ArticlePDF AvailableLiterature Review

Does the Stress of Laboratory Life and Experimentation on Animals Adversely Affect Research Data? A Critical Review

Authors:
  • Animal Free Research UK

Abstract

Recurrent acute and/or chronic stress can affect all vertebrate species, and can have serious consequences. It is increasingly and widely appreciated that laboratory animals experience significant and repeated stress, which is unavoidable and is caused by many aspects of laboratory life, such as captivity, transport, noise, handling, restraint and other procedures, as well as the experimental procedures applied to them. Such stress is difficult to mitigate, and lack of significant desensitisation/habituation can result in considerable psychological and physiological welfare problems, which are mediated by the activation of various neuroendocrine networks that have numerous and pervasive effects. Psychological damage can be reflected in stereotypical behaviours, including repetitive pacing and circling, and even self-harm. Physical consequences include adverse effects on immune function, inflammatory responses, metabolism, and disease susceptibility and progression. Further, some of these effects are epigenetic, and are therefore potentially transgenerational: the biology of animals whose parents/grandparents were wild-caught and/or have experienced chronic stress in laboratories could be altered, as compared to free- living individuals. It is argued that these effects must have consequences for the reliability of experimental data and their extrapolation to humans, and this may not be recognised sufficiently among those who use animals in experiments.
Introduction
In the December 2017 issue of ATLA, I published an
Editorial, in which I suggested that stress in ani-
mals in laboratories, resulting from their environ-
ment and experimental procedures, was not limited
to welfare concerns (1). It also adversely affects, sig-
nificantly and unavoidably, multiple biological sys-
tems and therefore affects the resultant
experimental data. This exacerbates inter-species
differences and makes extrapolation to humans even
more difficult and unreliable. In an effort to encour-
age and promote discussion of this issue, which is
underappreciated, I am expanding on that Editorial.
Stress may be thought of as “the sum of the biolog-
ical reactions to any adverse stimulus, physical,
mental, or emotional, internal or external, that
tends to disturb the homeostasis of an organism” (2).
It is increasingly acknowledged that diverse species
experience pain, stress and distress (see below), and
experience depression and anxiety disorders (see
Ferdowsian et al. [3]). This includes fish — notably
the ubiquitous laboratory zebrafish — which show
emotional fever/stress-induced hyperthermia in
response to a variety of stressors, including simple
handling (4, 5). Stress in fish can result in increased
aggressive behaviour (6), elevated anxiety, dimin-
ished weight-gain, and altered levels of dopamine
and serotonin metabolites in the brain (7), as well as
increased brain levels of extracellular adenosine,
which has neuromodulatory effects (8).
While all species experience stressors and stress
in their natural environments, psychological and
physiological problems can arise with exposure to
recurrent stressors, and/or when stress becomes
chronic and too difficult to cope with. This leads to
allostatic overload (excessive wear and tear on the
body), which may manifest in altered physiological
responses, some of which can be harmful (9) — gen-
erally thought of as a state of distress. How ever, the
US National Research Council (NRC) accepts that
there is “confusion in the scientific, regulatory, and
animal welfare communities” concerning the distinc-
tion between stress and distress; and that these
terms are often used interchangeably in animal wel-
Does the Stress of Laboratory Life and Experimentation on
Animals Adversely Affect Research Data? A Critical Review
Jarrod Bailey
Cruelty Free International, London, UK
Summary — Recurrent acute and/or chronic stress can affect all vertebrate species, and can have serious
consequences. It is increasingly and widely appreciated that laboratory animals experience significant and
repeated stress, which is unavoidable and is caused by many aspects of laboratory life, such as captivity,
transport, noise, handling, restraint and other procedures, as well as the experimental procedures
applied to them. Such stress is difficult to mitigate, and lack of significant desensitisation/habituation can
result in considerable psychological and physiological welfare problems, which are mediated by the
activation of various neuroendocrine networks that have numerous and pervasive effects. Psychological
damage can be reflected in stereotypical behaviours, including repetitive pacing and circling, and even
self-harm. Physical consequences include adverse effects on immune function, inflammatory responses,
metabolism, and disease susceptibility and progression. Further, some of these effects are epigenetic,
and are therefore potentially transgenerational: the biology of animals whose parents/grandparents were
wild-caught and/or have experienced chronic stress in laboratories could be altered, as compared to free-
living individuals. It is argued that these effects must have consequences for the reliability of
experimental data and their extrapolation to humans, and this may not be recognised sufficiently among
those who use animals in experiments.
Key words: animal welfare, cost–benefit analysis, data accuracy, glucocorticoids, psychological, stress,
translational medical research.
Address for correspondence: Jarrod Bailey, Cruelty Free International, 16a Crane Grove, London,
N7 8NN, UK.
E-mail: jarrod.bailey@crueltyfreeinternational.org
ATLA 46, 291–305, 2018 291
Comment
fare literature and that relevant available informa-
tion is “far from complete”, with distress remaining
“a complex and still poorly understood phenomenon”
(10). Nevertheless, it is generally understood that
distress manifests when an individual is unable to
cope and adapt successfully to one or more stressors,
resulting in compromised well-being due to the
inability to return to physiological and psychological
homeostasis (11). Notably, an individual can remain
in distress, even when a stressor is removed.
While different species and individuals have dif-
ferent stressors, variable ranges of stress to which
they can adapt, diverse spectra of tolerance, and
dissimilar manifestations and sequelae of exces-
sive stress, they share the same biological path-
ways and mechanisms that are adversely affected
by stress. Briefly, stressors stimulate the hypotha-
lamic–pituitary–adrenal (HPA) axis, the sympa-
thetic adrenal medullary axis, and the sympathetic
and parasympathetic nerve projections that
directly innervate secondary lymphoid organs (12–
14). Among other things, this results in the eleva-
tion of the ‘stress hormones’, cortisol and
corticosterone (CORT). Raised glucocorticoid (GC)
levels, alongside significant elevations of heart
rate, blood pressure and other hormone levels, are
acknowledged indicators of fear, stress and dis-
tress, and are used as biomarkers for stress in ter-
restrial vertebrates in laboratories (e.g. 15–17).
Direct consequences of these neuroendocrine
changes include deleterious effects on innate and
adaptive immunity, central nervous system
pathology, and cardiovascular and reproductive
perturbations (18, 19), leading to extensive and
diverse adverse health effects. Psychological
trauma, for instance, may result in altered health
or damaging patterns of behaviour. In humans,
immune perturbations manifest in poor responses
to vaccines, increased susceptibility to infections,
and accelerated disease progression (see Gurfein et
al. [18]). Further, the various mechanisms under-
lying such effects mean that stress has ramifica-
tions beyond the individuals experiencing it.
Successive generations, and individuals who have
experienced prenatal and/or early-life stress, may
be destined to suffer the consequences in adult-
hood.
Caveats and Framing the Argument
Chronic or long-term stress is not unique to ani-
mals in laboratories — it is part and parcel of life
for all species in whatever environment. I do not
intend to suggest otherwise, and it is accepted that
animals in the wild experience acute stressors reg-
ularly (such as lack of food and shelter, predation,
disease, and so on); not only is this entirely normal,
but it can be beneficial in terms of building allo-
static capacity. This Comment, however, argues
that stresses resulting from life in the laboratory
often have negative consequences — given the
opportunity, the animals would avoid many labo-
ratory stressors, and so they can hardly be consid-
ered benign. In addition, the degree, type,
frequency and duration of laboratory stressors are
different to those in the wild, and together could be
more chronic. This may result in greater adverse
consequences for animal welfare and for scientific
data quality. Experiencing repeated, unnatural
stressors, such as blood draws and gavage, cannot
be compared to the brief elevations in CORT
resulting from the natural diurnal/nocturnal
adrenal cycle. For instance, the former stressors
are very different from simply waking up, or being
hungry. In well-designed experiments, increases in
stress biomarkers are relative to baseline levels in
any case (see, for example, 16, 20).
That laboratory stress can — arguably, frequently
and unavoidably — have effects that compromise
animal welfare and experimental results is accepted
by the US NRC’s Committee on Recognition and
Alleviation of Distress in Laboratory Animals (10):
“In the longer term… a breakdown in an animal’s
ability to cope with its environment is likely to lead
to adverse emotional states and poor welfare. Some
of these cases may be quite minor and not give rise
to significant ethical concerns; but prolonged or
intense circumstances would compromise the ani-
mal’s welfare enough to warrant concern and also
significantly affect the research results.” Further:
“Strong evidence in rodents has shown that mild
stress of 2–3 months duration — a regimen that pro-
duces no signs of overt distress — alters the animals’
performance in tests of anxiety, depression, and
memory… Other findings indicate that rats’ habitu-
ation to a test environment can dramatically affect
their response to a toxic substance”. Stress can also
be beneficial: “Over a longer time frame, glucocorti-
coid production in response to infection helps restrict
the immune system, thus preventing deleterious
effects of inflammatory factors on tissues” (10).
While this latter statement is true, it also supports
the argument made here, that stress — even when
not ‘bad’ — adversely affects biological systems, and
consequently confounds research data.
Therefore, it is not only distress that leads to bio-
logical consequences affecting data reliability,
quality and relevance. It seems clear that the con-
sequences of stress are also an issue for welfare
and data quality. To quote again the US NRC:
“The impact of distress on both animal welfare and
research results is likely even more pronounced
than that of stress. Animals exposed to prolonged
severe stress experience underlying changes in
physiological functions (e.g. gastric lesions or
immunosuppression) that can interfere with exper-
imental manipulations; alter experimental vari-
ables such as behaviour, drug dosing and
clearance; change the progress of a disease; and
292 Comment
contribute to morbidity and mortality. A variety of
stressors can contribute to unintended distress,
from postoperative pain or infection to barren
housing conditions or the solitary confinement of
an individual of a social species. Stereotypies,
abnormal repetitive behaviours indicative of poor
well-being that are often observed in distressed
animals, are thought to reflect defective brain
function and to be a result of poor animal welfare.
Stereotypies are thus likely to interfere with
behavioural, neuroscience, and pharmacological
studies” (10); and “The impact of stress and dis-
tress on the quality of scientific research can result
in the generation of compromised data, which in
turn necessitates the use of more animals… Both
stress and distress represent potential complica-
tions in a wide range of experiments, and should be
proactively addressed by good experimental
design” (10). While I (and others) disagree that the
use of more animals and the careful design of
experiments can have an acceptably positive
impact on welfare, and on the human-relevance of
the data generated, the main points in this
Comment stand. Efforts to address this issue over
many years are acknowledged, but I argue that,
due to fundamental biology and inter-species dif-
ferences, the improvement of husbandry, veteri-
nary care, regulation, oversight, training, etc. can
never be sufficient to significantly address and
overcome these issues.
Stress Resulting from Laboratory Life
and Research, and its Biological
Impact
The issue for animals in laboratories is that life
can be inherently and excessively stressful — per-
haps much more than in their natural environ-
ments, from which laboratory conditions differ
greatly, even when enriched and accounting for
efforts to mitigate stressors. Laboratory conditions
preclude or limit many natural behaviours, hous-
ing tends to be much smaller than the animals’
natural ranges, and the animals are subjected to
frequent manipulations and handling, as well as
other alien factors that they try to resist and avoid
(16, 20). Stressful procedures and environmental
factors are numerous and varied. Briefly, they
include, but are not limited to: general handling
and manipulations, such as weighing and saline
injections (15, 16, 21–23); anaesthesia (15, 24–31);
restraint (21, 32–36); gavage (37–42); blood sam-
pling (15–17, 43–50); food and water restriction
(51, 52); non-natural environment (53–55) and
associated factors, such as noise and light (56–62);
social crowding and/or isolation (63–71); cage con-
ditions/cleaning/changing (18, 58, 72–78); trans-
port (19, 58, 79–83); observing procedures on, and
killing of, other animals (31, 84–87); and even
enrichment itself (17, 88, 89). It has been sug-
gested that captive-bred animals may not know
that these stressors are different to those in the
wild, though there might be some perception that
they are not similar to ‘natural’ stressors such as
limited food, inadequate shelter, predation and so
on. In any case, the point is that they are different,
but more importantly, they are also frequent, reg-
ular and inescapable.
Naturally, this has animal welfare and scientific
implications, acknowledged at least in some quar-
ters. For instance, stress from handling is accepted
as a source of “unexplained variation within and
between animal studies”, as it influences “both the
behaviour and physiology of animals” (23; see also
Balcombe et al. [16] and Meijer et al. [17]), and rel-
atively poor caging conditions “may contribute to
problems in translating murine research into
human studies” (18, 77, 78). However, it is acc -
epted by some that these factors are probably
widely underappreciated (90). It should be noted
that some consider enriched environments simply
as ‘less bad’ rather than ‘better’ than those that are
non-enriched. To illustrate, significant numbers of
animals experiencing enrichment still go on to
develop stereotypies (e.g. 91–98).
The Nature of Stress: Biological
Basis/Mechanisms of Stress and its
Adverse Effects
The physiological consequences of stress are varied
and powerful. This, in itself, is of concern for the
translation of animal data to humans, as they com-
pound and confound existing difficulties in transla-
tion due to species differences. First, however, a
consideration of the underlying mechanisms is
important, to demonstrate their fundamental nature
and potency.
Primary mediators of stress, such as GCs and cat-
echolamines, are released in response to stressors,
with various biological consequences. Such biological
effects generally go beyond species boundaries/limits
for mammalian species, though the mechanisms and
specific effects differ to varying degrees. These pri-
mary mediators are extremely powerful, because
they ultimately modulate the expression of many
genes. Secondary outcomes have been documented
“…in every physiological system, including the car-
diovascular system, metabolism, the central nervous
system, and the immune system”, and are con-
founded by the characteristics of the stressor(s), as
well as the attributes of the affected individual, such
as age, health, status, genetic background, past
experience, etc. (99).
The potency and ubiquity of the stress response
has been demonstrated in a number of in vitro stud-
ies, revealing that it generally blocks every impor-
tant cellular process, including DNA replication,
Comment 293
transcription, pre-mRNA processing, mRNA export,
and translation, until the cells recover (100).
Therefore, stress exerts its effects via varied molec-
ular mechanisms, with far-reaching consequences.
The principal ones are highlighted below.
Epigenetic mechanisms (histone acetylation and
DNA methylation) (101–107): Psychological
stress alters gene expression via histone acety-
lation and DNA methylation. Much occurs in
response to environmental triggers, e.g. diet,
drugs, toxins, and psychological stress, e.g. fear
conditioning and maternal care (103). Genes
involved in HPA axis function are especially
susceptible (108), e.g. in suicide victims with a
history of child abuse (109), and post-traumatic
stress disorder (PTSD) is strongly associated
with the epigenetic modification of genes
involved in immune function and inflammation
(110).
Alternative splicing/expression of regulatory
microRNAs: Alternative splicing of gene tran-
scripts and microRNAs (miRNAs), is a powerful
means of altering gene expression that can be
significantly affected by stress (111, 112). For
example, acute stress in humans altered the
splicing of 27 genes in peripheral leukocytes
(100).
Oxidative damage and ageing: Mental stress con-
tributes to oxidative stress in the body, and
therefore to oxidative damage (113). This effect
has been identified in students undergoing aca-
demic examinations (114), and in the lympho-
cytes of psychologically stressed individuals
(115). Oxidative stress is also associated with
PTSD and depression (116), contributes to the
ageing process (117), and is associated with neu-
rodegenerative disease, ophthalmologic disease,
cancer and cardiovascular disease (including
atherosclerosis, hypertension, cardiomyopathy,
chronic heart failure, myocardial ischaemia and
ventricular arrhythmias; 117, 118). There may be
confounding data on the effects of oxidative stress
from different species/strains of mice, which fur-
ther challenges the translation of data across
species (119).
Direct Physiological Consequences of
Stress
The physiological consequences of stress are
numerous and varied. Table 1 shows how promis-
cuous these consequences, effects and manifesta-
tions are in many species, including humans.
Given that these effects involve so many biological
pathways and systems, the effects on experimental
data must be significant.
Habituation/Desensitisation to Stress
Some argue that animals become habituated (120)
and/or desensitised (121) to stress, so implications
for welfare and experimental results may be over-
come (23, 122, 123). For instance, non-human
primates can be trained to approach test environ-
ments, and to present their arms for blood with-
drawal and so on, seemingly voluntarily. However,
it has been shown that repeat exposure to homo-
typic stressors of greater intensity and/or severity
does not result in habituation, and could actually
result in sensitisation of the CORT response (122).
Furthermore, CORT levels might only decrease for
certain types of stressor, persisting for other types
(35); where desensitisation has been shown, it is
only to a modest degree, in a small proportion of
the animals in the studies, and in small sample
sizes (70). While some studies have suggested that
enrichment might decrease stress, others have
shown a paradoxical increase in stress via CORT
levels (18). Similarly, some studies have suggested
that transferring scent-marked materials from old
to new cages reduces stress-related aggression,
while others found that it increases aggression
(74). Furthermore, mice do not habituate to stress
associated with simple handling, and indeed seem
to become sensitised to it (124–126). In instances
where CORT levels decrease, there is increasing
evidence that this does not necessarily mean an
attendant decrease in stress; other indicators, such
as the neutrophil–lymphocyte ratio, might be ‘bet-
ter’ indicators of chronic stress, which can occur in
the absence of increased serum CORT (127).
Adverse Physiological Sequelae of
Psychological Stress are Initiated
Prenatally or in Early Life, and are
Heritable
Captive-born animals can be exposed to stress pre-
natally via their wild-trapped mothers (128–131),
and as infants in laboratory environments, often
experiencing inadequate maternal contact and
care (132–134; cited by Camus et al. [135], and
Detter et al. [136]). If an animal’s parents or grand-
parents lived in laboratories, and/or were born of
parents that lived in laboratories and/or endured
being wild-caught, then their ancestors experi-
enced highly stressful lives and would have been
affected by the adverse consequences described
herein. Even if such an individual was subse-
quently afforded as stress-free a life as possible
(difficult, if not impossible, in a laboratory), the
consequences of their early lives, and the lives of
their ancestors, would lead to the same adverse
effects as if they had continued to experience
excessive stress as adults.
294 Comment
That early-life experiences affect adult psy-
chopathology is widely accepted. As Jean-Paul
Sartre put it, “Childhood decides” (see Murgatroyd
[102]). “A large body of data shows that stress dur-
ing pregnancy causes an increase of GCs in the
blood of the dams and the foetus, leading to alter-
ations of the structure and function of the develop-
ing brain… These alterations result in the
disturbance of the function of the neuroendocrine
system and different kinds of behaviour through-
out life” (137). Early-life/prenatal exposure to
stress leads to altered adrenocorticotrophic hor-
mone responsiveness, dysfunction of the HPA axis
(138, 139), and altered autonomic modulation of
immune function that may begin in utero (103).
Social isolation in several species leads to neuroen-
docrine changes, increased cortisol, and ensuing
behavioural problems (for references, see Cham -
pagne [101]). Maternal inflammation during preg-
nancy (from infection, or possibly stress) may
increase the risk of neurodevelopmental disorders
such as schizophrenia and cerebral palsy (140).
Physiological sequelae include cardiovascular dis-
ease and metabolic disorders such as diabetes
(141), compromised immune function, including
poor lymphocyte proliferation upon infection and
reduced placental transfer of antibodies during
pregnancy (140), autoimmune disorders, chronic
obstructive lung disease, asthma and obesity (see
Chang [142]). Pivotal to these adverse outcomes
are the aforementioned stress-related epigenetic
processes and oxidative damage. Methylation of
gene regulatory regions is partly involved (143),
the extent of which may be set during prenatal
development (141). Cord blood samples of infants
of mothers with late-pregnancy depression show
altered methylation of the GC receptor promoter,
which also predicts elevated salivary cortisol in
early life (144). Other modifications are inherited
and transgenerational in nature (145); for exam-
ple, poor prenatal nutrition affects GC receptor
methylation, affecting the growth and metabolism
of first and second-generation offspring (146), and
matrilineal transmission of the effects of diethyl-
stilbestrol (DES) occurs via hypomethylation, lead-
ing to increased cancer risk over two generations
(147).
Summary
The inherent, multi-faceted stress of laboratory life
— often excessive, relative to the more transient,
acute and ‘natural’ stresses experienced in the wild
— is evidenced by its often negative impact on the
well-being of the animals involved, both psycholog-
ically and physiologically. Because this harm is
mediated via established trans-species biological
mechanisms involving the HPA axis and the sym-
pathetic nervous system, and effected via oxidative
stress and epigenetic mechanisms, which affect
multiple biological pathways and systems, it can
have adverse and confounding effects on experi-
mental results. This modulation of many biochem-
ical pathways and gene expression can result in
downstream effects such as organ damage, cardio-
vascular diseases, attenuated immune function
and autoimmune disorders, premature ageing and
mortality, developmental abnormalities, elevated
tumour initiation and progression, and muscu-
loskeletal atrophy (16).
The absolute degree of translation of animal stud-
ies to humans is debatable, but undoubtedly,
“Studies using animal models are more translatable
to human disease when the animals’ welfare is
maximised” (127). Arguably, it is difficult to “max-
imise” welfare significantly, given the in herent,
widespread, substantial and largely intractable
nature of the stresses involved in animal research
and laboratory life. Notably, habituation and/or
desensitisation to many of the stressors is often not
possible, or at least not significant, and the impact
of these effects on experimental data — and their
extrapolation to humans — is likely to be signifi-
cant. The many sources of stress have been sum-
marised here, along with their effects on multiple
biological and physiological systems. The literature
warns that: “…animals subjected to the environ-
mental changes that occur during transportation…
react with changes in their physiology, such as body
weight, plasma hormonal levels, heart rate and
blood pressure changes… When measurements of
physiological parameters are performed using con-
ventional measurement techniques, the results
must be interpreted with caution as these conven-
tional techniques also have effects on the animals”
(148). Most importantly, “Suffering in animals can
result in physiological changes which may increase
the variability of experimental data” (149). Many
scientists are well aware of these effects and consid-
erations, and have cautioned against disregarding
them (150–152). Yet, while accepting the negative
effects of pain, stress and distress, and their influ-
ence on study outcome, such effects are often not
reported or are under-reported in scientific publica-
tions (90).
The impact of stress on immunological and
inflammatory responses seems particularly preva-
lent, and might be especially critical, seeing as
much animal experimentation involves infectious
agents and/or immune function (for a discussion
and references, see Bailey [153] and Bailey [154]).
Crucially, this impact exacerbates and compounds
existing immune differences between humans and
non-humans due to genetic differences. To illus-
trate, genomic duplications — one of the most sig-
nificant causes of genetic variation among
primates (155) and at the root of many aspects of
intra-species and inter-species diversity — differ-
entially affect many genes involved in immune and
Comment 295
Table 1: A summary of the physiological consequences and manifestations of stress, in many species, including humans
Consequences of general stress Notes
Stereotypies (abnormal, repetitive, invariant These correlate with basal plasma cortisol and corticotrophin-releasing hormone (CRH) in cerebrospinal fluid (166).
behaviours with no obvious function; 160, 161)
and self-harm (e.g. 162–165) in non-humans
Wide-ranging physiological symptoms in humans Anger, depression, anxiety, behavioural changes, food cravings, lack of appetite, frequent crying, difficulty sleeping, tiredness, lack of
concentration, chest pains, constipation, diarrhoea, cramps and muscle spasms, dizziness, fainting, nervous twitches, restlessness, sexual
dysfunctions, breathlessness, and a host of diseases and illnesses with probable associated psychogenic (as well as biological) causes (167).
Long-lasting neurophysiological changes These could “…have direct implications for electrophysiological, behavioural, and molecular studies” (see 69).
Isolated rats show “…structural and functional changes in the mesocorticolimbic dopaminergic system, exhibited hyperlocomotor
activity and impaired sensorimotor gating” (168).
Isolated pigs show “…sustained changes in behavioural, neuroendocrine and immune regulation” (169).
Socially isolated humans show increased risk of death, “…genome-wide transcriptional activity of impaired GC response genes and
increased activity of pro-inflammatory transcription control pathways”, and higher risk of developing “…conduct disorders,
personality disorders, major depression, PTSD, schizophrenia, and anxiety disorders” (see 170).
Multi-faceted modulation of the immune system This occurs in many, if not all, mammalian species (140, 171, 172). It is especially problematic for research involving immune function,
infectious diseases, etc. The expression of several hundred genes (many related to immune function) may be perturbed by simple
handling of animals (173; and see 33), which may be affected by the acute or chronic nature of the stressor (see 173). Chronic stress can:
shift neutrophil–lymphocyte ratios (127, 174);
affect expression of cytokines and cytokine receptors (175, 176), such as IL-2 and IL-6 (18);
alter gene splicing in peripheral leukocytes (10);
alter global and immune gene specific methylation (177); and
differentially affect brain activity and neurotransmitter release, macrophage activity and antibody production (33).
The same stressor in acute or chronic forms may have different effects:
In rodents, acute restraint increases delayed-type hypersensitivity (DTH) and leukocyte redeployment, but can increase or decrease
them when chronic (33, 178).
In humans, chronic, though not acute, stress increases susceptibility to colds (179).
These inconsistencies show that stress is mediated not only by GCs (180), and that accounting for the effects of stress in animal
research must be difficult, if not impossible.
Observed alterations in human immune function These include:
“attenuated responses to vaccination, poorer wound healing, exaggerated release of inflammatory mediators, & premature aging of
the immune system” (181);
increased plasma and CNS cytokine levels, impaired natural killer cell activity, lower T-lymphocyte counts (in PTSD/complex
PTSD patients; 182);
epigenetic changes exerting lifelong impact on immune and inflammatory function (in PTSD/complex PTSD patients; 182); and
greater inflammatory responses to vaccinations (in depressed humans; 183).
Neuropeptides involved in stress responses may accentuate pathophysiological sequelae in critically ill individuals (184). In healthy
humans, 49 different genetic pathways are affected by stress, including genes associated with immune function (185). Stressed students
undergoing examinations have significantly increased pro-inflammatory cytokines (186).
Activation of the HPA axis This accelerates ageing generally, with adverse effects on brain/central nervous system, immune system, skeletal muscle and bone tissue
(see 187–189). HPA dysregulation may lead to excessive inflammation via increases in the levels of circulatory inflammatory cytokines,
decreases in anti-inflammatory cytokines, and alterations in the expression of genes involved in immune activation of peripheral blood
cells (see 177).
296 Comment
Table 1: continued
Effects of specific stressors Notes
Disease susceptibility An increase in susceptibility has been noted across several species to:
general disease and somatic disorders (see 100, 103);
various cancers (190, 191);
pancreatitis and pancreatic tumours (192, 193);
gastrointestinal disorders (194);
thyroid pathology (195);
multiple sclerosis (171);
inflammatory bowel disease (142, 196);
cardiovascular disease (197–199);
accelerated ageing and age-related disorders (187); and
musculoskeletal injury (200).
Stressors can increase oxidative stress, triggering inflammatory pathways associated with type-2 diabetes, cardiovascular disease,
osteoporosis, arthritis, some cancers, and susceptibility to some infections (e.g. 201–205).
Handling Causes biological changes, affecting wellbeing and/or experimental results.
Enrichment intended to mitigate stress can substantially alter brain structure, function and physiology.
Light, noise, cage position/changing etc. all affect physiology, behaviour, anxiety and experimental data (206).
Effects of handling stress may often be “missed” by researchers (23).
Early-life stress in various species Results in:
abnormal brain development, leading to early-life psychopathologies and adult chronic mental illnesses (e.g. 207, 208);
epigenetic modifications associated with depression and suicidal behaviour in later life (e.g. 102, 209–211); and
elevated morbidity and mortality from chronic diseases of ageing, including vascular disease, autoimmune disorders, and premature
mortality (e.g. 141, 212).
Stress from maternal deprivation has negative effects on growth rates in rats, and adversely affects circadian clock and stress
responses (72).
Traumatic stress in humans Studies on PTSD patients have shown that:
acute stress affects glucose metabolism, inflammation and components of the immune system associated with type-2 diabetes (201);
serious long-term consequences include hypertension, heart attacks and stroke, as well as increased risk of obesity, Alzheimer’s
disease, and AIDS dementia complex (213).
Comment 297
inflammatory responses (156). Indels (genomic
insertions and deletions) also affect major histo-
compatibility complex (MHC) genes, which are
critical to immune responses, and are associated
with differences in response to infections, as well
as susceptibility to autoimmune diseases. These
are further confounded by sex-related and strain-
related differences. It has also been shown that the
susceptibility and responsiveness of mice to stres-
sors varies with the strain (157, 158). Stress also
affects sleep, and conversely sleep perturbations
exacerbate and sensitise individuals to stress, with
all the attendant consequences. These conse-
quences are also strain-dependent, and intimately
linked to the CRH/HPA system (159).
Additionally, the adverse consequences of stress
are multigenerational, as the associated epigenetic
mechanisms affect the germline. This is likely to
have significant consequences for animals, and
their offspring, in laboratories: if an animal’s par-
ents or grandparents experienced a stressful labo-
ratory life and experimental procedures, and/or if
the offspring experienced significant stress in early
life, then this will compound any further stress
that they experience as adults, in turn compound-
ing species differences and the translation of data
to humans.
Overall, these observations of detrimental phys-
iological effects and the general mechanisms
behind them have been detailed in many species
(including humans), and throughout the evolution-
ary scale from monkeys to rodents. The minutiae of
the genes and biochemical pathways responsible,
and their manifestations, may differ to some
degree, but there are common mechanisms and
adverse effects in all species examined to date. It
must be concluded that laboratory life for animals
used in experiments has serious and intractable
consequences for their welfare, and for the quality
and human relevance of the experimental data
obtained (which, in any case, are already of debat-
able applicability to humans, due to species differ-
ences).
Finally, I believe that this issue should be taken
much more seriously by legislators, regulators,
funders, practitioners and advocates of animal
experiments, and urge all involved to do so. The
information presented here could, and should, be a
valuable resource for project licence applicants,
ethics committees and the Home Office Inspect -
orate, for use in experimental design and harm–
benefit analyses, and to aid data interpretation.
Though it argues that relatively little can be done
to minimise many, if not all, stressors and stress,
it could inform attempts to do so — as well as con-
trolling variable factors and mitigating negative
consequences, etc. The information could also be
factored into existing guidance for the strategic
planning of animal experiments, since stress
impacts all areas of this planning, including study
objectives, species/strain selection, experimental
procedures, analgesia, training of staff, and so on.
Acknowledgements
This review was funded by Cruelty Free
International Trust, London, UK. It was based on
previous (not published) work conducted by the
same author in 2011, funded by the New England
Anti-Vivisection Society (NEAVS), Boston, USA, in
his role as its Science Director.
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Neurobiology 18, 1–22.
Comment 305
... Rats have been used extensively in various areas of scientific research, including fecundity and fertility studies, behavioural aspects and screening of compounds for teratogenic effects (André et al., 2018;Jeffrey et al., 2020). Hence, breeding of laboratory rats used in experiments has serious and intractable consequences on their welfare and the quality of the experimental data obtained from such experiment in terms of human relevance (Bailey, 2018). Breeding of rat model cut across varying conditions. ...
Article
Enrichment of laboratory rats' model in scientific research has severe consequences on their productivity and quality of the data output. Despite several reports on the welfare of Rattus norvegicus (albino rats), very few studies have considered comparing standard breeding of this species in enriched group (EG) and Un-enriched group (UG). This study demonstrated and compared standard breeding and maintenance of albino rat in the two groups. Six male and female rats (150 to 200 g) were hygienically housed, adequately fed, divided into 2 groups (EG and UG) and bred using 2 phases of trio system (one male and two females) for 12 weeks (84 days). Results revealed more litters in EG (more female than male) than in UG (more male than female). The rate of cannibalism was more pronounced in UG compared to EG. A significant (p<0.05) average weight increase of rats was observed in EG when compared with UG. However, no significant (p˃0.05) difference was observed in the quantity of feed and water consumption of the two groups. The temperature readings (clinical and mercury in glass thermometers) of both groups revealed no difference (p˃0.05). Thus, enriched group of laboratory rats appears to be more productive than un-enriched group during breeding period.
... Rats have been used extensively in various areas of scientific research, including fecundity and fertility studies, behavioural aspects and screening of compounds for teratogenic effects (André et al., 2018;Jeffrey et al., 2020). Hence, breeding of laboratory rats used in experiments has serious and intractable consequences on their welfare and the quality of the experimental data obtained from such experiment in terms of human relevance (Bailey, 2018). Breeding of rat model cut across varying conditions. ...
Article
Full-text available
Enrichment of laboratory rats' model in scientific research has severe consequences on their productivity and quality of the data output. Despite several reports on the welfare of Rattus norvegicus (albino rats), very few studies have considered comparing standard breeding of this species in enriched group (EG) and Un-enriched group (UG). This study demonstrated and compared standard breeding and maintenance of albino rat in the two groups. Six male and female rats (150 to 200 g) were hygienically housed, adequately fed, divided into 2 groups (EG and UG) and bred using 2 phases of trio system (one male and two females) for 12 weeks (84 days). Results revealed more litters in EG (more female than male) than in UG (more male than female). The rate of cannibalism was more pronounced in UG compared to EG. A significant (p<0.05) average weight increase of rats was observed in EG when compared with UG. However, no significant (p˃0.05) difference was observed in the quantity of feed and water consumption of the two groups. The temperature readings (clinical and mercury in glass thermometers) of both groups revealed no difference (p˃0.05). Thus, enriched group of laboratory rats appears to be more productive than un-enriched group during breeding period.
... Interpretations of experimental outcomes may differ depending on how housing conditions are framed. For example, we could reframe the shoebox cage as a treatment in which we are measuring the effects of persistent stressors related to an impoverished environment [124]. As described by Contreras and Rollin [33], (p. ...
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Environmental enrichment has been widely studied in rodents, but there is no consensus on what enrichment should look like or what it should achieve. Inconsistent use of the term “enrichment” creates challenges in drawing conclusions about the quality of an environment, which may slow housing improvements for laboratory animals. Many review articles have addressed environmental enrichment for laboratory rats and mice (Rattus norvegicus and Mus musculus). We conducted a metareview of 29 review articles to assess how enrichment has been defined and what are commonly described as its goals or requirements. Recommendations from each article were summarised to illustrate the conditions generally considered suitable for laboratory rodents. While there is no consensus on alternative terminology, many articles acknowledged that the blanket use of the terms “enriched” and “enrichment” should be avoided. Environmental enrichment was most often conceptualised as a method to increase natural behaviour and improve animal welfare. Authors also commonly outlined perceived risks and requirements of environmental enrichment. We discuss these perceptions, make suggestions for future research, and advocate for the adoption of more specific and value-neutral terminology.
... Along with the object recognition test 94 , it is among the most animal-friendly options for cognitive function assessment. Stress minimization during behavioural procedures is not only fundamental from an ethical point of view for animal welfare, but also for data reliability, as stress is a confounding variable that can put at risk the reproducibility of results 95,96 . Furthermore, the spontaneous alternation T-maze task does not require any sort of pre-training, making it possible to test animals through a single-day procedure, which can be useful in many experimental designs. ...
Article
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Spatial working memory can be assessed in mice through the spontaneous alternation T-maze test. The T-maze is a T-shaped apparatus featuring a stem (start arm) and two lateral goal arms (left and right arms). The procedure is based on the natural tendency of rodents to prefer exploring a novel arm over a familiar one, which induces them to alternate the choice of the goal arm across repeated trials. During the task, in order to successfully alternate choices across trials, an animal has to remember which arm had been visited in the previous trial, which makes spontaneous alternation T-maze an optimal test for spatial working memory. As this test relies on a spontaneous behaviour and does not require rewards, punishments or pre-training, it represents a particularly useful tool for cognitive evaluation, both time-saving and animal-friendly. We describe here in detail the apparatus and the protocol, providing representative results on wild-type healthy mice.
... Consequently, social stress can lead to negative consequences in both research and production (Ferdowsian and Beck, 2011). Because stress can impact both hormonal and behavioral measures, laboratory results can be altered (Bailey, 2018;Balcombe et al., 2004). Any unexplained data variation or unexpected physiological effects can impact the validity of experiments. ...
Article
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Many species use olfaction as a primary form of communication. Because of this, odor signals could be a useful tool to improve captive animal welfare by reducing aggression and promoting socio-positive behavior. However, to fully gauge the potential benefits of odor manipulations, the quality of existing literature must first be evaluated. Therefore, a systematic search and scoping review was conducted to summarize prevalent methods, treatment outcomes, and modulating factors in existing literature on the effect of mammalian, intraspecies odors on non-reproductive social behavior. Results from a systematic search of three databases were included if they were published in a peer reviewed journal, used a terrestrial mammalian species, and contained original data evaluating how an odor signal from the subject species directly affected non-reproductive social behavior. All articles were screened by two researchers, data were extracted by one, and reporting quality was assessed by both using the SYRCLE risk of bias tool. Sixty-three articles were included based on this criteria. Most subjects were sexually mature, male rodents. The most common odor treatment originated from urine and aggressive behavior was measured most often. Overall, urine and saliva treatments had a variable effect on aggression, while urine most often increased scent marking and social investigation behavior. Concerningly, most articles showed unclear or high risk of bias. Data from this review highlights a need for additional research on how odor signals from sources other than urine affect behavior and how socio-positive behaviors are affected in general. Further, it emphasizes the need for more transparent reporting as the current body of literature makes it difficult to determine each experiment’s quality and how much weight it should be given when interpreting outcomes pertaining to our overall understanding of olfactory communication.
... This makes it difficult to adjust to potential changes or to perform new measures unforeseen before recording. Placing markers on animals can be time-consuming and potentially disruptive to the animal (Bailey, 2018). Furthermore, errors in marker placement can arise due to movement of the skin over the joints during locomotion in animal models, especially in small rodents, but also larger models, such as the cat and dog (Goslow et al., 1973;Bauman and Chang, 2010). ...
Article
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Gait analysis in cats and other animals is generally performed with custom-made or commercially developed software to track reflective markers placed on bony landmarks. This often involves costly motion tracking systems. However, deep learning, and in particular DeepLabCut TM (DLC), allows motion tracking without requiring placing reflective markers or an expensive system. The purpose of this study was to validate the accuracy of DLC for gait analysis in the adult cat by comparing results obtained with DLC and a custom-made software (Expresso) that has been used in several cat studies. Four intact adult cats performed tied-belt (both belts at same speed) and split-belt (belts operating at different speeds) locomotion at different speeds and left-right speed differences on a split-belt treadmill. We calculated several kinematic variables, such as step/stride lengths and joint angles from the estimates made by the two software and assessed the agreement between the two measurements using intraclass correlation coefficient or Lin’s concordance correlation coefficient as well as Pearson’s correlation coefficients. The results showed that DLC is at least as precise as Expresso with good to excellent agreement for all variables. Indeed, all 12 variables showed an agreement above 0.75, considered good, while nine showed an agreement above 0.9, considered excellent. Therefore, deep learning, specifically DLC, is valid for measuring kinematic variables during locomotion in cats, without requiring reflective markers and using a relatively low-cost system.
... (Gylfe et al., 2000) found that Bbsl infection was reactivated in birds under simulated migratory stress, while it was not the case in the control group. If wild reservoirs are brought into the lab for experimental infection, results may be altered because the animals are under stress hence negatively affecting their immune function and possibly yielding higher infection rates (see (Bailey, 2018) for a review on this topic). ...
Article
Borrelia burgdorferi sensu lato (Bbsl) is a bacterial species complex that includes the etiological agents of the most frequently reported vector-borne disease in the Northern hemisphere, Lyme borreliosis. It currently comprises > 20 named and proposed genospecies that use vertebrate hosts and tick vectors for transmission in the Americas and Eurasia. Host (and vector) associations influence geographic distribution and speciation in Bbsl, which is of particular relevance to human health. To target gaps in knowledge for future efforts to understand broad patterns of the Bbsl-tick-host system and how they relate to human health, the present review aims to give a comprehensive summary of the literature on host association in Bbsl. Of 465 papers consulted (404 after exclusion criteria were applied), 96 sought to experimentally establish reservoir competence of 143 vertebrate host species for Bbsl. We recognize xenodiagnosis as the strongest method used, however it is infrequent (20% of studies) probably due to difficulties in maintaining tick vectors and/or wild host species in the lab. Some well-established associations were not experimentally confirmed according to our definition (ex: Borrelia garinii, Ixodes uriae and sea birds). We conclude that our current knowledge on host association in Bbsl is mostly derived from a subset of host, vector and bacterial species involved, providing an incomplete knowledge of the physiology, ecology and evolutionary history of these interactions. More studies are needed on all host, vector and bacterial species globally involved with a focus on non-rodent hosts and Asian Bbsl complex species, especially with experimental research that uses xenodiagnosis and genomics to analyze existing host associations in different ecosystems.
... Laboratory animals are often subjected to a large variety of environmental stressors, which compromises animal welfare and negatively affects animal physiology and psychology, thereby risking the quality of the research data [1][2][3][4]. Major sources of stress are experimental procedures, which may include painful or fear-evoking events. ...
Article
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In laboratory animal research, many procedures will be stressful for the animals, as they are forced to participate. Training animals to cooperate using clicker training (CT) or luring (LU) may reduce stress levels, and thereby increase animal welfare. In zoo animals, aquarium animals, and pets, CT is used to train animals to cooperate during medical procedures, whereas in experimental research, LU seem to be the preferred training method. This descriptive case study aims to present the behaviour of CT and LU pigs in a potentially fear-evoking behavioural test—the novel task participation test—in which the pigs walked a short runway on a novel walking surface. All eight pigs voluntarily participated, and only one LU pig showed body stretching combined with lack of tail wagging indicating reduced welfare. All CT pigs and one LU pig displayed tail wagging during the test, indicating a positive mental state. Hence, training pigs to cooperate during experimental procedures resulted in a smooth completion of the task with no signs of fear or anxiety in seven out of eight animals. We suggest that training laboratory pigs prior to experimental procedures or tests should be done to ensure low stress levels.
Article
Despite the international 3Rs principles that recommends replacing, reducing and refining the use of animals in medical experimentation, it remains difficult to obtain funding in Canada for medical research that respects these principles, particularly with regard to replacement. This observation led our team to review the literature on the arguments for and against animal experimentation in the fields of oncology and radiobiology. This article presents a synthesis of these arguments. Using the method created by McCullough and colleagues to conduct critical reviews of the ethics literature, we analysed 25 texts discussing the arguments for and against animal experimentation in oncology and radiobiology. Six broad categories of arguments for animal experimentation and eleven categories of arguments against it emerged from our analyses. Furthermore, the arguments against animal testing are more convincing from both an empirical and normative perspective. Also, most arguments obtained are transferable in other fields of medicine. In addition to the literature review, a critical reflection was conducted and other arguments were discussed. It seems that a conservative culture persists in medical research, despite the scientific evidence and ethical arguments to the contrary.
Article
Respiratory monitoring, using impedance with implanted telemetry in socially housed animals, was not possible until the recent development of digital signal transmission. The objective of this study was to evaluate digital telemetry monitoring of cardiopulmonary parameters (respiratory rate, tidal volume, minute volume, electrocardiography (DII), systemic arterial blood pressure, physical activity, and body temperature) in conscious, single-housed, non-rodent species commonly used in toxicology studies following administration of positive/negative controls (saline, dexmedetomidine, morphine, amphetamine, and doxapram), and also, the effects of various social housing arrangements in untreated female and/or male cynomolgus monkeys, Beagle dogs, and Göttingen minipigs (n = 4 per species). Aggressions were observed in socially housed male minipigs, however, which prevented pair-housed assessments in this species. All tested pharmacological agents significantly altered more than one organ system, highlighting important inter-organ dependencies when analyzing functional endpoints. Stress-related physiological changes were observed with single-housing or pair-housing with a new cage mate in cynomolgus monkeys and Beagle dogs, suggesting that stable social structures are preferable to limit variability, especially around dosing. Concomitant monitoring of cardiovascular and respiratory parameters from the same animals may help reduce the number of animals (3 Rs) needed to fulfill the S7A guidelines and allows for identification of organ system functional correlations. Globally, the data support the use of social housing in non-rodents for safety pharmacology multi-organ system (heart and lungs) monitoring investigations.
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Recurrent acute and/or chronic stress can affect all vertebrate species, and can have serious consequences. It is increasingly and widely appreciated that laboratory animals experience significant and repeated stress, which is unavoidable and is caused by many aspects of laboratory life, such as captivity, transport, noise, handling, restraint and other procedures, as well as the experimental procedures applied to them. Such stress is difficult to mitigate, and lack of significant desensitisation/habituation can result in considerable psychological and physiological welfare problems, which are mediated by the activation of various neuroendocrine networks that have numerous and pervasive effects. Psychological damage can be reflected in stereotypical behaviours, including repetitive pacing and circling, and even self-harm. Physical consequences include adverse effects on immune function, inflammatory responses, metabolism, and disease susceptibility and progression. Further, some of these effects are epigenetic, and are therefore potentially transgenerational: the biology of animals whose parents/grandparents were wild-caught and/or have experienced chronic stress in laboratories could be altered, as compared to free- living individuals. It is argued that these effects must have consequences for the reliability of experimental data and their extrapolation to humans, and this may not be recognised sufficiently among those who use animals in experiments.
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Assertions that the use of monkeys to investigate human diseases is valid scientifically are fre- quently based on a reported 90–93% genetic similarity between the species. Critical analyses of the rele- vance of monkey studies to human biology, however, indicate that this genetic similarity does not result in sufficient physiological similarity for monkeys to constitute good models for research, and that monkey data do not translate well to progress in clinical practice for humans. Salient examples include the failure of new drugs in clinical trials, the highly different infectivity and pathology of SIV/HIV, and poor extrapo- lation of research on Alzheimer’s disease, Parkinson’s disease and stroke. The major molecular differences underlying these inter-species phenotypic disparities have been revealed by comparative genomics and molecular biology — there are key differences in all aspects of gene expression and protein function, from chromosome and chromatin structure to post-translational modification. The collective effects of these dif- ferences are striking, extensive and widespread, and they show that the superficial similarity between human and monkey genetic sequences is of little benefit for biomedical research. The extrapolation of bio- medical data from monkeys to humans is therefore highly unreliable, and the use of monkeys must be con- sidered of questionable value, particularly given the breadth and potential of alternative methods of enquiry that are currently available to scientists.
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Unpredictable chronic mild stress (UCMS) and developmental social isolation are often utilized in laboratory animals to mimic unpredictable life stressors and early life adversity that may contribute to the development of major depressive disorder in humans. Zebrafish (Danio rerio) have been used to examine the effects of both developmental social isolation and UCMS. However, anxiety-like behavioral responses, social behavior, and neurochemical changes induced by stressors have not been well characterized. Furthermore, the possible interaction between UCMS and developmental isolation remains unexplored. In this study, we analyzed the effect of UCMS on developmentally isolated and socially reared zebrafish. The UCMS procedure entailed delivering unpredictably varying mild stressors twice a day for 15 consecutive days. To quantify social and anxiety-like behaviors, we measured the zebrafish's behavioral and neurochemical (dopaminergic and serotonergic) responses to an animated image of conspecifics in a novel tank. Our results suggest that UCMS increased anxiety-like behavioral responses, whereas developmental isolation altered motor responses during stimulus presentation. We also found that UCMS diminished weight gain and reduced whole-brain levels of dopamine and serotonin's metabolite 5-HIAA in developmentally isolated, but not socially reared zebrafish. Our findings reinforce the utility of combining developmental isolation with UCMS in zebrafish to model depressive-like behavior in humans.
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Assertions that the use of chimpanzees to investigate human diseases is valid scientifically are frequently based on a reported 98-99% genetic similarity between the species. Critical analyses of the relevance of chimpanzee studies to human biology, however, indicate that this genetic similarity does not result in sufficient physiological similarity for the chimpanzee to constitute a good model for research, and furthermore, that chimpanzee data do not translate well to progress in clinical practice for humans. Leading examples include the minimal citations of chimpanzee research that is relevant to human medicine, the highly different pathology of HIV/AIDS and hepatitis C virus infection in the two species, the lack of correlation in the efficacy of vaccines and treatments between chimpanzees and humans, and the fact that chimpanzees are not useful for research on human cancer. The major molecular differences underlying these inter-species phenotypic disparities have been revealed by comparative genomics and molecular biology - there are key differences in all aspects of gene expression and protein function, from chromosome and chromatin structure to post-translational modification. The collective effects of these differences are striking, extensive and widespread, and they show that the superficial similarity between human and chimpanzee genetic sequences is of little consequence for biomedical research. The extrapolation of biomedical data from the chimpanzee to the human is therefore highly unreliable, and the use of the chimpanzee must be considered of little value, particularly given the breadth and potential of alternative methods of enquiry that are currently available to science.
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Whether fishes are sentient beings remains an unresolved and controversial question. Among characteristics thought to reflect a low level of sentience in fishes is an inability to show stress-induced hyperthermia (SIH), a transient rise in body temperature shown in response to a variety of stressors. This is a real fever response, so is often referred to as 'emotional fever'. It has been suggested that the capacity for emotional fever evolved only in amniotes (mammals, birds and reptiles), in association with the evolution of consciousness in these groups. According to this view, lack of emotional fever in fishes reflects a lack of consciousness. We report here on a study in which six zebrafish groups with access to a temperature gradient were either left as undisturbed controls or subjected to a short period of confinement. The results were striking: compared to controls, stressed zebrafish spent significantly more time at higher temperatures, achieving an estimated rise in body temperature of about 2-4°C. Thus, zebrafish clearly have the capacity to show emotional fever. While the link between emotion and consciousness is still debated, this finding removes a key argument for lack of consciousness in fishes.