma corticosterone levels
(two-factor ANCOVA with treatment and sex as the factors, SVL as the
covariate, and plasma corticosterone levels 1 h post stimulus as the dependent
variable: SVL F[1,188]=0.99, P=0.32, all
interactions non-significant).
(4) How long does a response last, and do lizards acclimate to the stressor?
The duration of increased corticosterone levels following exposure to a
stressor depended on the lizard's sex as well as the procedure to which the
animal had been exposed (two-factor repeated measures ANOVA with treatment and
sex as the factors, and corticosterone levels at 1 h, 2 h and 14 days as the
repeated dependent variable: treatment F[3,54]=4.61,
P=0.006, sex F[1,54]=53.49, P<0.0001,
corticosterone levels over time F[2,108]=30.27,
P<0.0001, corticosterone levels over timexsex
F[2,108]=3.89, P=0.02, all other interactions
non-significant; see Fig. 3).
Plasma corticosterone levels in females increased 1 h after tail autotomy, but
fell back to control levels within 2 h and remained low 14 days later
(Fig. 3A). In contrast,
insertion of a microchip also caused a rise in plasma corticosterone levels of
females within 1 h, but this higher level was still evident not only after 2
h, but also after 14 days (compared to controls;
Fig. 3A). Finally, toe-clipping
did not cause any significant increase in plasma corticosterone levels of
female lizards (either at 1 h, 2 h or 14 days;
Fig. 3A). In contrast to
females, male lizards showed no significant increase in plasma corticosterone
levels in response to any of the three treatments (compared to the control)
over any of our test periods (Fig.
3B).
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(5) Do time of day and/or handling duration affect the stress response?
The time of day that blood samples were obtained significantly influenced
our results (two-factor ANCOVA with treatment and sex as the factors, minutes
since midnight as the covariate, and corticosterone levels 1 h after stimulus
as the dependent variable: minutes since midnight
F[1,169]=2.12, P=0.15, treatmentxminutes
since midnight F[9,169]=2.19, P=0.03, all other
interactions non-significant). However, a closer inspection of individual
regressions reveal that this significant interaction effect was due to a
single treatment; the response to tail autotomy was affected by time of day,
with marginally higher plasma corticosterone levels recorded later in the day
(regression: F[1,19]=4.54, P=0.047). Following
Bonferroni correction (
=0.005), this result was no longer statistically
significant. The duration of handling required to take a blood sample did not
significantly influence corticosterone levels (two-factor ANCOVA with
treatment and sex as the factors, handling time as the covariate, and
corticosterone levels 1 h after application of the stressor as dependent
variable: duration of handling F[1,186]=0.10,
P=0.76, all interactions non-significant).
(6) Is there a simpler way to measure the stress response?
At first sight, our results are encouraging for the idea that breathing
rates may offer a simple, cost-effective alternative to measuring plasma
corticosterone levels. The breathing rate of a lizard immediately after
application of a stressor was positively correlated with the animal's plasma
corticosterone levels 1-h post stimulus (correlation z-test on data from both
sexes combined: r=0.36, d.f.=183, P<0.0001;
Fig. 4). However, the
correlation between breathing rate and plasma corticosterone levels 1-h post
stimulus (1) was low (explaining only 13% of total variation in corticosterone
levels); and (2) differed between the sexes, with males showing lower
corticosterone levels than did females with the same breathing rate (1-factor
ANCOVA with sex as the factor, breathing rate as the covariate and
corticosterone levels as the dependent variable: sex
F[1,181]=102.11, P<0.0001, breathing rate
F[1,181]=34.17, P<0.0001, all interactions
non-significant; Fig. 4). In
addition, (3) patterns of response to stimuli suggested by breathing rate
differ from those suggested by the corticosterone data. For example, the sex
disparity seen for corticosterone levels was also seen for breathing rates in
response to some stimuli, but not others (2 factor ANOVA with sex and
treatment as the factors and breaths per minute as the dependent variable:
treatment F[9,164]=30.80, P<0.0001, sex
F[1,164]=0.80, P=0.37, treatmentxsex
F[9,164]=2.70, P=0.006, all other interactions
non-significant; Fig. 2B).
Similarly, although treatment significantly affected breathing rate as well as
corticosterone levels, the nature of those differences (e.g. which treatments
generated the greatest stress?) differed substantially between the two
measures (Fig. 2A,B).
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Validation of methodology
Perhaps the most surprising aspect of our results is that a study such as
this has not been conducted previously. Given that researchers often complain
about excessive regulation of their endeavours, and the arbitrary nature of
decisions by non-expert members of ethics committees, we might have expected
researchers to gather objective data to support their cases about the merits
of alternative research protocols. Our study is encouraging in this sense, in
that it suggests that a widely accepted and easily assayed measure of
physiological stress in vertebrates (plasma glucocorticoid levels) can offer a
straightforward index of the effects of experimental procedures on animal
welfare. The most significant caveat to this finding involves the meaning of
the term `stress'. Because this term has been defined in so many different
ways, a critic could argue that plasma glucocorticoid levels do not provide a
valid operational measure of the kind of stress that they perceive as
important in ethical issues. The only solution to this criticism, so far as we
can see, is a wider debate on the meaning of `stress' within the context of
animal-based research.
Unfortunately, the most obvious shortcut method that we attempted – recording breathing rates of our study animals – appears less informative in this respect than plasma corticosterone levels. Respiration rates offer a very sensitive assay: indeed, lizards often increase ventilation rates simply when the observer approaches them. However, the rates drop back to baseline levels rapidly also. Hence, the stress associated with an experimental manipulation may be too small to elevate plasma corticosteroid levels even if it induces a transitory increase in respiration rate (as was the case for handling, in the present study). Researchers might be able to use the easily gathered data on breathing rates to make comparisons among methods, sexes or species when initially selecting study systems, but we caution that the resulting data may bear little relationship to underlying levels of plasma corticosterone.
Degree of stress induced by alternative methods
It is encouraging to see that lizards experienced less stress as a result
of many of our experimental procedures than they did from biologically
realistic situations encountered in the field. However, the fact that the
degree of stress induced by a specific procedure was not intuitively obvious
means that many current regulations about research procedures may be based on
false premises. For example, the oft-criticized marking technique of
toe-clipping did not induce a major rise in corticosterone levels, whereas
lizards were affected to a much greater degree by an alternative and
supposedly less stressful method of implantation of a microchip, and by
superficially trivial manipulations such as housing in an unfamiliar
enclosure. In addition, physiological stress caused by microchip insertion
lasted for at least 14 days, in contrast to the animal's rapid recovery from
tail autotomy. Implanted microchips may cause constant irritation, impede
movement, and occasionally migrate around under the skin or into the body
cavity (Roark and Dorcas,
2000). In contrast, the much larger wound caused by tail autotomy
rapidly heals, presumably reflecting effective selection over many generations
to deal with this frequently occurring `natural' event.
It is possible to reconcile most of our results with other information on
the study species, even though we were unable to predict those response
patterns a priori. For example, the lack of response to predator
scent compared to the dramatic response to the presence of a larger competitor
species (Egernia saxatilis) accords well with prior behavioural
observations (Langkilde and Shine,
2005). Similarly, the lack of impact of handling time is
unsurprising, given that (1) all animals had a total duration of handling
(from disturbance to completion of blood sampling) of <147 s, and (2) based
on plasma corticosterone levels, simply handling an animal was not
particularly stressful. Finally, we observed none of the temporal variation in
plasma corticosterone levels reported for other species
(Chan and Callard, 1972;
Dauphin-Villernant and Xavier,
1987), but blood samples were obtained only during the lizards'
active period, when levels are least variable
(Jones and Bell, 2004).
Although post-hoc rationalization of our results is thus possible, we doubt
that the average animal ethics panel (nor, indeed, a group of expert
researchers familiar with the study species) would have predicted our results
a priori. Hence, using intuition as a basis for decisions about the
degree of stress induced by alternative practices is fraught with error.
Sex differences in plasma corticosterone levels and in stress responses
Why did our female lizards have corticosterone levels so much higher than
those of males, and respond more sensitively to experimental treatments?
Although published data are scarce, previous studies on reptiles generally
have reported the opposite situation: higher plasma corticosterone levels in
males than in females (Elsey et al.,
1990; Lance et al.,
2001; Mathies et al.,
2001). Higher levels of plasma corticosterone may enhance
organismal fitness by facilitating access to energy stores, and thus enhance
the animal's ability to escape rapidly from a predator, battle with
conspecifics, etc. (Lance et al.,
2001). Activities such as mate-searching, courtship, mating and
male–male combat may require elevated levels of energy expenditure, thus
favouring increased plasma corticosterone levels in males rather than females
(Lance et al., 2001).
Additionally, high levels of corticosterone in pregnant females of a
viviparous species (such as Eulamprus heatwolei) might have negative
effects on the developing offspring (Cree
et al., 2003). Neither of these ideas accords with our own data,
in that it was females not males that exhibited high levels of plasma
corticosterone; and many of the females that we used were pregnant during the
trials. Our analyses revealed no effect of pregnancy on plasma corticosterone
levels (T.L. and R.S., unpublished data). Nonetheless, our study animals are
not unique in females exhibiting higher corticosterone levels than males.
Corticosterone is also the major glucocorticoid in several mammalian species
including rodents, and in these groups, females typically exhibit higher
levels of plasma corticosterone than do males
(Handa et al., 1991;
Jones et al., 2005). This
situation has been interpreted as a proximate effect of sex hormones on
hypothalmic–pituitary–adrenal (HPA) function: estrogen enhances
the response of the HPA axis to stress, whereas testosterone suppresses it
(Greenberg and Wingfield,
1987; Handa et al.,
1991; Jones et al.,
2005).
In summary, our data suggest that direct measurement of plasma corticosterone levels offers a useful technique for quantifying the impact of research techniques on study organisms. In the viviparous lizards we studied, the magnitude and duration of elevation in plasma corticosterone levels differed strongly between the sexes and was influenced by experimental procedures. The range of protocols that we applied to our lizards encompassed many of the routine procedures used by behavioural ecologists, and in general the animals were affected no more strongly by these experimental manipulations than they were by simulated `natural' events (such as autotomy and encountering a heterospecific lizard). However, the relative impact of different techniques often diverged from our a priori expectations. When selecting our research methods to minimize stress on study organisms, we might be better advised to ask the animals directly (via techniques such as quantifying their plasma corticosterone levels) rather than relying on our own intuition as to what constitutes a `stressful' procedure.
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Acknowledgments |
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