ArticlePDF AvailableLiterature Review

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

December 2018
Alternatives to Laboratory Animals 46(5):291-305

DOI:10.1177/026119291804600501

Authors:

Animal Free Research UK

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.

Content uploaded by Jarrod Bailey

Content may be subject to copyright.

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.

References

1. Bailey, J. (2017). Does the stress inherent to labora-

tory life and experimentation on animals adversely

affect research data. ATLA 45, 299–301.

2. Anon. (2007). Stress. Saunders Comprehensive

Veterinary Dictionary, 3rd edn. Available at: http://

medical-dictionary.thefreedictionary.com/Stress

(Accessed 19.09.18).

3. Ferdowsian, H.R. & Beck, N. (2011). Ethical and

scientific considerations regarding animal testing

and research. PLoS One 6, e24059.

4. Rey, S., Huntingford, F.A., Boltaña, S., Vargas, R.,

Knowles, T.G. & Mackenzie, S. (2015). Fish can

show emotional fever: Stress-induced hyperthermia

in zebrafish. Proceedings of the Biological Sciences

B282, 20152266.

5. Tran, S., Nowicki, M., Fulcher, N., Chatterjee, D. &

Gerlai, R. (2016). Interaction between handling

induced stress and anxiolytic effects of ethanol in

zebrafish: A behavioral and neurochemical analy-

sis. Behavioural Brain Research 298, 278–285.

6. Rambo, C.L., Mocelin, R., Marcon, M., Villanova,

D., Koakoski, G., de Abreu, M.S., Oliveira, T.A.,

Barcellos, L.J.G., Piato, A.L. & Bonan, C.D. (2017).

Gender differences in aggression and cortisol levels

in zebrafish subjected to unpredictable chronic stress.

Physiology & Behavior 171, 50–54.

7. Fulcher, N., Tran, S., Shams, S., Chatterjee, D. &

Gerlai, R. (2017). Neurochemical and behavioral

responses to unpredictable chronic mild stress fol-

lowing developmental isolation: The zebrafish as a

model for major depression. Zebrafish 14, 23–34.

8. Zimmermann, F.F., Altenhofen, S., Kist, L.W., Leite,

C.E., Bogo, M.R., Cognato, G.P. & Bonan, C.D.

(2016). Unpredictable chronic stress alters adenosine

metabolism in zebrafish brain. Molecular Neuro -

biology 53, 2518–2528.

9. Maestripieri, D. & Hoffman, C.L. (2011). Chronic

stress, allostatic load, and aging in nonhuman pri-

mates. Development & Psychopathology 23, 1187–

1195.

10. National Research Council (US) Committee on

Recognition and Alleviation of Distress in Labor -

atory Animals (2008). Recognition and Alleviation of

Pain and Distress in Laboratory Animals, 198pp.

Wash ing ton, DC, USA: National Academies Press.

Avail able at: https://www.ncbi.nlm.nih.gov/books/

NBK4032/ (Accessed 13.09.17).

11. Moberg, G.P. & Mench, J.A. (eds) (2000). The Biology

of Animal Stress: Basic Principles and Implications

for Animal Welfare, 377pp. Wallingford, UK: CABI

Publishing.

298 Comment

12. Tracey, K.J. (2009). Reflex control of immunity.

Nature Reviews Immunology 9, 418–428.

13. Sternberg, E.M. (2006). Neural regulation of innate

immunity: A coordinated nonspecific host response

to pathogens. Nature Reviews Immunology 6, 318–

328.

14. Glaser, R. & Kiecolt-Glaser, J.K. (2005). Stress-

induced immune dysfunction: Implications for

health. Nature Reviews Immunology 5, 243–251.

15. Altholtz, L.Y., Fowler, K.A., Badura, L.L. & Kovacs,

M.S. (2006). Comparison of the stress response in

rats to repeated isoflurane or CO2:O2anesthesia

used for restraint during serial blood collection via

the jugular vein. Journal of the American Assoc -

iation for Laboratory Animal Science 45(3), 17–22.

16. Balcombe, J.P., Barnard, N.D. & Sandusky, C.

(2004). Laboratory routines cause animal stress.

Con temporary Topics in Laboratory Animal Science

43, 42–51.

17. Meijer, M.K., Sommer, R., Spruijt, B.M., van

Zutphen, L.F. & Baumans, V. (2007). Influence of

environmental enrichment and handling on the

acute stress response in individually housed mice.

Laboratory Animals 41, 161–173.

18. Gurfein, B.T., Stamm, A.W., Bacchetti, P., Dall man,

M.F., Nadkarni, N.A., Milush, J.M., Touma, C.,

Palme, R., Di Borgo, C.P., Fromentin, G., Lown-

Hecht, R., Konsman, J.P., Acree, M., Premenko-

Lanier, M., Darcel, N., Hecht, F.M. & Nixon, D.F.

(2012). The calm mouse: An animal model of stress

reduction. Molecular Medicine 18, 606–617.

19. Obernier, J.A. & Baldwin, R.L. (2006). Establishing

an appropriate period of acclimatization following

transportation of laboratory animals. ILAR Journal

47, 364–369.

20. Balcombe, J.P. (2006). Laboratory environments and

rodents’ behavioural needs: A review. Laboratory

Animals 40, 217–235.

21. Meijer, M.K., Spruijt, B.M., van Zutphen, L.F.M. &

Baumans, V. (2006). Effect of restraint and injec-

tion methods on heart rate and body temperature in

mice. Laboratory Animals 40, 382–391.

22. Novak, M.A., Hamel, A.F., Kelly, B.J., Dettmer,

A.M. & Meyer, J.S. (2013). Stress, the HPA axis,

and nonhuman primate well-being: A review.

Applied Animal Behaviour Science 143, 135–149.

23. Gouveia, K. & Hurst, J.L. (2013). Reducing mouse

anxiety during handling: Effect of experience with

handling tunnels. PLoS One 8, e66401.

24. Conlee, K.M., Stephens, M.L., Rowan, A.N. & King,

L.A. (2005). Carbon dioxide for euthanasia: Con -

cerns regarding pain and distress, with special ref-

erence to mice and rats. Laboratory Animals 39,

137–161.

25. Wong, D., Makowska, I.J. & Weary, D.M. (2013).

Rat aversion to isoflurane versus carbon dioxide.

Biology Letters 9, 20121000.

26. Moody, C.M. & Weary, D.M. (2014). Mouse aversion

to isoflurane versus carbon dioxide gas. Applied

Animal Behaviour Science 158, 95–101.

27. Fyer, M.R., Uy, J., Martinez, J., Goetz, R., Klein,

D.F., Fyer, A., Liebowitz, M.R. & Gorman, J. (1987).

CO2challenge of patients with panic disorder.

American Journal of Psychiatry 144, 1080–1082.

28. Danneman, P.J., Stein, S. & Walshaw, S.O. (1997).

Humane and practical implications of using carbon

dioxide mixed with oxygen for anesthesia or

euthanasia of rats. Laboratory Animal Science 47,

376–385.

29. Anestis, S.F. (2009). Urinary cortisol responses to

unusual events in captive chimpanzees (Pan trog -

lodytes). Stress 12, 49–57.

30. Whitten, P.L., Stavisky, R., Aureli, F. & Russell, E.

(1998). Response of fecal cortisol to stress in captive

chimpanzees (Pan troglodytes). American Journal

of Primatology 44, 57–69.

31. Springer, D.A. & Baker, K.C. (2007). Effect of keta-

mine anesthesia on daily food intake in Macaca

mulatta and Cercopithecus aethiops. American

Journal of Primatology 69, 1080–1092.

32. Martin, C. (2014). Contributions and complexities

from the use of in vivo animal models to improve

understanding of human neuroimaging signals.

Frontiers in Neuroscience 8, 211.

33. Bowers, S.L., Bilbo, S.D., Dhabhar, F.S. & Nelson,

R.J. (2008). Stressor-specific alterations in corticos-

terone and immune responses in mice. Brain,

Behavior, & Immunity 22, 105–113.

34. Maninger, N., Mason, W., Ruys, J., Mendoza, S. &

Maninger, C. (2010). Acute and chronic stress

increase DHEAS concentrations in rhesus monkeys.

Psychoneuroendocrinology 35, 1055–1062.

35. Lee, J.I., Shin, J.S., Lee, J.E., Jung, W.Y., Lee, G.,

Kim, M.S., Park, C.G. & Kim, S.J. (2013). Changes

of N/L ratio and cortisol levels associated with

experimental training in untrained rhesus mac -

aques. Journal of Medical Primatology 42, 10–14.

36. Reinhardt, V., Liss, C. & Stevens, C. (1995).

Restraint methods of laboratory non-human pri-

mates: A critical review. Animal Welfare 4, 221–

238.

37. de Meijer, V.E., Le, H.D., Meisel, J.A. & Puder, M.

(2010). Repetitive orogastric gavage affects the phe-

notype of diet-induced obese mice. Physiology &

Behavior 100, 387–393.

38. Õkva, K., Tamoseviciute, E., Ciziute, A., Pokk, P.,

Ruksenas, O. & Nevalainen, T. (2006). Refinements

for intragastric gavage in rats. Scandinavian Jour -

nal of Laboratory Animal Sciences 33, 243–252.

39. Gonzales, C., Zaleska, M.M., Riddell, D.R., Atchison,

K.P., Robshaw, A., Zhou, H. & Sukoff Rizzo, S.J.

(2014). Alternative method of oral administration by

peanut butter pellet formulation results in target

engagement of BACE1 and attenuation of gavage-

induced stress responses in mice. Pharmacology,

Biochemistry & Behavior 126, 28–35.

40. Bonnichsen, M., Dragsted, N. & Hansen, A.K.

(2005). The welfare impact of gavaging laboratory

rats. Animal Welfare 14, 223–227.

41. Brown, A.P., Dinger, N. & Levine, B.S. (2000).

Stress produced by gavage administration in the

rat. Contemporary Topics in Laboratory Animal

Science 39, 17–21.

42. Walker, M.K., Boberg, J.R., Walsh, M.T., Wolf, V.,

Trujillo, A., Duke, M.S., Palme, R. & Felton, L.A.

(2012). A less stressful alternative to oral gavage for

pharmacological and toxicological studies in mice.

Toxicology & Applied Pharmacology 260, 65–69.

43. Morton, D.B., Abbot, D., Barclay, R., Close, B.S.,

Ewbank, R., Gask, D., Heath, M., Mattic, S., Poole,

T., Seamer, J., Southee, J., Thompson, A., Trussell,

B., West, C. & Jennings, M. (1993). Removal of

blood from laboratory mammals and birds. First

report of the BVA/FRAME/RSPCA/UFAW Joint

Working Group on Refinement. Laboratory Ani -

mals 27, 1–22.

44. Sarlis, N.J. (1991). Chronic blood sampling tech-

niques in stress experiments in the rat — A mini

Comment 299

review. Animal Technology 42, 51–59.

45. Capitanio, J.P., Mendoza, S.P. & McChesney, M.

(1996). Influences of blood sampling procedures on

basal hypothalamic-pituitary-adrenal hormone lev-

els and leukocyte values in rhesus macaques

(Macaca mulatta). Journal of Medical Primatology

25, 26–33.

46. Teilmann, A.C., Kalliokoski, O., Sorensen, D.B.,

Hau, J. & Abelson, K.S. (2014). Manual versus

automated blood sampling: Impact of repeated

blood sampling on stress parameters and behavior

in male NMRI mice. Laboratory Animals 48, 278–

291.

47. Teilmann, A.C., Nygaard Madsen, A., Holst, B., Hau,

J., Rozell, B. & Abelson, K.S. (2014). Physiological

and pathological impact of blood sampling by retro-

bulbar sinus puncture and facial vein phlebotomy in

laboratory mice. PLoS One 9, e113225.

48. Burnett, J.E. (2011). Dried blood spot sampling:

Practical considerations and recommendation for

use with preclinical studies. Bioanalysis 3, 1099–

1107.

49. Wickremsinhe, E.R. & Perkins, E.J. (2015). Using

dried blood spot sampling to improve data quality

and reduce animal use in mouse pharmacokinetic

studies. Journal of the American Association for Lab -

oratory Animal Science 54(2), 139–144.

50. Langkilde, T. & Shine, R. (2006). How much stress

do researchers inflict on their study animals? A

case study using a scincid lizard, Eulamprus heat-

wolei. Journal of Experimental Biology 209, 1035–

1043.

51. Jennings, M., Prescott, M.J., Buchanan-Smith, H.M.,

Gamble, M.R., Gore, M., Hawkins, P., Hubrecht, R.,

Hudson, S., Jennings, M., Keeley, J.R., Morris, K.,

Morton, D.B., Owen, S., Pearce, P.C., Prescott, M.J.,

Robb, D., Rumble, R.J., Wolfensohn, S. & Buist, D.

(2009). Refinements in husbandry, care and common

procedures for non-human primates: Ninth report of

the BVAAWF/FRAME/RSPCA/UFAW Joint Working

Group on Refinement. Laboratory Animals 43, Suppl.

1, 1–47.

52. Willems, R.A. (2009). Regulatory issues regarding

the use of food and water restriction in laboratory

animals. Lab Animal 38, 325–328.

53. Calisi, R.M. & Bentley, G.E. (2009). Lab and field

experiments: Are they the same animal? Hormones

& Behavior 56, 1–10.

54. Meijer, T. & Schwabl, H. (1989). Hormonal patterns

in breeding and nonbreeding kestrels, Falco tinnun-

culus: Field and laboratory studies. General &

Comparative Endocrinology 74, 148–160.

55. Marra, P.P., Kevin, T.L. & Bruce, L.T. (1995).

Plasma corticosterone levels in two species of

Zonotrichia sparrows under captive and free-living

conditions. Wilson Bulletin 107, 305–296.

56. Barrett, A.M. & Stockham, M.A. (1963). The effect

of housing conditions and simple experimental pro-

cedures upon the corticosterone level in the plasma

of rats. Journal of Endocrinology 26, 97–105.

57. Cavigelli, S.A., Guhad, F.A., Ceballos, R.M., Whetzel,

C.A., Nevalainen, T., Lang, C.M. & Klein, L.C. (2006).

Fecal corticoid metabolites in aged male and female

rats after husbandry-related disturbances in the

colony room. Journal of the American Association for

Laboratory Animal Science 45(6), 17–21.

58. Castelhano-Carlos, M.J. & Baumans, V. (2009). The

impact of light, noise, cage cleaning and in-house

transport on welfare and stress of laboratory rats.

Laboratory Animals 43, 311–327.

59. Baldwin, A.L. (2007). Effects of noise on rodent

physiology. International Journal of Comparative

Psychology 20, 134–144.

60. Pines, M.K., Kaplan, G. & Rogers, L.J. (2004).

Stressors of common marmosets (Callithrix jacchus)

in the captive environment: Effects on behaviour and

cortisol levels. Folia Primatologica 75, 317–318.

61. Schreuder, M.F., Fodor, M., van Wijk, J.A. &

Delemarre-van de Waal, H.A. (2007). Weekend ver-

sus working day: Differences in telemetric blood

pressure in male Wistar rats. Laboratory Animals

41, 86–91.

62. Patterson-Kane, E.G. & Farnworth, M.J. (2006).

Noise exposure, music, and animals in the labora-

tory: A commentary based on Laboratory Animal

Refinement and Enrichment Forum (LAREF) dis-

cussions. Journal of Applied Animal Welfare Science

9, 327–332.

63. Kirillov, O.I., Khasina, E.I. & Durkina, V.B. (2003).

[Effect of stress on postnatal growth in weight of rat

body and adrenal gland]. Ontogenez 34, 371–376.

64. Bernátová, I., Púzserová, A., Navarová, J., Csizmad -

iová, Z. & Zeman, M. (2007). Crowding-induced alter-

ations in vascular system of Wistar-Kyoto rats: Role

of nitric oxide. Physiological Research 56, 667–669.

65. Armario, A., Castellanos, J.M. & Balasch, J. (1984).

Effect of crowding on emotional reactivity in male

rats. Neuroendocrinology 39, 330–333.

66. Cyr, N.E. & Romero, L.M. (2008). Fecal glucocorti-

coid metabolites of experimentally stressed captive

and free-living starlings: Implications for conserva-

tion research. General & Comparative Endocrin -

ology 158, 20–28.

67. Wingfield, J.C. & Kitaysky, A.S. (2002). Endocrine

responses to unpredictable environmental events:

Stress or anti-stress hormones. Integrative & Com -

parative Biology 42, 600–609.

68. Romero, L.M. & Wingfield, J.C. (1999). Alterations

in hypothalamic-pituitary-adrenal function associ-

ated with captivity in Gambel’s white-crowned

sparrows (Zonotrichia leucophrys gambelii). Com -

parative Biochemistry & Physiology. Part B, Bio -

chemistry & Molecular Biology 122, 13–20.

69. Ferland, C.L. & Schrader, L.A. (2011). Cage mate

separation in pair-housed male rats evokes an

acute stress corticosterone response. Neuroscience

Letters 489, 154–158.

70. Clay, A.W., Bloomsmith, M.A., Marr, M.J. & Maple,

T.L. (2009). Habituation and desensitization as

methods for reducing fearful behavior in singly

housed rhesus macaques. American Journal of

Primatology 71, 30–39.

71. Lilly, A.A., Mehlman, P.T. & Higley, J.D. (1999).

Trait-like immunological and hematological meas-

ures in female rhesus across varied environmental

conditions. American Journal of Primatology 48,

197–223.

72. Allen, K.P., Dwinell, M.R., Zappa, A., Temple, A. &

Thulin, J. (2013). Comparison of 2 rat breeding

schemes using conventional caging. Journal of the

American Association of Laboratory Animal Science

52(2), 142–145.

73. Burn, C.C., Peters, A., Day, M.J. & Mason, G.J.

(2006). Long-term effects of cage-cleaning frequency

and bedding type on laboratory rat health, welfare,

and handleability: A cross-laboratory study.

Laboratory Animals 40, 353–370.

74. Meller, A., Kasanen, I., Ruksenas, O., Apanaviciene,

300 Comment

N., Baturaite, Z., Voipio, H.M. & Nevalainen, T.

(2011). Refining cage change routines: Comparison of

cardiovascular responses to three different ways of

cage change in rats. Laboratory Animals 45, 167–

173.

75. Line, S.W., Markowitz, H., Morgan, K.N. & Strong,

S. (1991). Effects of cage size and environmental

enrichment on behavioral and physiological res -

ponses of rhesus macaques to the stress of daily

events. In Through the Looking Glass (ed. M.A.

Novak & A.J. Petto), pp. 160–179. Washington, DC,

USA: American Psychological Association.

76. Clarke, A.S., Mason, W.A. & Mendoza, S.P. (1994).

Heart rate patterns under stress in three species of

macaques. American Journal of Primatology 33,

133–148.

77. Wurbel, H. (2001). Ideal homes? Housing effects on

rodent brain and behaviour. Trends in Neuro -

sciences 24, 207–211.

78. Wolfer, D.P., Litvin, O., Morf, S., Nitsch, R.M.,

Lipp, H.P. & Würbel, H. (2004). Laboratory animal

welfare: Cage enrichment and mouse behaviour.

Nature, London 432, 821–822.

79. Burn, C.C., Deacon, R.M. & Mason, G.J. (2008).

Marked for life? Effects of early cage-cleaning

frequency, delivery batch, and identification tail-

marking on rat anxiety profiles. Developmental

Psych o biology 50, 266–277.

80. Schapiro, S.J., Lambeth, S.P., Jacobsen, K.R.,

Williams, L.E., Nehete, B.N. & Nehete, P.N. (2012).

Physiological and welfare consequences of trans-

port, relocation, and acclimatization of chimpanzees

(Pan troglodytes). Applied Animal Behaviour Science

137, 183–193.

81. Wolfensohn, S.E. (1997). Brief review of scientific

studies of the welfare implications of transporting

primates. Laboratory Animals 31, 303–305.

82. Honess, P.E., Johnson, P.J. & Wolfensohn, S.E.

(2004). A study of behavioural responses of non-

human primates to air transport and re-housing.

Laboratory Animals 38, 119–132.

83. Ferreira, C.S., Vasconcellos, R.S., Pedreira, R.S.,

Silva, F.L., Sá, F.C., Kroll, F.S., Maria, A.P., Vent -

urini, K.S. & Carciofi, A.C. (2014). Alterations to

oxidative stress markers in dogs after a short-term

stress during transport. Journal of Nutritional

Science 3, e27.

84. Line, S.W., Morgan, K.N., Markowitz, H. & Strong,

S. (1989). Heart rate and activity of rhesus monkeys

in response to routine events. Laboratory Primate

Newsletter 28, 9–12.

85 Sharp, J., Zammit, T., Azar, T. & Lawson, D. (2003).

Are “by-stander” female Sprague-Dawley rats

affected by experimental procedures. Contemporary

Topics in Laboratory Animal Science 42, 19–27.

86. Gilmore, A.J., Billing, R.L. & Einstein, R. (2008).

The effects on heart rate and temperature of mice

and vas deferens responses to noradrenaline when

their cage mates are subjected to daily restraint

stress. Laboratory Animals 42, 140–148.

87. Bowers, C.L., Crockett, C.M. & Bowden, D.M.

(1998). Differences in stress reactivity of laboratory

macaques measured by heart period and respira-

tory sinus arrhythmia. American Journal of Prima -

tology 45, 245–261.

88. Baumans, V. (2005). Environmental enrichment for

laboratory rodents and rabbits: Requirements of

rodents, rabbits, and research. ILAR Journal 46,

162–170.

89. Hutchinson, E., Avery, A. & Vandewoude, S.

(2005). Environmental enrichment for laboratory

rodents. ILAR Journal 46, 148–161.

90. Reinhardt, V. & Reinhardt, A. (2000). Blood collec-

tion procedure of laboratory primates: A neglected

variable in biomedical research. Journal of Applied

Animal Welfare Science 3, 321–333.

91. Olsson, A. & Dahlborn, K. (2002). Improving hous-

ing conditions for laboratory mice: A review of

‘environmental enrichment’. Laboratory Animals

36, 243–270.

92. Zimmermann, A., Stauffacher, M., Langhans, W. &

Würbel, H. (2001). Enrichment-dependent differ-

ences in novelty exploration in rats can be explained

by habituation. Behavioural Brain Research 121,

11–20.

93. Würbel, H., Chapman, R. & Rutland, C. (1998).

Effect of feed and environmental enrichment on

development of stereotypic wire-gnawing in labora-

tory mice. Applied Animal Behaviour Science 60,

69–81.

94. Powell, S.B., Newman, H.A., McDonald, T.A.,

Bugenhagen, P. & Lewis, M.H. (2000). Develop -

ment of spontaneous stereotyped behavior in deer

mice: Effects of early and late exposure to a more

complex environment. Developmental Psychobiol -

ogy 37, 100–108.

95. Callard, M.D., Bursten, S.N. & Price, E.O. (2000).

Repetitive backflipping behaviour in captive roof

rats (Rattus rattus) and the effects of cage enrich-

ment. Animal Welfare 9, 139–152.

96. Balcombe, J. (2010). Laboratory rodent welfare:

Thinking outside the cage. Journal of Applied Ani -

mal Welfare Science 13, 77–88.

97. Leach, M.C., Ambrose, N., Bowell, V.J. & Morton,

D.B. (2000). The development of a novel form of

mouse cage enrichment. Journal of Applied

Animal Wel fare Science 3, 81–91.

98. Würbel, H., Freire, R. & Nicol, C.J. (1998). Pre -

vention of stereotypic wire-gnawing in laboratory

mice: Effects on behaviour and implications for

stereotypy as a coping response. Behavioural Pro -

cesses 42, 61–72.

99. McEwen, B.S. & Seeman, T. (1999). Protective and

damaging effects of mediators of stress. Elabor -

ating and testing the concepts of allostasis and allo-

static load. Annals of the New York Academy of

Sciences 896, 30–47.

100. Kurokawa, K., Kuwano, Y., Tominaga, K., Kawai,

T., Katsuura, S., Yamagishi, N., Satake, Y., Kajita,

K., Tanahashi, T. & Rokutan, K. (2010). Brief nat-

uralistic stress induces an alternative splice vari-

ant of SMG-1 lacking exon 63 in peripheral

leukocytes. Neuroscience Letters 484, 128–132.

101. Champagne, F.A. (2010). Epigenetic influence of

social experiences across the lifespan. Develop mental

Psychobiology 52, 299–311.

102. Murgatroyd, C. & Spengler, D. (2011). Epigenetic

programming of the HPA axis: Early life decides.

Stress 14, 581–589.

103. Wright, R. (2011). Epidemiology of stress and

asthma: From constricting communities and fragile

families to epigenetics. Immunology & Allergy

Clinics of North America 31, 19–39.

104. Hoffmann, A., Zimmermann, C.A. & Spengler, D.

(2015). Molecular epigenetic switches in neurode-

velopment in health and disease. Frontiers in

Behavioral Neuroscience 9, 120.

105. Grace, C.E., Kim, S.J. & Rogers, J.M. (2011). Mat -

Comment 301

ernal influences on epigenetic programming of the

developing hypothalamic–pituitary–adrenal axis.

Birth Defects Research. Part A, Clinical & Mol -

ecular Teratology 91, 797–805.

106. Gudsnuk, K. & Champagne, F.A. (2012). Epi -

genetic influence of stress and the social environ-

ment. ILAR Journal 53, 279–288.

107. Champagne, F.A. (2012). Interplay between social

experiences and the genome: Epigenetic conse-

quences for behavior. Advances in Genetics 77, 33–

57.

108. Wright, R.J. & Enlow, M.B. (2008). Maternal stress

and perinatal programming in the expression of

atopy. Expert Review of Clinical Immunology 4, 535–

538.

109. McGowan, P.O., Sasaki, A., D’Alessio, A.C., Dymov,

S., Labonte, B., Szyf, M., Turecki, G. & Meaney, M.J.

(2009). Epigenetic regulation of the glucocorticoid

receptor in human brain associates with childhood

abuse. Nature Neuroscience 12, 342–348.

110. Uddin, M., Aiello, A.E., Wildman, D.E., Koenen,

K.C., Pawelec, G., de los Santos, R., Goldmann, E.

& Galea, S. (2010). Epigenetic and immune func-

tion profiles associated with posttraumatic stress

disorder. Proceedings of the National Academy of

Sciences of the USA 107, 9470–9475.

111. Biamonti, G. & Caceres, J.F. (2009). Cellular stress

and RNA splicing. Trends in Biochemical Sciences

34, 146–153.

112. Katsuura, S., Kuwano, Y., Yamagishi, N., Kuro -

kawa, K., Kajita, K., Akaike, Y., Nishida, K.,

Masuda, K., Tanahashi, T. & Rokutan, K. (2012).

MicroRNAs miR-144/144* and miR-16 in peripheral

blood are potential biomarkers for naturalistic stress

in healthy Japanese medical students. Neuroscience

Letters 516, 79–84.

113. Hapuarachchi, J.R., Chalmers, A.H., Winefield,

A.H. & Blake-Mortimer, J.S. (2003). Changes in

clinically relevant metabolites with psychological

stress parameters. Behavioral Medicine 29, 52–59.

114. Sivonova, M., Zitnanova, I., Hlincikova, L., Skod -

acek, I., Trebaticka, J. & Durackova, Z. (2004).

Oxidative stress in university students during

examinations. Stress 7, 183–188.

115. Knickelbein, K.Z., Flint, M., Jenkins, F. & Baum,

A. (2008). Psychological stress and oxidative

damage in lymphocytes of aerobically fit and

unfit individuals. Journal of Applied Biobehavioral

Research 13, 1–19.

116. Maes, M. (2001). Psychological stress and the

inflam matory response system. Clinical Science 101,

193–194.

117. Videan, E.N., Heward, C.B., Chowdhury, K., Plum -

mer, J., Su, Y. & Cutler, R.G. (2009). Com parison of

biomarkers of oxidative stress and cardiovascular

disease in humans and chimpanzees (Pan

troglodytes). Comparative Medicine 59, 287–296.

118. Wang, L., Muxin, G., Nishida, H., Shirakawa, C.,

Sato, S. & Konishi, T. (2007). Psychological stress-

induced oxidative stress as a model of sub-healthy

condition and the effect of TCM. Evidence-Based

Complementary & Alternative Medicine 4, 195–

202.

119. Lewis, K.N., Andziak, B., Yang, T. & Buffenstein,

R. (2013). The naked mole-rat response to oxidative

stress: Just deal with it. Antioxidants & Redox

Signaling 19, 1388–1399.

120. Webster, J. (2002). Animal Welfare: A Cool Eye To -

wards Eden, 284pp. Oxford, UK: Blackwell Science.

121. Chance, P. (2003). Learning and Behavior (5th

Edition). Belmont, CA, USA: Thomson Wadworth.

122. Barnum, C.J., Blandino, P.J. & Deak, T. (2007).

Adaptation in the corticosterone and hyperthermic

responses to stress following repeated stressor

exposure. Journal of Neuroendocrinology 19, 632–

642.

123. Gruen, M.E., Thomson, A.E., Clary, G.P., Hamilton,

A.K., Hudson, L.C., Meeker, R.B. & Sherman, B.L.

(2013). Conditioning laboratory cats to handling and

transport. Lab Animal 42, 385–389.

124. Longordo, F., Fan, J., Steimer, T., Kopp, C. &

Luthi, A. (2011). Do mice habituate to “gentle han-

dling?” A comparison of resting behavior, corticos-

terone levels and synaptic function in handled and

undisturbed C57BL/6J mice. Sleep 34, 679–681.

125. Kramer, K., van de Weerd, H., Mulder, A., Van

Heijningen, C., Baumans, V., Remie, R., Voss, H.P.

& van Zutphen, B.F. (2004). Effect of conditioning

on the increase of heart rate and body temperature

provoked by handling in the mouse. ATLA 32,

Suppl. 1A, 177–181.

126. Clement, J.G., Mills, P. & Brockway, B. (1989). Use

of telemetry to record body temperature and activ-

ity in mice. Journal of Pharmacological Methods

21, 129–140.

127. Swan, M.P. & Hickman, D.L. (2014). Evaluation of

the neutrophil–lymphocyte ratio as a measure of

distress in rats. Lab Animal 43, 276–282.

128. Anon. (2009). The Revision of the UK Directive on

the Protection of Animals Used for Scientific Pur -

poses, Volume II: Evidence, 252pp. London, UK:

The Stationery Office. Available at: http://www.

publications.parliament.uk/pa/ld200809/ldselect/

ldeucom/164/164ii.pdf (Accessed 14.09.18).

129. Anon. (2006). Action to be Taken Against Laos,

Vietnam and Cambodia Monkey Business. London,

UK: Cruelty Free International. Available at:

https://www.crueltyfreeinternational.org/breaking-

news/action-be-taken-against-laos-vietnam-and-

cambodia-monkey-business (Accessed 19.09.18).

130. Anon. (2008). Cambodia: The Trade in Primates for

Research. London, UK: Cruelty Free International.

131. Anon. (2010). Mauritius: The Trade in Primates for

Research. London, UK: Cruelty Free International.

132. Suomi, S.J. (1991). Primate separation models of

affective disorders. In Neurobiology of Learning,

Emotion and Affect (ed. J. Madden), pp. 195–214.

New York, NY, USA: Lippincott Williams and Wil -

kins.

133. Nakamichi, M., Cho, F. & Minami, T. (1990).

Mother–infant interactions of wild-born, individu-

ally-caged cynomolgus monkeys (Macaca fascicu-

laris) during the first 14 weeks of infant life.

Primates 31, 213–224.

134. Maestripieri, D., Martel, F.L., Nevison, C.M.,

Simpson, M.J. & Keverne, E.B. (1991). Anxiety in

rhesus monkey infants in relation to interactions

with their mother and other social companions.

Developmental Psychobiology 24, 571–581.

135. Camus, S.M., Rochais, C., Blois-Heulin, C., Li, Q.,

Hausberger, M. & Bezard, E. (2013). Birth origin

differentially affects depressive-like behaviours:

Are captive-born cynomolgus monkeys more vul-

nerable to depression than their wild-born counter-

parts? PLoS One 8, e67711.

136. Dettmer, A.M., Novak, M.A., Suomi, S.J. & Meyer,

J.S. (2012). Physiological and behavioral adapta-

tion to relocation stress in differentially reared rhe-

302 Comment

sus monkeys: Hair cortisol as a biomarker for anx-

iety-related responses. Psychoneuroendocrinol ogy

37, 191–199.

137. Butkevich, I., Mikhailenko, V., Semionov, P.,

Bagaeva, T., Otellin, V. & Aloisi, A.M. (2009). Eff -

ects of maternal corticosterone and stress on

behavioral and hormonal indices of formalin pain

in male and female offspring of different ages.

Hormones & Behavior 55, 149–157.

138. Heim, C., Newport, D.J., Wagner, D., Wilcox, M.M.,

Miller, A.H. & Nemeroff, C.B. (2002). The role of

early adverse experience and adulthood stress in the

prediction of neuroendocrine stress reactivity in

women: A multiple regression analysis. Depression &

Anxiety 15, 117–125.

139. Heim, C., Mletzko, T., Purselle, D., Musselman,

D.L. & Nemeroff, C.B. (2008). The dexamethasone/

corticotropin-releasing factor test in men with

major depression: Role of childhood trauma.

Biological Psychiatry 63, 398–405.

140. Christian, L.M. (2012). Psychoneuroimmunology

in pregnancy: Immune pathways linking stress

with maternal health, adverse birth outcomes, and

fetal development. Neuroscience & Biobehavioral

Reviews 36, 350–361.

141. Kinnally, E.L., Feinberg, C., Kim, D., Ferguson, K.,

Leibel, R., Coplan, J.D. & John, M.J. (2011). DNA

methylation as a risk factor in the effects of early

life stress. Brain, Behavior, & Immunity 25, 1548–

1553.

142. Chang, L. (2011). The role of stress on physiologic

responses and clinical symptoms in irritable bowel

syndrome. Gastroenterology 140, 761–765.

143. Bird, A.P. (1986). CpG-rich islands and the func-

tion of DNA methylation. Nature, London 321,

209–213.

144. Oberlander, T.F., Weinberg, J., Papsdorf, M.,

Grunau, R., Misri, S. & Devlin, A.M. (2008). Pre -

natal exposure to maternal depression, neonatal

methylation of human glucocorticoid receptor gene

(NR3C1) and infant cortisol stress responses.

Epigenetics 3, 97–106.

145. Fukuda, S. & Taga, T. (2005). Cell fate determina-

tion regulated by a transcriptional signal network

in the developing mouse brain. Anatomical Science

International 80, 12–18.

146. Zambrano, E., Martinez-Samayoa, P.M., Bautista,

C.J., Deas, M., Guillen, L., Rodriguez-Gonzalez,

G.L., Guzman, C., Larrea, F. & Nathanielsz, P.W.

(2005). Sex differences in transgenerational alter-

ations of growth and metabolism in progeny (F2) of

female offspring (F1) of rats fed a low protein diet

during pregnancy and lactation. Journal of Phys -

iology 566, 225–236.

147. Newbold, R.R., Padilla-Banks, E. & Jefferson, W.N.

(2006). Adverse effects of the model environmental

estrogen diethylstilbestrol are transmitted to sub-

sequent generations. Endocrinology 147, S11–S17.

148. Capdevila, S., Giral, M., Ruiz de la Torre, J.L.,

Russell, R.J. & Kramer, K. (2007). Acclimatization

of rats after ground transportation to a new animal

facility. Laboratory Animals 41, 255–261.

149. Anon. (2007). NC3Rs blood sampling microsite

launched. Laboratory Animals 41, 407.

150. Mason, J.W., Wool, M.S., Wherry, F.E., Pennington,

L.L., Brady, J.V. & Beer, B. (1968). Plasma growth

hormone response to avoidance sessions in the mon-

key. Psychosomatic Medicine 30, Suppl., 760–773.

151. Roberts, R.A., Soames, A.R., James, N.H., Gill, J.H.

& Wheeldon, E.B. (1995). Dosing-induced stress

causes hepatocyte apoptosis in rats primed by the

rodent nongenotoxic hepatocarcinogen cyproterone

acetate. Toxicology & Applied Pharmacology 135,

192–199.

152. Brenner, G.J., Cohen, N., Ader, R. & Moynihan,

J.A. (1990). Increased pulmonary metastases and

natural killer cell activity in mice following han-

dling. Life Sciences 47, 1813–1819.

153. Bailey, J. (2011). Lessons from chimpanzee-based

research on human disease: The implications of

genetic differences. ATLA 39, 527–540.

154. Bailey, J. (2014). Monkey-based research on human

disease: The implications of genetic differences.

ATLA 42, 287–317.

155. Armengol, G., Knuutila, S., Lozano, J.J., Madrigal,

I. & Caballin, M.R. (2010). Identification of human

specific gene duplications relative to other pri-

mates by array CGH and quantitative PCR.

Genomics 95, 203–209.

156. Perry, G.H., Yang, F., Marques-Bonet, T., Murphy,

C., Fitzgerald, T., Lee, A.S., Hyland, C., Stone, A.C.,

Hurles, M.E., Tyler-Smith, C., Eichler, E.E., Carter,

N.P., Lee, C. & Redon, R. (2008). Copy number vari-

ation and evolution in humans and chimpanzees.

Genome Research 18, 1698–1710.

157. Tang, X., Orchard, S.M. & Sanford, L.D. (2002).

Home cage activity and behavioral performance in

inbred and hybrid mice. Behavioural Brain

Research 136, 555–569.

158. Tang, X. & Sanford, L.D. (2002). Telemetric record-

ing of sleep and home cage activity in mice. Sleep

25, 691–699.

159. Sanford, L.D., Yang, L., Wellman, L.L., Dong, E. &

Tang, X. (2008). Mouse strain differences in the

effects of corticotropin releasing hormone (CRH) on

sleep and wakefulness. Brain Research 1190, 94–

104.

160. Mason, G.J. (1991). Stereotypies and suffering. Be -

hav ioural Processes 25, 103–115.

161. Prescott, M.J., Morton, D.B., Anderson, D., Buck -

well, A., Heath, M.S., Hubrecht, R., Jennings, M.,

Robb, M.D., Ruane, M.B. & Swallow, M.J. (2004).

Refining dog husbandry and care. Laboratory Ani -

mals 38, 1–94.

162. Hubrecht, R. (1995). The welfare of dogs in human

care. In The Domestic Dog: Its Evolution, Behav -

iour, and Interactions with People (ed. J. Serpell),

pp. 179–198. Cambridge, UK: Cambridge Univers -

ity Press.

163. Bourgeois, S.R., Vazquez, M. & Brasky, K. (2007).

Combination therapy reduces self-injurious behav-

ior in a chimpanzee (Pan troglodytes troglodytes): A

case report. Journal of Applied Animal Welfare

Science 10, 123–140.

164. Brüne, M., Brüne-Cohrs, U., McGrew, W.C. & Preu -

schoft, S. (2006). Psychopathology in great apes:

Concepts, treatment options and possible homologies

to human psychiatric disorders. Neuro science &

Biobehavioral Reviews 30, 1246–1259.

165. Lutz, C., Well, A. & Novak, M. (2003). Stereotypic

and self-injurious behavior in rhesus macaques: A

survey and retrospective analysis of environment

and early experience. American Journal of Prima -

tology 60, 1–15.

166. Novak, M.A., Meyer, J.S., Lutz, C. & S., T. (2007).

Stress and the performance of primate stereotyp-

ies. In Stereotypic Animal Behaviour: Fundamen -

tals and Applications for Welfare (ed. G. Mason &

Comment 303

J. Rushen), p. 248. Wallingford, UK: CAB Inter -

national.

167. Anon. (2009). Stress. [Nursing Times, 23.02.09].

Available at: https://www.nursingtimes.net/stress/

1995960.article (Accessed 17.09.18).

168. Wang, Y.C., Ho, U.C., Ko, M.C., Liao, C.C. & Lee,

L.J. (2012). Differential neuronal changes in medial

prefrontal cortex, basolateral amygdala and nucleus

accumbens after postweaning social isolation. Brain

Structure & Function 217, 337–351.

169. Tuchscherer, M., Kanitz, E., Puppe, B., Tuch sch -

erer, A. & Viergutz, T. (2009). Changes in endo -

crine and immune responses of neonatal pigs

exposed to a psychosocial stressor. Research in

Veterinary Science 87, 380–388.

170. Kaushal, N., Nair, D., Gozal, D. & Ramesh, V.

(2012). Socially isolated mice exhibit a blunted

homeostatic sleep response to acute sleep depriva-

tion compared to socially paired mice. Brain

Research 1454, 65–79.

171. Sorenson, M., Janusek, L. & Mathews, H. (2011).

Psychological stress and cytokine production in

multiple sclerosis: Correlation with disease symp-

tomatology. Biological Research for Nursing 15,

226–233.

172. Dhabhar, F.S. (2009). Enhancing versus suppres-

sive effects of stress on immune function: Implica -

tions for immunoprotection and immunopathology.

Neuroimmunomodulation 16, 300–317.

173. He, Y.D., Karbowski, C.M., Werner, J., Everds, N.,

Di Palma, C., Chen, Y., Higgins-Garn, M., Tran, S.,

Afshari, C.A. & Hamadeh, H.K. (2014). Common

handling procedures conducted in preclinical safety

studies result in minimal hepatic gene expression

changes in Sprague-Dawley rats. PLoS One 9,

e88750.

174. Li, H., Chen, L., Zhang, Y., Lesage, G., Zhang, Y.,

Wu, Y., Hanley, G., Sun, S. & Yin, D. (2011). Chronic

stress promotes lymphocyte reduction through TLR2

mediated PI3K signaling in a beta-arrestin 2

dependent manner. Journal of Neuro immunology

233, 73–79.

175. Xiang, L., Rehm, K., Marshall, G. & Xiang, D.B.

(2011). Effects of acute stress-induced immuno -

modulation on Th1/Th2 cytokine and catechol -

amine receptor expression in human peripheral

blood cells. Neuropsychobiology 65, 12–19.

176. Huang, C.J., Stewart, J.K., Franco, R.L., Evans,

R.K., Lee, Z.P., Cruz, T.D., Webb, H.E. & Acevedo,

E.O. (2011). LPS-stimulated tumor necrosis factor-

alpha and interleukin-6 mRNA and cytokine

responses following acute psychological stress.

Psychoneuroendocrinology 36, 1553–1561.

177. Smith, A., Conneely, K., Kilaru, V., Weiss, T.,

Bradley, B., Cubells, J., Ressler, K., Kilaru, M. &

Bradley, T. (2011). Differential immune system DNA

methylation and cytokine regulation in post-trau-

matic stress disorder. American Journal of Medical

Genetics. Part B, Neuropsychiatric Gen etics 156,

700–708.

178. Dhabhar, F.S. & McEwen, B.S. (1997). Acute stress

enhances while chronic stress suppresses cell-

mediated immunity in vivo: A potential role for

leukocyte trafficking. Brain, Behavior, & Imm unity

11, 286–306.

179. Cohen, S., Frank, E., Doyle, W.J., Skoner, D.P.,

Rabin, B.S. & Gwaltney, J.M. (1998). Types of

stressors that increase susceptibility to the com-

mon cold in healthy adults. Health Psychology 17,

214–223.

180. Blecha, F., Kelley, K.W. & Satterlee, D.G. (1982).

Adrenal involvement in the expression of delayed-

type hypersensitivity to SRBC and contact sensitiv-

ity to DNFB in stressed mice. Proceedings of the

Society for Experimental Biology & Medicine 169,

247–252.

181. Gouin, J.P., Hantsoo, L. & Kiecolt-Glaser, J.K.

(2008). Immune dysregulation and chronic stress

among older adults: A review. Neuroimmuno

modulation 15, 251–259.

182. Pace, T.W. & Heim, C.M. (2011). A short review on

the psychoneuroimmunology of posttraumatic

stress disorder: From risk factors to medical comor-

bidities. Brain, Behavior, & Immunity 25, 6–13.

183. Glaser, R., Robles, T.F., Sheridan, J., Malarkey,

W.B. & Kiecolt-Glaser, J.K. (2003). Mild depressive

symptoms are associated with amplified and pro-

longed inflammatory responses after influenza

virus vaccination in older adults. Archive of

General Psychiatry 60, 1009–1014.

184. Papathanassoglou, E.D., Giannakopoulou, M.,

Mpouzika, M., Bozas, E. & Karabinis, A. (2010).

Potential effects of stress in critical illness through

the role of stress neuropeptides. Nursing in Crit ical

Care 15, 204–216.

185. Nater, U.M., Whistler, T., Lonergan, W., Mletzko,

T., Vernon, S.D. & Heim, C. (2009). Impact of acute

psychosocial stress on peripheral blood gene

expression pathways in healthy men. Biological

Psychology 82, 125–132.

186. Kamezaki, Y., Katsuura, S., Kuwano, Y., Tana -

hashi, T. & Rokutan, K. (2012). Circulating cyto -

kine signatures in healthy medical students

exposed to academic examination stress. Psycho -

physiology 49, 991–997.

187. Hasan, K.M., Rahman, M.S., Arif, K.M. & Sobhani,

M.E. (2011). Psychological stress and aging: Role of

glucocorticoids (GCs). Age 34, 1421–1433.

188. Kitajima, T., Ariizumi, K., Bergstresser, P.R. &

Takashima, A. (1996). A novel mechanism of gluco-

corticoid-induced immune suppression: The

inhibiton of T cell-mediated terminal maturation of

a murine dendritic cell line. Journal of Clinical

Investigation 98, 142–147.

189. Porter, N.M. & Landfield, P.W. (1998). Stress hor-

mones and brain aging: Adding injury to insult?

Nature Neuroscience 1, 3–4.

190. Sun, X., Zhong, X., Liu, Z., Cai, H., Fan, Q., Wang,

Q., Liu, Q., Song, Y., He, S., Zhang, X. & Lu, Z.

(2010). Proteomic analysis of chronic restraint

stress-induced Gan (肝)-stagnancy syndrome in rats.

Chinese Journal of Integrative Medicine 16, 510–517.

191. Gidron, Y. & De, Z. (2010). Influence of stress and

health-behaviour on miRNA expression. Molecular

Medicine Reports 3, 455–457.

192. Binker, M.G., Binker-Cosen, A.A., Richards, D.,

Gaisano, H.Y., de Cosen, R.H. & Cosen-Binker, L.I.

(2010). Chronic stress sensitizes rats to pancreati-

tis induced by cerulein: Role of TNF-a. World

Journal of Gastroenterology 16, 5565–5581.

193. Schuller, H.M., Al-Wadei, H.A., Ullah, M.F. &

Plummer, H.K. (2012). Regulation of pancreatic can-

cer by neuropsychological stress responses: A novel

target for intervention. Carcinogenesis 33, 191–196.

194. Hong, S., Owyang, C., Hong, Z. & Owyang, W.

(2011). Corticosterone mediates reciprocal changes

in CB1 and TRPV1 receptors in primary sensory

neurons in the chronically stressed rat. Gastro -

304 Comment

enterology 140, 627–637.

195. Nadol’nik, L.I. (2010). [Stress and the thyroid

gland]. Biomeditsinskaia Khimiia 56, 443–456.

196. Rampton, D. (2011). The influence of stress on the

development and severity of immune-mediated dis-

eases. Journal of Rheumatology Supplement 88,

43–47.

197. Menezes, A., Lavie, C., Milani, R., O’Keefe, J. &

O’Keefe, L. (2011). Psychological risk factors and

cardiovascular disease: Is it all in your head?

Postgraduate Medicine 123, 165–176.

198. Gavrilovic, L., Spasojevic, N. & Dronjak, S. (2010).

Subsequent stress increases gene expression of cat-

echolamine synthetic enzymes in cardiac ventricles

of chronic-stressed rats. Endocrine 37, 425–429.

199. Eitel, I., von Knobelsdorff-Brenkenhoff, F., Bern -

hardt, P., Carbone, I., Muellerleile, K., Aldrovandi,

A., Francone, M., Desch, S., Gutberlet, M., Strohm,

O., Schuler, G., Schulz-Menger, J., Thiele, H. &

Friedrich, M.G. (2011). Clinical characteristics and

cardiovascular magnetic resonance findings in

stress (takotsubo) cardiomyopathy. JAMA 306,

277–286.

200. Allen, D., McCall, G., Loh, A., Madden, M. &

Mehan, R. (2010). Acute daily psychological stress

causes increased atrophic gene expression and

myostatin-dependent muscle atrophy. American

Journal of Physiology. Regulatory, Integrative &

Comparative Physiology 299, R889–R898.

201. Nowotny, B., Cavka, M., Herder, C., Loffler, H.,

Poschen, U., Joksimovic, L., Kempf, K., Krug, A.W.,

Koenig, W., Martin, S. & Kruse, J. (2010). Effects of

acute psychological stress on glucose metabolism

and subclinical inflammation in patients with post-

traumatic stress disorder. Hormone & Metabolic

Research 42, 746–753.

202. Marotta, F., Naito, Y., Padrini, F., Xuewei, X., Jain,

S., Soresi, V., Zhou, L., Catanzaro, R., Zhong, K.,

Polimeni, A. & Chui, D.H. (2011). Redox balance sig-

nalling in occupational stress: Modification by

nutraceutical intervention. Journal of Biological

Regulators & Homeostatic Agents 25, 221–229.

203. Jankord, R., Zhang, R., Flak, J.N., Solomon, M.B.,

Albertz, J. & Herman, J.P. (2010). Stress activation

of IL-6 neurons in the hypothalamus. Ameri can

Journal of Physiology. Regulatory, Integrative &

Comparative Physiology 299, R343–R351.

204. Videan, E.N., Fritz, J. & Murphy, J. (2008). Effects

of aging on hematology and serum clinical chem-

istry in chimpanzees (Pan troglodytes). American

Journal of Primatology 70, 327–338.

205. Lammey, M., Baskin, G., Gigliotti, A., Lee, D.R.,

Ely, J. & Sleeper, M. (2008). Interstitial myocardial

fibrosis in a captive chimpanzee (Pan troglodytes)

population. Comparative Medicine 58, 389–394.

206. Rasmussen, S., Miller, M.M., Filipski, S.B. & Tol -

wani, R.J. (2011). Cage change influences serum

corticosterone and anxiety-like behaviors in the

mouse. Journal of the American Association for

Laboratory Animal Science 50(4), 479–483.

207. Wei, L., Simen, A., Mane, S. & Kaffman, A. (2012).

Early life stress inhibits expression of a novel

innate immune pathway in the developing hip-

pocampus. Neuropsychopharmacology 37, 567–580.

208. Jackowski, A., Perera, T.D., Abdallah, C.G., Garrido,

G., Tang, C.Y., Martinez, J., Mathew, S.J., Gorman,

J.M., Rosenblum, L.A., Smith, E.L., Dwork, A.J.,

Shungu, D.C., Kaffman, A., Gelernter, J., Coplan,

J.D. & Kaufman, J. (2011). Early-life stress, corpus

callosum development, hippocampal volumetrics,

and anxious behavior in male nonhuman primates.

Psychiatry Research 192, 37–44.

209. Mehta, M. & Schmauss, C. (2011). Strain-specific

cognitive deficits in adult mice exposed to early life

stress. Behavioral Neuroscience 125, 29–36.

210. Labonte, B. & Turecki, G. (2010). The epigenetics of

suicide: Explaining the biological effects of early

life environmental adversity. Archives of Suicide

Research 14, 291–310.

211. Schroeder, M., Krebs, M.O., Bleich, S. & Frieling,

H. (2010). Epigenetics and depression: Current

challenges and new therapeutic options. Current

Opinion in Psychiatry 23, 588–592.

212. Miller, G., Chen, E. & Chen, P. (2011). Psych -

ological stress in childhood and susceptibility to the

chronic diseases of aging: Moving toward a model

of behavioral and biological mechanisms. Psychol -

ogical Bulletin 137, 959–997.

213. Raber, J. (1998). Detrimental effects of chronic

hypothalamic–pituitary–adrenal axis activation.

From obesity to memory deficits. Molecular

Neurobiology 18, 1–22.

Comment 305

Recent Advances in the Behavioral Ecology of European Plethodontid Salamanders

Article

Full-text available

Nov 2023

There is a recent growing interest in the study of evolutionary and behavioral ecology of amphibians. Among salamanders, Plethodontidae is the most speciose family, with more than 500 species, while in Europe, there are only 8 species, all belonging to the genus Speleomantes. European plethodontids recently received increasing attention with regard to the study of their natural history, ecology and behavior; however, the lack of standardized data, especially for the latter, hampers comparative analysis with the species from the New World. We here synthetized the recent advances in Speleomantes behavioral ecology, considering as a starting point the comprehensive monography of Lanza and colleagues published in 2006. We identified the behavioral categories that were investigated the most, but we also highlighted knowledge gaps and provided directions for future studies. By reviewing the scientific literature published within the period 2006–2022, we observed a significant increase in the number of published articles on Speleomantes behavior, overall obtaining 36 articles. Behavioral studies on Speleomantes focused mainly on trophic behavior (42%), and on intraspecific behavior (33%), while studies on pheromonal communication and interspecific behavioral interactions were lacking. In addition, most of the studies were observational (83%), while the experimental method was rarely used. After providing a synthesis of the current knowledge, we suggest some relevant topics that need to be considered in future research on the behavioral ecology of European plethodontids, highlighting the importance of a more integrative approach in which both field observations and planned experiments are used.

Making a case for the free exploratory paradigm: animal welfare-friendly assays that enhance heterozygosity and ecological validity

Article

Full-text available

Aug 2023

High and Hyper: Fentanyl Induces Psychomotor Side-Effects in Healthy Pigs

Article

Full-text available

May 2023

Analgesic effects of fentanyl have been investigated using behavior. The behavioral effects of fentanyl and possible serotonergic influence are largely unknown. We therefore investigated behavioral effects of fentanyl, with or without the serotonin antagonist ketanserin, in pigs. Fourteen mixed-breed pigs, weighing 17–25 kg were included in a randomised blinded prospective, balanced three-group study. Ten pigs received first 5 and then 10 µg/kg of fentanyl intravenously. Ketanserin at 1 mg/kg or saline was given intravenously as a third injection. Four control pigs received three injections of saline. Behavior was video-recorded. The distance moved was automatically measured by commercially available software, and behaviors manually scored in retrospect. Fentanyl inhibited resting and playing, and induced different repetitive behaviors. The mean (SD) distance moved in the control group and fentanyl group was 21.3 (13.0) and 57.8 (20.8) metres respectively (p < 0.05 for pairwise comparison). A stiff gait pattern was seen after fentanyl injection for median (range) 4.2 (2.8–5.1) minutes per 10 min, which was reduced to 0 (0–4) s after ketanserin administration. Conclusion: fentanyl-induced motor and behavioral effects, and serotonergic transmission may be involved in some of them. The psychomotor side effects of fentanyl could potentially interfere with post-operative pain evaluation in pigs.

The impact of stress and anesthesia on animal models of infectious disease

Article

Full-text available

Feb 2023

Stress and general anesthesia have an impact on the functional response of the organism due to the detrimental effects on cardiovascular, immunological, and metabolic function, which could limit the organism's response to an infectious event. Animal studies have formed an essential step in understanding and mitigating infectious diseases, as the complexities of physiology and immunity cannot yet be replicated in vivo. Using animals in research continues to come under increasing societal scrutiny, and it is therefore crucial that the welfare of animals used in disease research is optimized to meet both societal expectations and improve scientific outcomes. Everyday management and procedures in animal studies are known to cause stress, which can not only cause poorer welfare outcomes, but also introduces variables in disease studies. Whilst general anesthesia is necessary at times to reduce stress and enhance animal welfare in disease research, evidence of physiological and immunological disruption caused by general anesthesia is increasing. To better understand and quantify the effects of stress and anesthesia on disease study and welfare outcomes, utilizing the most appropriate animal monitoring strategies is imperative. This article aims to analyze recent scientific evidence about the impact of stress and anesthesia as uncontrolled variables, as well as reviewing monitoring strategies and technologies in animal models during infectious diseases.

The proof is in the poo: Non‐invasive method to detect endoparasitic infection

Article

Full-text available

Jul 2023

Almost every animal trait is strongly associated with parasitic infection or the potential exposure to parasites. Despite this importance, one of the greatest challenges that researchers still face is to accurately determine the status and severity of the endoparasitic infection without killing and dissecting the host. Thus, the precise detection of infection with minimal handling of the individual will improve experimental designs in live animal research. Here, we quantified extracellular DNA from two species of endoparasitic worm that grow within the host body cavity, hairworms (phylum Nematomorpha) and mermithids (phylum Nematoda), from the frass of their insect host, a cave wētā (Orthoptera: Rhaphidophoridae) and an earwig (Dermaptera: Forficulidae), respectively. Frass collection was done at two successive time periods, to test if parasitic growth correlated with relative DNA quantity in the frass. We developed and optimised two highly specific TaqMan assays, one for each parasite‐specific DNA amplification. We were able to detect infection prevalence with 100% accuracy in individuals identified as infected through post‐study dissections. An additional infection in earwigs was detected with the TaqMan assay alone, likely because some worms were either too small or degraded to observe during dissection. No difference in DNA quantity was detected between sampling periods, although future protocols could be refined to support such a trend. This study demonstrates that a non‐invasive and minimally stressful method can be used to detect endoparasitic infection with greater accuracy than dissection alone, helping improve protocols for live animal studies.

Why workshops work: Examining the efficacy of training trainers to train goats

Article

Full-text available

Nov 2023
ANIM WELFARE

Experimental procedures involving farm animals are often associated with stress due to restraining. Stress can be reduced through use of positive reinforcement training, which then serves as refinement according to the 3Rs principles. Trainer skills, however, may influence the feasibility and success of animal training. The potential influence of trainer skills as well as the education of animal trainers are rarely described in literature but are necessary information for the implementation of positive reinforcement training as a refinement measure. To investigate the effect of educational programmes on animal trainers, we compared the training success of two groups of participants in training goats to elicit a behaviour that would allow simulated venipuncture. One group was educated in a two-day workshop while the other was provided with specific literature for self-instructed learning. Training success was evaluated using an assessment protocol developed for this study. A greater training success in the WORKSHOP GROUP, reflected by objective and subjective measures, was clearly supported statistically. In addition, 73 versus only 13% of the participants of the WORKSHOP GROUP and the self-instructed BOOK GROUP, respectively, stated that they could completely implement the knowledge gained in the course of this study. Our results indicate that more intensively educated trainers can train animals more successfully. In conclusion, if animal training is implemented as refinement, animal caretakers should receive instruction for positive reinforcement training.

Physiological differences between wild and captive animals: a century-old dilemma

Article

Nov 2023
J EXP BIOL

Laboratory-based research dominates the fields of comparative physiology and biomechanics. The power of lab work has long been recognized by experimental biologists. For example, in 1932, Georgy Gause published an influential paper in Journal of Experimental Biology describing a series of clever lab experiments that provided the first empirical test of competitive exclusion theory, laying the foundation for a field that remains active today. At the time, Gause wrestled with the dilemma of conducting experiments in the lab or the field, ultimately deciding that progress could be best achieved by taking advantage of the high level of control offered by lab experiments. However, physiological experiments often yield different, and even contradictory, results when conducted in lab versus field settings. This is especially concerning in the Anthropocene, as standard laboratory techniques are increasingly relied upon to predict how wild animals will respond to environmental disturbances to inform decisions in conservation and management. In this Commentary, we discuss several hypothesized mechanisms that could explain disparities between experimental biology in the lab and in the field. We propose strategies for understanding why these differences occur and how we can use these results to improve our understanding of the physiology of wild animals. Nearly a century beyond Gause's work, we still know remarkably little about what makes captive animals different from wild ones. Discovering these mechanisms should be an important goal for experimental biologists in the future.

Brief exposure to captivity in a songbird is associated with reduced diversity and altered composition of the gut microbiome

Article

Aug 2023
FEMS MICROBIOL ECOL

The gut microbiome is important for host fitness and is influenced by many factors including the host's environment. Captive environments could potentially influence the richness and composition of the microbiome and understanding these effects could be useful information for the care and study of millions of animals in captivity. While previous studies have found that the microbiome often changes due to captivity, they have not examined how quickly these changes can occur. We predicted that the richness of the gut microbiome of wild-caught birds would decrease with brief exposure to captivity and that their microbiome communities would become more homogeneous. To test these predictions, we captured wild house sparrows (Passer domesticus) and collected fecal samples to measure their gut microbiomes immediately after capture ('wild sample') and again 5-10 days after capture ('captive sample'). There were significant differences in beta diversity between the wild and captive samples, and captive microbiome communities were more homogenous but only when using non-phylogenetic measures. Alpha diversity of the birds' microbiomes also decreased in captivity. The functional profiles of the microbiome changed, possibly reflecting differences in stress or the birds' diets before and during captivity. Overall, we found significant changes in the richness and composition of the microbiome after only a short exposure to captivity. These findings highlight the necessity of considering microbiome changes in captive animals for research and conservation purposes.

Anesthesia and analgesia in laboratory pigs

Chapter

Jan 2023

Refined Tamoxifen Administration in Mice by Encouraging Voluntary Consumption of Palatable Formulations

Preprint

Full-text available

Apr 2023

Drug administration in preclinical rodent models is essential for research and development of novel therapies. Compassionate administration methods have been developed, but these are mostly incompatible with water-insoluble drugs such as tamoxifen or do not allow for precise timing or dosing of the drugs. For more than two decades, tamoxifen has been administered by oral gavage or injection to CreERT2/loxP gene-modified mouse models to spatiotemporally control gene expression, with the numbers of such models steadily increasing in recent years. Animal-friendly procedures for accurately administering tamoxifen or other water-insoluble drugs would therefore have an important impact on animal welfare. Based on a previously published micropipette feeding protocol, we developed palatable formulations to encourage voluntary consumption of tamoxifen. We evaluated the acceptance of the new formulations by mice during training and treatment and assessed the efficacy of tamoxifen-mediated induction of CreERT2/loxP dependent reporter genes. Both sweetened milk and syrup-based formulations encouraged mice to consume tamoxifen voluntarily, but only sweetened milk formulations were statistically non-inferior to oral gavage in inducing CreERT2-mediated gene expression. Serum concentrations of tamoxifen metabolites, quantified using an in-house developed cell assay, confirmed the lower efficacies of syrup- as compared to sweetened milk-based formulations. We found dosing with a micropipette to be more accurate, with the added advantage that the method requires little training for the experimenter. The new palatable solutions encourage voluntary consumption of tamoxifen without loss of efficacy compared to oral gavage and thus represent a refined administration method.

Does the Stress Inherent to Laboratory Life and Experimentation on Animals Adversely Affect Research Data?

Article

Full-text available

Dec 2017

Jarrod Bailey

Monkey-based Research on Human Disease: The Implications of Genetic Differences

Article

Full-text available

Nov 2014

Jarrod Bailey

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.

Neurochemical and Behavioral Responses to Unpredictable Chronic Mild Stress Following Developmental Isolation: The Zebrafish as a Model for Major Depression

Article

Full-text available

Jul 2016

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.

Lessons from Chimpanzee-based Research on Human Disease: The Implications of Genetic Differences

Article

Full-text available

Dec 2011

Jarrod Bailey

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.

Effects of Noise on Rodent Physiology

Article

Full-text available

Dec 2007

Ann Baldwin

Fish can show emotional fever: Stress-induced hyperthermia in zebrafish

Article

Full-text available

Nov 2015
Proc Biol Sci

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.

The welfare of dogs in human care

Chapter