KUOPION YLIOPISTON JULKAISUJA C. LUONNONTIETEET JA YMPÄRISTÖTIETEET 258
KUOPIO UNIVERSITY PUBLICATIONS C. NATURAL AND ENVIRONMENTAL SCIENCES 258
NIINA KEMPPINEN
Refinement and Reduction Outcomes of
Cage Furniture and Restricted Feeding
in Laboratory Rats
Doctoral dissertation
To be presented by permission of the Faculty of Natural and Environmental Sciences
of the University of Kuopio for public examination in Auditorium ML3, Medistudia building,
University of Kuopio, on Friday 20 th November 2009, at 12 noon
Department of Biosciences
University of Kuopio
Laboratory Animal Centre
University of Helsinki
JOKA
KUOPIO 2009
Distributor :
Kuopio University Library
P.O. Box 1627
FI-70211 KUOPIO
FINLAND
Tel. +358 40 355 3430
Fax +358 17 163 410
http://www.uku.fi/kirjasto/julkaisutoiminta/julkmyyn.shtml
Series Editor :
Professor Pertti Pasanen, Ph.D.
Department of Environmental Science
Author’s address: Laboratory Animal Centre
P.O. Box 56
FI-00014 University of Helsinki
Tel. +358 050 415 4444
E-mail: [email protected]
Supervisors:
Professor Timo Nevalainen
National Laboratory Animal Center
University of Kuopio
Professor Jaakko Mononen
Department of Biosciences
University of Kuopio
Eila Kaliste, Ph.D.
State Provincial Office of Southern Finland
Tarja Kohila, Ph.D.
Laboratory Animal Centre
University of Helsinki
Reviewers:
Klas Abelson, Ph.D.
Department of Experimental Medicine
University of Copenhagen, Denmark
Docent Ulla-Marjut Jaakkola
Central Animal Laboratory
University of Turku
Opponent:
Docent Hanna-Marja Voipio
Laboratory Animal Centre
University of Oulu
ISBN 978-951-27-1196-3
ISBN 978-951-27-1291-5 (PDF)
ISSN 1235-0486
Kopijyvä
Kuopio 2009
Finland
Kemppinen, Niina. Refinement and Reduction Outcomes of Cage Furniture and Restricted Feeding in
Laboratory Rats. Kuopio University Publications C. Natural and Environmental Sciences 258. 2009.
140 p.
ISBN 978-951-27-1196-3
ISBN 978-951-27-1291-5 (PDF)
ISSN 1235-0486
ABSTRACT
The most recent European housing and care regulations for laboratory rats mandate provision of a
structured environment and group housing. Dividing structures and shelters in the cage offer rats
opportunities to seek or avoid contact with other group members and hence are regarded as beneficial
to animal welfare.
In order to compare the physical environment of the IVC and the open cage, temperature, relative
humidity, lighting and sound levels of the cages were measured. BN/RijHsd and F344/NHsd male rats
were used, and they were housed in IVC- or open cages of the same type, three rats per cage, one of
them carrying a telemetric transponder. Four groups and a crossover design were used: two groups
with a maze made of crossing two aspen boards, a rectangular aspen tube group and controls. In one
maze, drilled holes were loaded snugly with food pellets; rats had to gnaw wood to gain access to
their food. Rats and food were weighed before and after each study period. The means of locomotor
activity and means and coefficient of variations for mean arterial pressure (MAP) and heart rate (HR)
were calculated for days 2, 6, 10 and 14 in each period. As a way of determining which of the
statistically significant MAP and HR mean changes were biologically meaningful, the corresponding
night-day differences of the controls were used in this two step assessment. On day 8 of each two
week period, the rats were changed to clean cages and on day 11 exposed to IG-gavage. The means of
activity, mean arterial pressure and heart rate were processed for the first hour subsequent to the
procedure and thereafter separately in the light and dark periods and for the two cage types. Baseline
values for each rat, for both dark and light and cage types were calculated from recordings made 24 h
earlier; and these were subtracted from the corresponding response values.
In the study of the physical environment of the cage types, there were differences in all measured
parameters. In F344 rats, diet board was more effective in controlling weight, but when combining the
strains, all comparisons with diet board were significant. In both cages, the F344 rats were generally
more active than the BN rats during the dark phase, but not during the light phase. In the IVCs, both
board types lowered MAP of F344 rats throughout the two week period and at the end of that period.
Plain board was found to be the better of the two; hence dividing walls with or without restricted
feeding seem beneficial for the welfare of F344 rats. None of the MAP or HR differences in BN rats
were biologically significant. The MAP CV results showed that cage furniture may be used to achieve
a considerable reduction value in blood pressure studies, but the outcome is strain-specific. Neither of
the strains exhibited any statistically significant differences in faecal corticosterone or IgA excretion
to these items. Based on the MAP results, the tube appeared to be a poor choice for F344 rats,
whereas for BN rats, all furniture items seemed beneficial, with both board types apparently superior
to the tube. In general, F344 rats had higher faecal corticosterone levels than BN rats with the reverse
being true for secretory IgA values.
In conclusion, LA and cardiovascular parameters seemed appropriate ways to evaluate the impact of
cage furniture on physiological parameters, and covered structures such as tubes do not seem to
provide any refinement value in these two rat strains.
Universal Decimal Classification: 599.323.4, 57.082.2, 636.028, 636.083.312.5, 636.084.42
National Library of Medicine Classification: QY 50, QY 54, QY 56, QY 60.R6
CAB Thesaurus: animal welfare; animal housing; cages; furniture; walls; tubes; ventilation;
laboratory animals; rats; Rattus norvegicus; rat feeding; restricted feeding; food restriction; food
intake; body weight; weight gain; animal behaviour; cardiovascular system; blood pressure; heart rate;
corticosterone; IgA; faeces; temperature; relative humidity; lighting; sounds; locomotion
ACKNOWLEDGMENTS
The studies in this thesis were performed in the Laboratory Animal Centre of the University of
Helsinki. The support and encouragement of people at the University of Helsinki and at the
University of Kuopio have made it possible to complete this thesis. This study has been financially
supported by the Academy of Finland, the Finnish Ministry of Education, ECLAM and ESLAV
Foundation, the North-Savo Cultural Foundation, the Finnish Research School for Animal Welfare,
the Oskar Öflund Foundation and the University of Kuopio.
I express my gratitude especially to the following persons:
I owe my deepest gratitude and appreciation to my supervisor Professor Timo Nevalainen, who
without I may never would have the opportunity to complete this thesis. Thank you, Timo for your
support and for all the knowledge about the laboratory animal science that I have received from you
during these years.
My warmest thanks belong also to my other supervisors, Professor Jaakko Mononen, Docent Eila
Kaliste and PhD Tarja Kohila as well as to PhD Satu Mering, the follow-up member of my studies.
Thank you for your encouragement and your comments concerning the thesis.
I want to acknowledge the reviewers of my thesis, Docent Ulla-Marjut Jaakkola (Laboratory Animal
Centre, the University of Turku, Finland) and PhD Klas Abelson (the University of Copenhagen) for
their careful review and constructive comments concerning the thesis.
I wish to thank to all persons that have been involved in my work: PhD Erkki Björk for analyzing
the sound levels from the cages, Professor Jann Hau for organizing the analysis of the faecal
samples, and statistician Kari Mauranen for helping with the statistics. I am also thankful to Ewen
MacDonald for editing the language of this thesis.
I warmly thank Proffesor Eero Lehtonen, MD, PhD, the director of the Laboratory Animal Centre of
the University of Helsinki, for support and possibility to work with the thesis.
I owe my special thanks for Tapvei Ltd and Veijo Tirkkonen for providing all of the cage items
needed in my research.
To my colleague and friend, Anna Meller, DVM, I am very grateful for all help concerning my
thesis. Thank you Anna, I have learnt so much from working with you during these years.
I highly appreciate the support that I have received from the people in Finnish Laboratory Animal
Science Association (FinLAS) and Scandinavian Society for Laboratory Animal Science
(ScandLAS). In particular, the board members in both societies have encouraged me greatly in my
thesis. It has been an honour to be your secretary.
I express my gratitude to Mr Heikki Pekonen for help with the surgical procedures and to Ms Virpi
Nousiainen for daily caretaking of the rats and performing the IG-gavages during the experiments. I
thank my workmates in the Viikki unit of the Laboratory Animal Centre of the University of
Helsinki. Jaana, Pia, Irmeli, Anki, Pirjo, Maria and Mari, thank you all for understanding. Without
your presence working would have been boring.
Finally, I want to thank all my dear friends and relatives for enriching my life outside of the work
place. My special thanks go to my parents and my sister for their support and understanding.
Niina Kemppinen
Helsinki, October 2009
ABBREVIATIONS
2Rs
3Rs
ACTH
ANS
BALB/c
BN
BPM
C57BL/6J
CRF
CR
CV
DBA/2
DPB
EC
ECG
F344
FP7
GR
HEPA
HPA
HR
IG
IgA
IVC
LA
MAP
MR
PE
PP
ppm
QTL
RH
SBP
SEM
SNS
Tb
Reduction, Refinement
Reduction, Refinement, Replacement
Adrenocorticotrophic hormone
Autonomic nervous system
Inbred mouse strain
Brown Norway – an inbred rat strain
Beats per minute
Inbred mouse strain
Corticotrophin releasing factor
Caloric restriction
Coefficient of variation
Inbred mouse strain
Diastolic blood pressure
European Commission
Electrocardiogram
Fischer 344 – an inbred rat strain
Seventh Framework Programme
Glucocorticoid receptor
High efficiency particulate air
Hypothalamic pituitary adrenal
Heart rate
Intragastric
Immunoglobulin A
Individually ventilated cage
Locomotor activity
Mean arterial pressure
Mineralocorticoid receptor
Point estimate
Pulse pressure
Parts per million
Quantitative trait locus
Relative humidity
Systolic blood pressure
Standard error of mean
Sympathetic nervous system
Body temperature
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following publications, referred to in the text by their chapter numbers.
Chapter II Niina Kemppinen, Anna Meller, Erkki Björk, Tarja Kohila & Timo Nevalainen.
2008. Exposure in the shoebox: Comparison of physical environment of IVCs and open rat cages.
Scandinavian Journal of Laboratory Animal Science 35:97-103.
Chapter III Niina Kemppinen, Anna Meller, Kari Mauranen, Tarja Kohila & Timo Nevalainen.
2008. Work for food – A solution to restricted food intake in group housed rats? Scandinavian
Journal of Laboratory Animal Science 35:81-90.
Chapter IV Niina Kemppinen, Anna Meller, Kari Mauranen, Tarja Kohila & Timo Nevalainen.
2009. The effect of dividing walls, a tunnel and restricted feeding on cardiovascular responses to
cage change and gavage in rats (Rattus norvegicus). Journal of the American Association for
Laboratory Animal Science 48:157-165.
Chapter V Niina Kemppinen, Jann Hau, Anna Meller, Kari Mauranen, Tarja Kohila & Timo
Nevalainen. 2010. Impact of aspen furniture and restricted feeding on activity, blood pressure, heart
rate, and faecal corticosterone and immunoglobulin A excretion in rats (Rattus norvegicus) housed
in individually ventilated cages. Published online Laboratory Animals, doi: 10.1258/la.2009.
009058.
Chapter VI Niina Kemppinen, Anna Meller, Jann Hau, Kari Mauranen, Tarja Kohila & Timo
Nevalainen. Refinement value of aspen furniture and restricted feeding for rats in open rat cages.
Submitted to Scandinavian Journal of Laboratory Animal Science.
CONTENTS
CHAPTER I: GENERAL INTRODUCTION................................................................... 13 1.1 Alternative methods to the use of animals in research ........................................ 15 1.2 Animal welfare .................................................................................................... 18 1.3 Individually Ventilated Caging (IVC) ................................................................. 19 1.4 Stress and stress indicators in laboratory rats ...................................................... 23 1.5 Telemetry as a study method in laboratory animal welfare ................................ 27 1.6 Restricted feeding in rats ..................................................................................... 28 1.7 Cage furniture in laboratory rats.......................................................................... 32 1.8 Cage change and IG-gavage ................................................................................ 35 1.9. Strain differences in rat ...................................................................................... 38 1.10 Aims of the present study .................................................................................. 42 1.11 References ......................................................................................................... 43 CHAPTER II: Exposure in the shoebox: Comparison of physical environment of IVCs
and open rat cages.............................................................................................................. 53 CHAPTER III: Work for food – A solution to restricted food intake in group housed rats?
........................................................................................................................................... 63 CHAPTER IV: The effect of dividing walls, a tunnel and restricted feeding on
cardiovascular responses to cage change and gavage in rats (Rattus norvegicus). ........... 75 CHAPTER V: Impact of aspen furniture and restricted feeding on activity, blood
pressure, heart rate, and faecal corticosterone and immunoglobulin A excretion in rats
(Rattus norvegicus) housed in individually ventilated cages. ........................................... 87 CHAPTER VI: Refinement and reduction value of aspen furniture and restricted feeding
of rats in conventional cages. ............................................................................................ 99 CHAPTER VII: GENERAL DISCUSSION ................................................................... 121 7.1 IVC vs. open cage.............................................................................................. 123 7.2 Diet board as a restricted feeding method in rats .............................................. 125 7.3 Cage change and IG-gavage .............................................................................. 126 7.4 Cage furniture items and stress indicators in rats .............................................. 129 7.5 Differences in rat strains .................................................................................... 132 7.6 The Two R value of the results.......................................................................... 132 7.7 Conclusions ....................................................................................................... 137 7.8 References ......................................................................................................... 138 CHAPTER I
GENERAL INTRODUCTION
CHAPTER I
GENERAL INTRODUCTION
1.1 Alternative methods to the use of
animals in research
It is realistic to state that laboratory animals
will still be used in research in the near future
(Festing 2004); indeed there are several
indications that their use may even increase.
For example in Europe, the European
Commission (EC) intends to double funding
for basic research (EU 2007a); this will
undoubtedly have consequences on the
quantity and quality of animals being used.
Similar trends in funding can be seen
elsewhere where governments and funding
agencies place their trust and financial
backing on science as a way to solve many
global problems. Concomitantly, the use of
genetically altered animals is increasing all
over the world. In Europe, the new Chemicals
directive
(Registration,
Evaluation,
Authorisation and Restriction of Chemicals,
REACH) is estimated to add 8-30 million
animals to the numbers already being used.
The latest statistics for 2002 and 2005 from
the EC also supports this view; the number of
animals used has increased from ten to 12
million in three years (EC 2005, EC 2007b).
The continuing need for the use of animals
in research places an even greater emphasis on
animal welfare and novel ways to measure it.
Animal welfare refers to implementation and
measurement of ways to verify efficiency
adhered to the alternative methods. Contrary
to the common belief, the principals behind
the alternative definitions are not new. These
principles were introduced already 1831 when
British physiologist Marshall Hall proposed
five principles that should govern animal
experimentation (Paton 1984):
1. An experiment should never be
performed if the necessary information
could be obtained by observations.
2. No experiment should be performed
without a clearly defined and obtainable
objective.
3. Scientists should be well-informed
about the work of their predecessors and
peers in order to avoid unnecessary
repetition of an experiment.
4. Justifiable experiments should be
carried out with the least possible infliction
of suffering (often through the use of
lower, less sentient animals).
5. Every
experiment
should
be
performed under circumstances that would
provide the clearest possible results,
thereby diminishing the need for repetition
of experiments.
Marshall Hall’s principles were translated
to 3Rs by Russell and Burch in their 1959
book
“The
Principles
of
Humane
Experimental Technique”. The concept of the
3Rs aims at replacing, reducing or the refining
use of laboratory animals. According to them,
“Replacement” means “the substitution for
conscious living higher animals with
insentient material", “Reduction” means
“reduction in the numbers of animal used to
obtain information of a given amount and
precision”, and “Refinement” means “any
decrease in the incidence or severity of
inhumane procedures applied to those animals
which still have to be used” (Russell & Burch
1959).
Today these 3R principles constitute the
alternatives to the use of animals and have
been incorporated into most regulatory
documents and mentioned in a plethora of
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
15
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
guidelines and recommendations (Council of
Europe 1986, Declaration of Bologna 1999,
EC 2006) dealing with the use of animals in
research and in funding of such activity, e.g.
ethical rules in the Seventh Framework
Programme (FP7) (EC 2007a).
It is increasingly evident that animal-based
research is not always done in the best
possible way. There is doubt with respect to
the extent to which the refined methods
developed in laboratory animal science are
actually applied to studies using animals.
Richardson & Flecknell (2005) found that
postoperative pain control was used in less
than 20 % of potentially painful research
procedures; Olsson et al. (2007) discovered
only a few references to refinement in studies
using neurodegenerative rodent models.
Furthermore, recent systematic reviews raise
questions over the benefit of preclinical
research on animals for the development of
clinical applications, on the grounds of
inappropriate design and methodology
(Dirnagl 2006) or incorrect timing (Pound et
al. 2004) of animal-based research in relation
to the clinical studies.
While the Replacement is the ultimate
objective, complete avoidance of the use of
sentient beings does not, unfortunately, appear
to be possible (Festing 2004). Until this
ultimate objective has been achieved, animals
will continue to be used in those situations
where no Replacement is available. It is
considered morally wrong and even cruel not
to extend the principles of Reduction and
Refinement (the 2Rs) to those animals during
the meantime.
Overly large variation of result parameters
is undesirable in research because it is bound
16
to increase the number animals needed in a
study, that is against the desired aims of
reduction (Festing et al. 2002). Refinement
includes methods that are designed to improve
housing or procedures of the animals. The
scientists quite correctly expect reliable results
and here both refinement and reduction have
an essential role to play. It is not only the
improvements in welfare but also a uniform
nature of welfare that are important both to
the scientists and the animals.
In 2005, COST (European Cooperation in
Science and Technology) Action B24
submitted the 2Rs Initiative to the FP7. In this
Initiative, it was acknowledged that although
Replacement is the ultimate objective - i.e. to
completely avoid the use of sentient beings unfortunately this is not yet possible. Until
this objective has been achieved, animals will
continue to be used where no replacement is
available. The inclusion of 2Rs funding into
the FP7 was supported by 50 International and
European universities, research institutes,
scientific associations and animal welfare
organizations,
but
implementation
of
measures has not taken place.
Refinement and Reduction alternatives are
interrelated; studies may do well in refinement
while major compromises occur in reduction
or the opposite (Nevalainen 2004). The
optimum and the prohibited directions are
clear but in the remaining choice
combinations one has to make a value
judgement on whether refinement or reduction
is more important. In Figure 1.1, the
relationship of the 2Rs is illustrated with two
perpendicular axes; in addition to fewer
animals and refined welfare, we should aim at
better science resulting from animal use.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
General introduction
Refined welfare
Less harm
Fewer
animals
More
animals
Better science
Compromised
welfare
Figure 1.1 Reduction (x-axis) and Refinement (y-axis) dimensions for assessment of any care routine
or study procedure on animals. Arrows point to a direction of less harm to the animals, and to a desired
concomitant change in research quality, both essential elements to be addressed in animal studies.
Russell and Burch (1959) also argued that
application of the 3Rs should not be
detrimental to the scientific outcome of
animal studies. This aspect should also be
addressed by systematic research, with the
aim of establishing which of the 3R
alternatives results in better science and even
the opposite. It is quite clear that all of the 3R
methods may not be free of adverse
consequences on science, and those may be
associated with consequential wastage of
animals.
The implementation of the 3Rs can actually
lead to improvements in scientific quality. A
systematic scientific approach to find out
which of the alternatives indeed results in
better science is necessary. In other words, the
scope of ethics in animal studies should
expand to encompass scientific reasoning. The
twin aims of ethical and scientific integrity
can and indeed should be addressed with
education based on targeted research focussed
on the topic.
The Directive (86/609/EEC) on the
protection of animals used for experimental
and other scientific purposes states that the
Commission and the EU Member States must
actively encourage and support the
development, validation and acceptance of
methods which could reduce, refine or replace
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
17
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
the use of laboratory animals. This has not
been fully implemented; and the ongoing
revision of the Directive is anticipated to be
more stringent on the requirement of
implementing the alternative methods, i.e. the
3Rs (EU 2008).
Closer integration of laboratory animal
science and research using animals is a
necessity. Currently there is European
research activity on implementation of the
3Rs in animal testing (European Centre for
Validation of Alternative Methods (ECVAM),
FP7), the main focus being on replacement;
this covers only about 25 % of animal use and
one of the 3Rs, e.g. excluding fundamental
and applied research. The belief is that the
establishment of scientifically proven 3R
methodology combined with mandatory
application
will
improve
Europe's
competitiveness.
1.2 Animal welfare
The definition of animal welfare varies in
many ways depending on the author’s point of
view, and as Newberry (1995) has said, “the
concept of animal welfare is a vague notion
that evades precise definition and is used
inconsistently in the literature”. Clark et al.
(1997a) also considered the animal welfare a
vague concept, which can neither be viewed
in a purely objective manner nor simply
described, defined, or assessed. Furthermore,
Clark et al. (1997a) used the term “animal
well-being” because it is more widely used in
the United States rather than the alternative
“animal welfare”.
The classical definition for animal welfare
is an individual’s state as regards its attempts
to cope with its environment (Broom 1986).
Moreover, it has been stated that coping of the
18
animal refers to both how much work has to
be done in order to cope with the environment
and the extent to which coping attempts are
successful (Broom & Johnson 1993). In
analogy to this coping definition, Webster
(1995) has defined the welfare of the animal
as “determined by its capacity to avoid
suffering and sustain fitness”.
Fraser et al. (1997) listed three ethical
concerns that reflect the quality of life of
animals and can also be used as bases for
animal welfare definitions. First, animals
should lead natural lives through the
development and use of their natural
adaptations and capabilities, including natural
behaviour. These authors suggested that the
genetically encoded “nature” of an animal can
be viewed as the set of adaptations that an
animal possesses as a result of its evolutionary
history, and the set of genetically encoded
instructions that guide the animal’s normal
development. Second, animals should feel
well by being free from prolonged and intense
fear, pain and other negative states, and by
experiencing normal pleasures, i.e. animals
have feelings. Third, animals should function
well, in the sense that they experience
satisfactory health, growth and normal
functioning of physiological and behavioural
systems. In animal welfare definitions, the
biological functioning of animals is often
linked to certain concepts such as fitness and
stress.
All the three concerns can be seen in the
“five freedoms” proposed by the Farm Animal
Welfare Council of United Kingdom in 1993
(Webster 2003). Five freedoms categorise the
different elements necessary for good welfare
and husbandry provisions and their
promotion. These freedoms are:
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
General introduction
1) freedom from thirst, hunger and
malnutrition
2) freedom from discomfort
3) freedom from pain, injury and disease
4) freedom to display most normal
patterns of behaviour
5) freedom from fear and distress
Animal welfare has received considerable
attention also in the EC legislation. In the
Amsterdam Treaty (EU 1997), the protocol
on protection and welfare of animals confirms
the EC’s intension to ensure improved
protection and respect for the welfare of
animals as sentient beings. It also states that
while formulating and implementing the EC's
agriculture, transport, internal market and
research policies, the EC and the Member
States shall pay full regard to the welfare
requirements of animals.
Since the animal welfare is a complex
concept with many definitions, there are no
simple methods to assess it. Different methods
measure only different components of the
welfare rather than the animal welfare itself
(Rushen & de Passillé 1992). According to
Clark et al. (1997b) classic and practical
assessment of animal welfare includes a
combination
of
animal
appearance,
performance,
behaviour,
productivity,
disability, injury, disease, longevity, mortality
and of the state of an animal’s environment.
Thus it is essential to use a variety of welfare
indicators if an adequate assessment of animal
housing and management systems (Broom
1991).
The animal welfare has been assessed e.g.
with preference and behavioural tests, number
of wounds, disease and reproductive levels,
and adrenal activity (Broom 1988, 1991).
Preference tests can show what animals
choose and how hard they will work for a
preferred event or object. Behavioural
observations tell us whether the animals are
able to carry out normal behaviour in their
environment. Injuries, declined reproductive
capabilities and high disease frequencies have
been used as incidences of reduced fitness of
animals. Adrenal activity responses are brief
and the responses of the adrenal cortex decline
after a few hours and thus they are usable for
assessment of short-term welfare problems.
However, if adverse conditions continue for
many hours, also bursts of glucocorticoid
production can be detected (Broom 1988).
1.3 Individually Ventilated Caging (IVC)
Until recently the prevailing housing
system of laboratory rodents has been open,
conventional cages. In these cages, animals
are in direct contact with the ambient room air
and thus also with the other animals in the
room, which renders transmission of
infectious agents, gas emissions, e.g.
ammonia, and allergens, to room air and other
cages. The development of pressurized
individually ventilated housing system for
laboratory rodents began already 1963 at the
Jackson Laboratory in USA (Clough et al.
1995). During the last decade, the scientific
community has witnessed the massive
introduction of IVC-caging, where each
individual cage receives its own HEPA
filtered air flow, primarily designed for animal
health status maintenance and occupational
safety for the personnel.
The IVCs have clear advantages over open
cages; they provide protection against
infections to animals, drastically decrease
emissions from animals and cages to and
compensate for poor ventilation in the room
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
19
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
(Brandstetter et al. 2005). Even though the
actual cages may be the same as those used in
open cage systems, the physical environment
inside is not (Clough et al. 1995, Teixeira et
al. 2006).
Air to the IVC system can be supplied
either from central ventilation or directly from
the room, and the same applies to the exhaust
air. The latter arrangement is typical to older
facilities or those in transition from open
cages to IVCs. This division has consequences
on effective ventilation and other physical
environmental characteristics inside the cage.
Table 1.1 is a summary of studies on physical
environment of IVC and resulting effects both
on mouse and rat welfare.
The IVC systems are efficient in isolating
the animals from other animals possibly
harbouring animal pathogens because all
incoming air is HEPA-filtered. It has been
shown that the IVC system effectively
prevents the transmission of particles from
room to cage air and also between cages
(Clough et al. 1995, Myers et al. 2003).
Hence it is no wonder that IVC systems are
gaining popularity despite the rather high
investment costs associated with their
purchase.
There are also clear occupational health
benefits to the personnel associated to IVC
equipment, i.e. a reduction in both the levels
of airborne allergens and ammonia (Keller et
al. 1983, Lipman et al. 1992 Lipman 1999,
Renström et al. 2001, Teixeira et al. 2006).
Airborne allergens with IVC caging appear to
be about 1.5% of the levels applicable
irrespective of the source of incoming air.
Ammonia levels – if air is circulated from
room air and back – depend on the ventilation
efficacy of the room. A low ammonia content
20
is important for both human and animal
health; e.g. 5 ppm concentration in the cages
leads to a weaker defence capability of the
respiratory tract (Dalhamn 1956).
The air changes in the IVC cages can be
considerably higher with the same or even
lower air flow than in animal rooms simply
because the combined cage volume is always
much less than that of the room. This is
considerable improvement compared to open
cages, where the room ventilation rate of 5-20
changes per h reduced levels of CO2,
ammonia, relative humidity (RH) and
temperature inside the mouse cage (Reeb et
al. 1997). Common ventilation rates inside
IVC-cage vary between 25-120 changes per h,
but also extremely high, such as over 600 air
changes per h, have been examined (Teixeira
et al. 2006). Preference studies on both
BALB/c mice and Sprague-Dawley (SD) rats
showed preference to lower range of the
common air change rates (Baumans et al.
2002, Krohn et al. 2003b). However, mice
preferred cages with a somewhat higher air
change if the cage was provided with a
covered air supply and nesting material; this
combination appeared to lower the stress
effect associated to high ventilation rates
(Baumans et al. 2002).
The high ventilation in IVCs results in
better air quality, but has also a drying effect
on bedding. The latter allows lower cage
changing frequency in IVC than in the open
cages. Different cage ventilation rates (30-100
changes per h) have been evaluated with three
different cage changing intervals (7, 14 and 21
days) in C57BL/6J mice (Reeb et al. 1998,
Reeb-Whitaker et al. 2001). Relative humidity
and concentrations of ammonia and carbon
dioxide (CO2) were lower at higher
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
General introduction
Table 1.1 Summary of mouse and rat studies on effects of the IVC-system.
Reference
Species:
strain/stock
Baumans et al. 2002
Brandstetter et al. 2005
Brielmeier et al. 2006
Clough et al. 1995
Mouse: BALB/c
Unspecified
Mouse
Mouse
Compton et al. 2004
Mouse: Swiss
Webster
Mouse:
C57BL/6J
Mouse
Rat: Wistar
Cruden 2007
Gordon et al. 2001
Hasegawa 1997
Hawkins et al. 2003
Höglund & Renström
2001
Keller 1983
Krohn & Hansen 2002
Krohn et al. 2003b
Lipman et al.1992
Lipman 1999
Myers et al. 2003
Unspecified
Mouse: NMRI
Reeb-Whitaker et al.
2001
Renström et al. 2001
Silverman et al. 2008
Teixeira et al. 2006
Mouse: CF1
Mouse: NMRI
Rat: SD
Mouse: ICR
Unspecified
Mouse: SCID
and TNF
Mouse:
C57BL/6J
Mouse:
C57BL/6J
Mouse:
C57BL/6J
Mouse: NMRI
Mouse: ICR
Rat: Wistar
Tsai et al. 2003
Tu et al. 1997
Mouse: DBA2
Unspecified
Reeb et al. 1997
Reeb et al. 1998
Topic
Influence of intracage ventilation rate
General evaluation of IVC
Microbiological monitoring in IVC
Light intensity, sound level, ventilation rate,
temperature, relative humidity
Microbiological monitoring in IVC
Ammonia, CO2 and O2 concentrations in IVC with
different bedding materials
Containment of mouse allergens in IVC
Impact of air change rate on CO2 and O2
concentrations in IVC
IVC and animal welfare
Cage environment in two IVC-systems
Ammonia level
CO2 concentration in unventilated IVC
Impact of low levels of CO2 in IVC
Effect of cage ventilation on microenvironment
Overview of IVC-system
Transmission of infectious agents in IVC
Impact of room ventilation on mouse cage ventilation
and microenvironment
Microenvironment in IVC: effect of ventilation, mice
population and bedding change frequency
Cage change frequency in IVC
Allergens and ergonomics of IVC
Ammonia and carbon dioxide in IVC
Physical environment of IVC and effects on
inflammatory airway diseases
Breeding performance in IVC
Ventilation in IVC
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
21
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
ventilation rates, whereas the lowest
ventilation rate with weekly cage changing
caused excessive pup mortality. Furthermore,
plasma corticosterone levels in mice were
lower when the cages were changed less
frequently. A cage change every two weeks
with a ventilation rate of 60 air changes per
hour seemed to provide ideal conditions for
mouse health and housing. The same applies
to RH, CO2 level and also to the lower
temperature in rats housed in forced-air
ventilated cages with high ventilation rates,
this study concluded the optimum ventilation
rate to be 60 air changes per h with rats as
well (Hasegawa et al. 1997).
The IVC system is dependent on a
continuous supply of electricity. If the power
supply fails or the sealed cage is detached
from the IVC-rack, CO2 and other gaseous
emissions start to build up. In IVCs without
ventilation, the concentration of CO2 increases
by 2-8 % within two hours depending on the
type of the cage, when mouse body weight per
cage volume in each cage was 20 g/l (Krohn
& Hansen. 2002). The exposure of 3 or 5 %
CO2 has been shown to lower systolic blood
pressure and heart rate of SD male rats (Krohn
et al. 2003c).
The move from traditional open cages to
IVCs causes changes in physical environment
inside the cage. It can be expected that at least
temperature, RH, acoustic environment and
light intensity are altered compared to open
cages in this transition, especially if both
systems are in the same space and room air is
circulated through the IVC-system. Obviously
the climatic conditions in the cage depend on
those of the surrounding room as well as the
air supply source and exhaust of the cage-rack
(Scheer et al. not dated). Clough et al. (1995)
22
noted that the physical environment in IVCs
compared to open cages, displayed higher
temperatures than in the room, higher RH,
lower light intensity and elevated background
sound level when the air fan was in the room.
The ambient temperature has a variety of
effects on core temperature, weight gain,
delivery rate, litter size, food and water intake,
organ weights, haematological values, blood
pressure, heart rate (HR), activity, O2
consumption and CO2 elimination in mice and
rats (Pool & Stephenson 1977, Yamauchi et
al. 1981, Gwosdow & Besch 1985, Swoap et
al. 2004). The elevation in ambient
temperature decreases activity in male Wistar
rats (Pool & Stephenson 1977) and mean
arterial pressure (MAP) and HR in female SD
rats and NIH Swiss mice (Swoap et al. 2004).
At high temperatures, inactivity is the
animals’ first attempt to reduce heat
production and thus to counteract the rising
body temperature.
An ambient temperature above 30 °C
decreases delivery rate, litter size and weaning
rate in Wistar rats (Yamauchi et al. 1981).
Although higher ambient temperatures in the
IVCs have been reported (Clough et al. 1995),
the elevation of 1-4 °C is not necessarily
detrimental to reproduction; as shown by Tsai
et al. (2003) who examined the long-term
reproduction performance between DBA/2
mice housed in open cages and IVCs in the
same room. However, in the IVCs the
coefficients of variation were higher for most
of the measured parameters (e.g. total number
of litters or pups/dam and breeding index).
This suggests that individual mice need more
time to adapt to the IVCs than to the open
cages.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
General introduction
Environmental noise has been shown to
exert effects on cardiovascular function,
hormones, reproduction, sleep and body and
organ weights in laboratory rodents (Rabat
2007, Turner et al. 2007). Chronic intermittent
noise elevates plasma corticosterone and rats
do not seem to habituate to the noise
(Strausbaugh et al. 2003). Animals have
different hearing ability than humans; rodents
e.g. are able to hear ultrasounds (> 20 kHz)
non-audible to humans (Heffner & Heffner
2007). It has been postulated that IVC system
may be associated with these ultrasounds, but
Krohn et al. (2003b) were unable to detect
ultrasounds originating from the ventilation of
the IVC system. Rats have a different hearing
sensitivity than humans and therefore the Rweighting (Björk et al. 2000) should be used
in studies on the acoustic environment. Rats
seem to adapt to repeated sound stimuli
(Voipio 1997), but cage material, working
style and hearing sensitivity all may change
the sound pressure level in the rodent cage
(Voipio et al. 2006). This study also detected
higher sound exposure levels caused by
stainless steel than polycarbonate cages, but
calm and hurried working style made no
difference with either of the materials. When
the results were adjusted for rat and human
hearing capabilities, differences were found in
procedures conducted with both cage
materials and both working styles. As a
common trend, H-weighted (tailored for
human audiogram) sound exposure levels
were about 10 dB higher than those with Rweighting.
The cages in the IVC system have extra
lids and this often leads to dimmer light inside
the cages, although the illumination of the
IVCs has been measured in only a few studies
(Clough et al. 1995). Dark cages are better for
albino rodents because prolonged periods of
bright lighting have been shown to cause
retinal damage in these animals (Gorn &
Kuwabara 1967, Stotzer et al. 1970, Weisse et
al. 1974). The intensity of illumination is
crucial to retinal damage in rats; 8000 lx
evokes photoreceptor damage in a couple of
hours, whereas 194 lx does the same over a
longer time period (Kuwabara & Gorn 1968,
O´Steen et al. 1972). Furthermore, albino and
pigmented rats are different in terms of visual
acuity; pigmented Dark Agouti (inbred) and
Long-Evans (outbred), and wild rats have
grating thresholds around 1.0 cycle/degree
(c/d), whereas in the albino rats Fisher344
(inbred), Sprague-Dawley (SD, outbred) and
Wistar (outbred) the corresponding value is
0.5 c/d. Interestingly, the highest visual acuity
has been found in F1-hybrid of F344 x BN
with grating threshold of 1.5 c/d (Prusky et al.
2002). The study of Birch & Jacobs (1979)
found the same results in acuity in albino and
hooded pigmented rats, but in the hooded rats,
the luminance level had no effect on spatial
acuity.
1.4 Stress and stress indicators in
laboratory rats
Stress is the outcome of external or internal
factors – stressors – which can alter biological
equilibrium (Pekow 2005). Stress induces
changes in animals’ physiology, behaviour
and biochemistry (Moberg & Mench 2000).
Behavioural changes consist of grooming,
appetite,
activity,
aggression,
facial
expression, vocalization, appearance, posture
and response to handling; physiological
changes e.g. temperature, HR and blood
pressure, respiration, weight loss, blood cell
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
23
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
count and cell structure; and biochemical
changes e.g. levels of corticosteroids,
catecholamines, thyroxin, prolactin, betaendorphin, ACTH, glucagon, insulin and
vasopressin (Pekow 2005). There are many
ways with which stress can be assessed e.g.
animals’ behaviour, cardiovascular parameters
and activity, and biochemical assays such as
corticosterone determined from blood or
faecal samples.
Stress may affect animal welfare if
consequent adaptation has biological costs
(Pekow 2005). Stress can be defined in three
ways, according to its impact on animal wellbeing; neutral stress, eustress or distress.
Neutral stress induces adaptive effects that are
not harmful or beneficial for animals, eustress
initiates a response that enhances animal wellbeing, and distress induces a harmful adaptive
response.
During acute stress, the autonomic nervous
system (ANS) is activated and hormones are
released from the brain, peripheral nervous
system and other organs. The sympathetic
nervous system (SNS) evokes release of the
catecholamines, adrenaline and noradrenaline,
from adrenal medulla. The catecholamines
increase HR and blood flow, and release
glucagon from pancreas which improves
glucose availability in blood.
In the brain, hypothalamic pituitary adrenal
(HPA) –axis response to stressors is
manifested by secretion of corticotrophin
releasing factor (CRF). CRF triggers anterior
pituitary to release adrenocorticotropic
hormone (ACTH), which then stimulates
release of glucocorticoids (corticosterone in
rats and mice) from adrenal cortex (Matteri et
al. 2000, Pekow 2005). In rats, CRF has been
shown to play an important role during mild
24
stress situations associated with increases in
blood pressure, HR, body temperature (Tb)
and locomotor activity (LA), but CRF does
not contribute to cardiovascular and body
temperature regulation in normal non-stress
situations (Morimoto et al. 1993). The
adrenals can also be activated during
beneficial activities like mating, but in general
it indicates that the animal is experiencing
some difficulties in trying to cope, and levels
of the adrenal products or the activities of
adrenal enzymes, which are involved in the
synthesis of catecholamine (e.g. adrenaline,
noradrenaline and dopamine), are useful
welfare indicators (Broom 1991).
HPA-axis activation to stressors is
restrained by negative feedback exerted by
glucocorticoids; an interaction with two types
of receptors in the brain: mineralocorticoid
receptors (MRs) and glucocorticoid receptors
(GRs). Since corticosterone binds to MRs
with much greater affinity than to GRs, MRs
are mainly occupied in rats during periods of
inactivity and in the morning hours, whereas
GRs are occupied mainly during the dark
period and after exposure to stress (Armario
2006). Furthermore, van den Buuse et al.
(2002) showed MRs and GRs mediate
differential and opposing actions on
corticosterone in regulating sympathetic
cardiovascular stress responses in rats.
Stress can be also chronic, seen in three
different situations (Armario 2006):
a) continuous exposure to stressors for at
least a few days or weeks
b) repeated exposure, e.g. on daily basis, to
the same stressor for one or several weeks
c) chronic exposure to combination of
different stressors which change in some way
from day to day.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
General introduction
1.4.1 Cardiovascular parameters
In mammals, the SNS plays an important
role in the maintenance of cardiac output to
meet the demands placed on the organism by
increasing both HR and cardiac contractility.
The baroreceptor reflex regulates blood
pressure and HR under normal conditions
(d’Uscio et al. 2000). In freely moving
animals, the cardiac neural drive responsible
for a substantial fraction of spontaneous HR
variability depends on both vagal and
sympathetic activity, while blood pressure
variability reflects only the vagal influence
(Ferrari et al. 1987).
In laboratory rats, blood pressure and HR
increase in stressful situations, e.g. when they
are subjected to even common procedures
(Sharp et al. 2002a, 2002b, 2003a, Azar et al.
2005). Van den Buuse et al. (2001) showed
that novelty stress in rats, e.g. when they are
exposed to an open field situation, caused an
increase in blood pressure and HR, and
concluded that these responses were
attributable to increased SNS activity.
Restricted feeding has been shown to lower
blood pressure (Young 1978, Einhorn et al.
1982, van Ness et al. 1997) and HR (Herlihy
et al. 1992) in normotensive and
spontaneously hypertensive rats (SHR). It
seems that the food restriction lowers the
activity of the SNS thus decreasing blood
pressure (Young 1978, Einhorn et al. 1982,
van Ness et al. 1997). Furthermore, restricted
feeding appears to lower HR in old F344 rats,
which has been suggested to result from
enhanced baroreflex responsiveness (Herlihy
et al. 1992).
1.4.2 Corticosterone and IgA
In rats, activation of the HPA axis
increases the serum corticosterone level in
response to stressful stimulation; this response
is not, however, as rapid as that of ANS.
Corticosterone release has been detected in
serum as early as three minutes after ACTH
injection (Siswanto et al. 2008), but if
exposure to stressors continues for 15 min or
more, the maximum serum corticosterone
level is detected after 30-60 minutes (Armario
2006). Corticosteroid metabolites are excreted
both into urine and faeces, but compared to
serum corticosterone, they have not been
detected in the samples until 6-10 hours later
in urine and 4-12 hours later in faeces after
stressful event (Bamberg et al. 2001, Royo et
al. 2004, Lepschy et al. 2006, Siswanto et al.
2008, Abelson et al. 2009,).
The level of serum corticosterone has been
shown to increase in rats in many stressful
situations, such as after cage cleaning and
obtaining a vaginal smear (Honma et al.
1984), blood sampling (Sabatino et al. 1991,
Haemisch et al. 1999), during immobilization
(Sternberg et al. 1992, Dhabhar et al. 1995,
Sarrieau et al. 1998, Schrijver et al. 2002,
Márquez et al. 2004, Tamashiro et al. 2004),
acoustic startle exposure (Glowa et al. 1992)
and forced swimming test (Sternberg et al.
1992, Armario et al. 1995) and a couple of
hours before a meal when rats are fed
restrictly (Honma et al. 1984, Sabatino et al.
1991, Duclos et al. 2005). Circulating
corticosterone levels have been shown to
increase equally in dominant and subordinate
male rats in response to 1 hour immobilization
(Tamashiro
et
al.
2004).
Plasma
corticosterone secretion in rats seems to
decrease with repeated immobilization and
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
25
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
blood sampling, i.e. evidence of some
adaptation to these procedures (Haemisch et
al. 1999, Márquez et al. 2004).
In rats, the corticosterone excretion has
been estimated to occur 16-80 % via faeces
and 25-80 % via urine, and there are
differences in excretion during the time of the
day and between males and females (Bamberg
et al. 2001, Erikson et al. 2004, Lepschy et al.
2007, Abelson et al. 2009). Assessment of
stress sensitive molecules from faecal samples
has a number of advantages: e.g. animals are
most often not disturbed by sampling.
Laboratory rodents defecate several times a
day (Cavigelli et al. 2005) which enables
monitoring of an individual animal for several
consecutive days or months. Furthermore,
there is no need to handle animals, there is a
delay before corticosteroids appear in the
faecal pellets; all this ensures that the
corticosteroid levels in the samples are not
affected by the sampling procedure (Bamberg
et al. 2001, Möstl & Palme 2002, Cavigelli et
al. 2005).
Prolonged stress may also lead to
immunosuppression; e.g. the levels of
secretory immunoglobulin A (IgA) in saliva
have been used to assess welfare status in
different housing conditions (Guhad & Hau
1996). Another possibility is to quantify the
IgA from faecal samples (Eriksson et al. 2004,
Pihl & Hau 2003, Royo et al, 2004). Royo et
al. (2004) stated that stress-induced changes in
IgA concentrations occur more slowly than
changes in corticosteroids and consequently
faecal IgA may be more useful for assessing
long-term
well-being,
while
faecal
corticosterone is better at monitoring acute
stress events.
The study of Eriksson et al. (2004) showed
26
that the excretion of the corticosterone and
IgA into faeces and urine did vary between
day and night but was rather similar during
the daytime. In the dark phase, the amounts
excreted in the urine increased dramatically
whereas faecal corticosterone excretion
exhibited only a moderate increase. The same
has been shown with faecal IgA (Royo et al.
2004), but some studies have shown higher
faecal corticosterone secretion in the morning
samples compared to the evening samples
(Bamberg et al. 2001, Pihl & Hau 2003, Royo
et al. 2004, Cavigelli et al. 2005, Lepschy et
al. 2007). Furthermore, in female rats, the
corticosterone levels vary with the estrus
cycle; daily faecal corticoid levels were
lowest on the day of estrus and rose
progressively during metestrus, diestrus and
proestrus (Cavigelli et al. 2005). Furthermore,
the basal serum corticosterone levels have
been reported to be higher in the evening
compared to the morning samples (Dhabhar et
al. 1993).
Corticosteroid measurements have been
used in a few studies in rats to assess the value
of furniture items, but the results have been
often conflicting. The study of Belz et al.
(2003) showed that single-housed male and
female SD rats with environmental
enrichment had lower baseline plasma
corticosterone levels than rats in standard
cages, whereas another study with male
Wistar rats detected significantly higher
corticosterone levels in the animals housed in
enriched cages (Moncek et al. 2004).
However, in the latter study, multiple
combinations of various items were used and
the effect of each single item could not be
differentiated. Nevertheless, the combination
did not seem to improve the housing
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
General introduction
environment of the rats in terms of lowering
their corticosterone levels.
The use of cortisol or corticosterone as an
indicator of animal welfare in different
housing methods has been criticized since
cortisol and corticosterone levels are not
always closely related to the mental or
emotional states of animals and since the
measures of a single hormone ignore the
complex physiological reactions of animals to
their environments (Rushen & de Passillé
1992). Furthermore, many studies in farm
animals have also reported conflicting results;
adrenal responsiveness after chronic stress has
increased, decreased or there has been no
change (Rushen 1991).
1.5 Telemetry as a study method in
laboratory animal welfare
The traditional methods to measure blood
pressure in the conscious animals are likely to
cause an increase in the measurable blood
pressure (Irvine et al. 1997). However,
Abernathy et al. (1995) detected no difference
between the tail cuff method and an implanted
transmitter in the values of blood pressure and
HR. The radiotelemetry transmitter allows
measurement of the cardiovascular parameters
in freely moving animals and hence values
obtained are devoid of concomitant handling
or restraint. For this reason, the results
obtained with telemetry are more accurate. In
addition to blood pressure and HR, telemetry
allows measurement of many other
physiological parameters such as ECG, Tb,
and also LA.
Telemetry has become a widely used
method in laboratory animal welfare science
since it has many advantages over the more
conventional techniques. Kramer et al. (2001)
listed four advantages of radiotelemetry:
1. reduction of distress of conscious,
freely moving laboratory animals
2. elimination of stress related to the use
of restrainers
3. reduction of animals used
4. around-the-clock data collection
On the other hand, there are also potential
harms which need to be considered when
using telemetry: surgical implantation,
physical impact of the device on the animal,
and distress if animals are housed individually
(Morton et al. 2003). In particular, the use of
appropriate methods of anaesthesia and
postoperative care with a proper analgesia are
important, hence surgery procedural details
should be described in detail in the scientific
papers conducted with telemetry (Morton et
al. 2003).
Modern
transponders
are
totally
implantable and animals can be group housed,
but at present, only one animal can carry a
transponder in the cage, because in most of
the available devices the signal is transmitted
at only one frequency. However, the devices
with several frequencies are now entering the
market. The report of the Hawkins et al.
(2004) recommends keeping animals group
housed in telemetry studies unless there are
clear contraindications to this choice.
Cardiovascular parameters e.g. HR and
blood pressure increase after handling and
restraint, (Irvine et al. 1997, Kramer et al.
2000, Batūraitė et al. 2005) indicating that
those parameters can be considered as stress
indicators. Indeed telemetry has been used to
assess an animal's response to the handling or
experimental procedures, e.g. a rat’s response
to the IG-gavage or cage changing procedures,
but also to different kinds of housing, such as
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
27
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
flooring, enrichment and timed and restricted
feeding. The articles examined telemetry in
studies related to animal welfare in rats are
listed in Table 1.2.
In undisturbed conditions, blood pressure,
HR and LA of laboratory rats follow a
circadian rhythm; all exhibiting lower values
in the light phase (Saleh & Winget 1977,
Witte et al. 1998, van den Buuse 1994, 1999,
van den Brant 1999). The diurnal rhythm is
controlled by the circadian oscillator, which is
located in the suprachiasmatic nuclei in the
hypothalamus. Induced lesions to the
circadian oscillator have been shown to
interfere with many physiological activities,
e.g. blood pressure, HR and LA (Saleh &
Winget 1977, Janssen et al. 1994, Sano et al.
1995), wake rhythm and sleep (Ibuka &
Kawamura 1975, Sei et al. 1995, Sei et al.
1997) and timed feeding (van den Buuse
1999). Indeed, most of the studies on the
circadian rhythm as related to cardiovascular
parameters and LA have been done with
telemetry.
Telemetry has also been used to assess
blood pressure, HR, LA and body temperature
of various rat strains (van den Buuse 1994,
van den Brant 1999), effect of ambient
temperature (Swoap et al. 2004) and to
compare adult and old rats (Zhang &
Sannajust 2000). Van den Brant (1999)
showed that when the SBP of the wild
“ancestor” rat is compared to inbred rats, the
strains can be divided into two categories:
“hypotensive“
and
“hypertensive”.
Differences between strains have also been
detected in HR and LA (van den Buuse 1994,
van den Brant 1999).
28
1.6 Restricted feeding in rats
The overwhelming majority of laboratory
rodents are fed ad libitum, i.e. food is
available all the time. However, there is
evidence that ad libitum feeding causes
obesity and increases the incidence of
neoplasia, kidney, heart and other organ
diseases in rats (Yu et al. 1982, Roe 1994,
Roe et al. 1995, Lipman et al. 1999, Hubert et
al. 2000). Rats on restricted feeding live
longer; their survival curve shifts to the right
compared to ad libitum fed rats, with the
difference being about one year (Yu et al.
1982, Yu et al. 1985, Hubert et al. 2000).
However,
different
physiological
and
behavioural consequences are associated with
food restriction (Toth & Gardiner 2000).
Keenan et al. (1999) argued that ad libitum
feeding is the worst standardized factor in
rodent bioassays. In long-term studies, the
longevity of the animals is compromized due
to neoplasia and degenerative diseases, and
this interferes with the sensitivity of the study
and necessitates more animals being used.
Masoro (2005) has reviewed the caloric
restriction (CR) studies on aging and
suggested that the obesity and CR are a
consequence of a combination of different
mechanisms. However, it has been proven that
the use of restricted feeding in carcinogenicity
tests can reduce the incidence of tumours in
the control animals, which lowers the number
of animals needed to obtain significant results
(Beynen 1992).
Laboratory rats should be housed in groups
(Council of Europe 2006, EU 2007b), which
is also the preferred method. However, when
animals are group housed, there is no practical
or effective way to restrict the food intake of
all individuals within the group at the same
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
General introduction
Table 1.2 Summary of telemetry studies on refinement and reduction in rats. Abbreviations: MAP =
mean arterial pressure, SBP = systolic blood pressure, DBP = diastolic blood pressure, PP = pulse
pressure, HR = heart rate, LA = locomotor activity, Tb = body temperature.
Study
Strain/stock
Alban et al. 2001
Azar et al. 2005
Azar et al. 2008
Batūraitė et al. 2005
Bonnichsen et al. 2005
Duke et al. 2001
Gärtner et al. 1980
Harkin et al. 2002
Irvine et al. 1997
Krohn et al. 2003a
Lawson et al. 2000
Lemaire & Morméde
1995
Schnecko et al. 1998
Schreuder et al. 2007
Wistar
SHR
SD, SHR
SD, Wistar
SD
SD
SD
SD
WKY, SHR
SD
SHR
Wistar, LongEvans, BHR
SD
Wistar
Sharp et al. 2002a
Sharp et al. 2002b
Sharp et al. 2002c
Sharp et al. 2003a
Sharp et al. 2003b
SD
SD
SD
SD
SD
Sharp et al. 2003c
Sharp et al. 2005a
SD
SD
Sharp et al. 2005b
Sharp et al. 2006
SD
SD
Swoap 2001
Hypertensive
Koletsky rats
SD
Wistar
Van den Buuse 1999
Õkva et al. 2006
Topic
Telemetric parameters
IG-gavage doses
Common procedures
Housing in dim light
Handling and lifting
IG-gavage
Cage change
Handling and procedures
Different stressors
Restraint
Cage flooring
Enrichment
Chronic social stress
BT
MAP, HR
HR
SBP, DBP, MAP, HR, LA
HR
MAP, HR
HR
HR, BT, Activity
SBP, DBP, HR
SBP, DBP, HR, Tb
SBP, DBP, HR, Activity
SBP, DBP, HR, Activity
Common procedures
Working-days vs.
weekend
Common procedures
Common procedures
Witnessing procedures
Common procedures
Common procedures for
“by-stander”
Post-procedure cage size
Adaptation to
manipulation
Enrichment
CO2, Ar and N2 for
euthanasia
Restricted feeding
SBP, DBP, HR, Activity
SBP, DBP, MAP, HR,
Activity
MAP, HR, Activity
MAP, HR
MAP, HR
MAP, HR
HR
Timed feeding
IG-gavage
MAP, HR. Activity
SBP, DBP, HR
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
MAP, HR, Activity
MAP, HR, Activity
SBP, HR, Activity
MAP, HR
MAP, HR, Activity
29
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
time. In the group-housing situation, the
dominant animal eats more than the others
(Beynen 1992, Ritskes-Hoitinga & Chwalibog
2003). In solitary animals arranging restricted
feeding is technically straightforward, but it
may change the diurnal rhythm of the animals.
There are two approaches used in food or
caloric restriction studies in rats (Claassen
1994). In meal feeding rats have access to
food for only a few hours during the day
(Saito et al. 1975, Stephan 1984, Strubbe,
&Alingh Prins 1986, Roe et al. 1995, van den
Buuse 1999). In another approach, a certain
amount of food as a single portion is offered
daily (Vermeulen et al. 1997, Markowska
1999, Hubert et al. 2000). One common
feature of these methods is that rats have to be
housed on their own. Furthermore, with the
single-meal feeding, plasma corticosterone
levels have been shown to increase for a
couple of hours before the meal and decrease
soon thereafter (Honma et al. 1984, Sabatino
et al. 1991). Thus, these kinds of caloric
restriction methods seem to be stressful to
rats.
Restricted feeding has been shown to
decrease blood pressure (Young 1978,
Einhorn et al. 1982, van Ness et al. 1997) and
HR (Herlihy et al. 1992) in rats. It was
concluded that food restriction had reduced
the activity of the SNS causing a hypotensive
effect (Young 1978, Einhorn et al. 1982, van
Ness et al. 1997), enhanced baroreflex
responsiveness as well as decreasing HR in
old F344 rats (Herlihy et al. 1992).
Nocturnal animals, such as rats, forage and
eat mainly during the dark phase. In
laboratory animal facilities, rats eat most of
their daily food (70-95 %) during the dark
phase if the food is available ad libitum
30
(Zucker 1971, Spiteri 1982, Strubbe et al.
1986b, Strubbe & Alingh Prins 1986); in fact
eating during the dark seems to be genetically
determined in rats (Ritskes-Hoitinga &
Strubbe 2004). Normal feeding activity of rats
consists of two peaks during the dark, the first
one at the beginning of the dark phase and the
other at the end (Spiteri 1982, Strubbe et al.
1986a), and it has been shown that when ad
libitum feeding was reinstated after a
restricted feeding schedule, the rats will
instantly revert to their original feeding
pattern (Spiteri 1982, Strubbe et al. 1986b).
When rats are fed once a day or the food
deprivation period has been longer than six
hours, locomotion behaviour (Yu et al. 1985,
Vermeulen et al. 1997) and running wheel
activity (Duclos et al. 2005) increase; both
likely as consequences of food searching
behaviour.
Normal feeding activity of rats follows the
circadian rhythm (Stephan 1984, Strubbe et
al. 1987, Sano et al. 1995, Ritskes-Hoitinga &
Chwalibog 2003). When rats are fed with a
single meal or they have certain amount of
food once a day, this normally happens during
the light phase because of the facility's
working hours. In this situation, animals
consume all of the offered food right away,
which interferes with species-specific eating
patterns
and
compromises
digestive
physiology. In rats, this may lead to major
phase
changes
in
biochemical
and
physiological functions of the digestive
system in dark active animals. For example,
changes have been observed in the levels of
serum insulin and glucose (Strubbe & Alingh
Prins 1986, Strubbe et al. 1987, Rubin et al.
1988), mucosal enzymes of small intestine
(Saito et al. 1975) and bile flow (Ho &
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
General introduction
Drummond 1975). Last, but not least, the
altered eating times have been shown to exert
an impact on blood pressure, heart rate and
behavioural activity in rats (van den Buuse
1999, Curtis et al. 2003).
One way to combine group housing and
restricted feeding is to provide foraging items.
The review of Johnson & Patterson-Kane
(2003) lists the theoretical backgrounds of
foraging items for rats. They state four
reasons for providing foraging items for
laboratory rats:
1. foraging ethology
2. optimal foraging theory
3. contrafreeloading
4. foraging items used in other species
The ethological argument originates from
the fact that in their natural environment, rats
need to search, identify, procure and handle
material in order to acquire food. According
to the optimal foraging theory, in rats there are
other motives in addition to hunger to
encourage foraging, e.g. net energy intake
(Johnson & Patterson-Kane 2003).
Contrafreeloading is a phenomenon where
animals would rather work for food than eat
from a freely available food source. Carder &
Berkowitz (1970) and Neuringer (1969)
reported that even if the rats had free access to
food, they would rather earn their food as long
as the work demand was low. Coburn & Tarte
(1976) found that rats living in an
impoverished environment pressed a lever
more often than rats living in an enriched
environment, and suggested this to result from
the increased possibility for activity. The
review of Inglis et al. (1997) notes that
contrafreeloading can occur in many different
species, not only in rats. Inglis et al. (1997)
also list the factors that have an effect on the
level of contrafreeloading: prior training, level
of food deprivation, required effort, stimulus
change, environmental uncertainty, rearing
conditions, manipulation of the environment
and the nature of the foraging task.
Foraging items have been widely used in
other species and previous studies have
demonstrated the success of foraging items in
improving welfare (Johnson & PattersonKane 2003). Non-device foraging items
provide variety in the food and the location of
the food in the enclosure, e.g. frozen food,
changes in food size, scatter feeding, hiding
the food and live food. With foraging devices,
the animal must manipulate the item in order
to access the food, i.e. food balls and puzzle
feeders (Johnson & Patterson-Kane 2003).
There are also studies intended to develop
foraging items for laboratory rats. In the study
of Johnson et al. (2004) rats had access to
their diet only via a one cm wide slot or
alternatively they had a “foraging device”,
where the pellets were under gravel. With the
slot feeding, rats ate longer but consume less
food surprisingly with no effect on weight.
The rats preferred eating from the “foraging
device”, gained more weight than ad libitum
fed controls eventhough work was required to
access to food.
Cover & Barron (1998) introduced a diet
optimization feeder including seven carousel
wells for every weekday and a semiautomated filling station. This feeder provided
only a certain amount of daily food for rats,
which can evoke meal-feeding problems as
discussed above. A fourth method studied
incorporation of largely indigestible sugar
beet pulp fibre into the chow; weight gain
reduction was achieved, but the method also
resulted in an enlarged gastrointestinal tract -
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
31
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
especially caecum (Eller et al. 2004).
1.7 Cage furniture in laboratory rats
In laboratory rats, various items made of
diverse materials have been used to furnish
animal cages, as can be seen in Table 1.3. In
most of the studies, the cage items have been
called “enrichment”, which is defined in many
ways depending on the perspective of the
definer.
Newberry
(1995)
defined
environmental
enrichment
as
“an
improvement in the biological functioning of
captive animals resulting from modifications
to their environment”. Evidence of improved
biological functioning was defined e.g.
increased lifetime for reproduction, increased
inclusive fitness, or improved health.
Chamove (1989) used behaviour to define
enrichment; the aim of enrichments is to
increase “desirable” behaviour (e.g. foraging)
and reduce “undesirable” behaviour (e.g.
stereotyped behaviour or hair-pulling); i.e.
enrichment allows the animals to exhibit
species specific behaviours. Purves (1997)
stated the enrichment improves the life of
laboratory rodents, and creates less variability
in experimental outcomes, and thus reduces
the number of animal needed.
Newberry (1995) listed problems in the
enrichment studies; the control environment in
different studies varies from wire bottom
cages with one animal to large pens with
several animals, and added objects from single
item to diverse combination of items.
Furthermore, the term “enrichment” means an
improvement, but according to Newberry
(1995), the term is applied to different types
of environmental change (e.g. social, physical,
sensory) rather than the outcome of studies.
Environmental enrichment has been shown
32
to have significant effects on growth and
behaviour of male rats (Zaias et al. 2008), and
enhance habituation of exploratory activity in
response to novelty and improved spatial
learning and memory (Schrijver et al. 2002).
The early study of Cummins et al. (1977)
showed differences in brain development in
Wistar rats that were housed with sensory
enrichment or a deprived environment. Based
on these results they proposed a
developmental model for environmental
enrichment. One essential feature of the model
is that there exists an element of neural
development associated with cells that fully
mature only in response to sensory
stimulation. The neural development is
represented on a percentage scale (y-axis),
where 100 % means that the development
cannot proceed anymore, and the sensory
stimulation is from minimal to optimal scale
(x-axis).
Rats prefer a cage with a shelter
(Townsend 1997, Manser et al. 1998b,
Patterson-Kane et al. 2001, Patterson-Kane et
al. 2003). The reason for this preference may
be that shelters provide protection from light
for rats (Manser et al. 1998a, Eskola et al.
1999). Rats with a furnished environment are
more active than those without a shelter (van
der Harst et al. 2003) and its presence in the
cage decreases fearfulness (Townsend 1997).
The European regulations on laboratory
rodents mandate the provision of sufficient
nest material for nest building; if that is not
possible, a nest box should be provided
(Council of Europe 2006, EU 2007b). Since
rats are poor nest-builders (Jegstrup et al.
2005), they must be provided with cage
furniture for this purpose. Furniture with a
cover and dividing walls in the cage area may
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
General introduction
Table 1.3 Summary of studies on cage items used in rats.
Study
Strain/stock
Belz et al. 2003
Bradshaw &
Poling 1991
SD
SD
Burke et al. 2007
SD
Chmiel &
Long-Evans
Noonan 1996
Eskola et al. 1999 Wistar
Holm & Ladewig
2007
SD
Johnson et al.
2004
Wistar
Lawson et al.
2000
Manser et al.
1998a
Manser et al.
1998b
Moncek et al.
2004
Orok-Edem &
Key 1994
Patterson-Kane et
al. 2001
SHR
Patterson-Kane
2003
SD
SD
Wistar
Lewis
Hooded
Norway
Wistar
Cage items
Kong Toy®, Nestlet®
Paper towels, wood chips,
plastic pipe, wood
platform
Plastic hiding hut,
polyvinyl chloride tube,
plastic climbing platform,
one toy, four wooden
chews
15 items e.g. different
kinds of balls
A block with drilled holes,
rectangular tube
Aspen tube, aspen
gnawing sticks and woodwool nesting material
Modified food hopper,
gnawing sticks, “foraging
device”
PVC drainpipe, golf balls,
running wheel
Nest box, nesting material
Topic/Parameters
Plasma ACTH, corticosterone
Direct observations with the
items
Open-field activity,
electrophysiological and
morphometric outcome
measures with moderate
thoracic spinal core injury
Direct observations with the
items
Behaviour, gnawing volume,
growth of rats
Level pressing activity in rats
in object enriched or nonenriched environment
Behaviour, food consumption,
weight gain, choices in
preference test
SBP, DBP, HR, activity
Time spent with objects in
preference tests
Nest box, nesting material The weight rats lifted to reach
the cage with studied objects
Toys, tunnel, swing,
Adrenal weight, ACTH,
running wheel
corticosterone,
Tongue depressor, hanging Behavioural activities
wooden block
40 different toys and
Choices in preference tests
pieces of equipment e.g.
large marble, wooden
block, chicken wire ball,
shredded paper, tin nest
box
Seven different kind of
Choices in preference tests
shelters
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
33
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
Study
Strain/stock
Cage items
Topic/Parameters
Schrijver et al.
2002
Lister Hooded
Sharp et al. 2005
SD, SHR
Results of behavioural tests
(open field, L/D box, Morris
water maze), plasma ACTH
and corticosterone
SBP, HR, Activity
Townsend 1997
Wistar
Van den Harst et
al. 2003
Wistar
Williams et al.
2008
Lister-Hooded
Zaias et al. 2008
SD
A thick layer of bedding
material, shelves, wooden
branches, hay, a rope,
plastic tunnels, a hut
Simulated burrow,
gnawing and food foraging
items, shredding and
nesting items.
Shelter made of mouse
cage
Shelter, tunnel-shaped
compartment, gnawing
sticks
A shelter, a box shape, a
post and a car – all made
of Lego®
Plastic tunnels and balls,
paper nestlets
be useful to allow the rats to collaborate or
avoid contact with other group members
(Stauffacher 2002). A cage divider can be
used for separating individual animals but also
enabling social interaction at the same time
(Boggiano et al. 2008). The regulations are
the same for all laboratory rodents, i.e. rats,
mice, gerbils, hamsters and guinea pigs.
Nonetheless, all rodent species, and strains
and stocks within a species display differences
(see Table 1.5) and specific needs, which
require
detailed
consideration
and
implementation. Nonetheless, the study of
Sharp et al. 2005 has stated that it is difficult
to make generalized recommendations for the
animal care community with regard to rat
enrichment programs.
The cage item has been made of different
materials, such as plastic, wood and
34
Behaviour
Behaviour
The frequency of first
introduction and time spent
with objects in preference tests
Body weight, food
consumption, activity
cardboard. The items made of wood are
beneficial because then they are usually the
same material as the bedding. Cage items that
are made of organic material have been shown
to emit volatile compounds, like pinenes, but
these emissions can be reduced by autoclaving
prior to use (Nevalainen & Vartiainen 1996).
Bedding made of soft wood (spruce, cedar,
pine) has been reported to increase
hexobarbital oxidase activity and shorten
sleep time in mice (Vesell 1968). The
presence of volatile compounds in bedding
can induce hepatic microsomal enzymes and
hence modify pharmacological effects, e.g.
duration of sleeping time (Ferguson, 1966,
Vesell, 1968, Wade et al., 1968; Sabine, 1975,
Cunliffe-Beamer et al.,1981, Nielsen et al.,
1984, Weichbrod et al., 1988), but also these
compounds can influence some aspects of
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
General introduction
endocytosis (Buddaraju & Van Dyke, 2003).
Aspen tubes as a cage items have been shown
to endure several bouts of sanitation without
increasing microbiological burden (Voipio et
al. 2008). Old cast-off bottles have also been
used as cage items for laboratory rodents and
are considered to have no effect on the
animals. However, bottles are normally made
of polycarbonate, which has been reported to
leach bisphenol A towards end of their useful
life (Howdeshell et al. 2003), which is a
compound with estrogenic activity (Krishnan
et al. 1993) and thus old polycarbonate bottles
are not recommended to be used as cage
items.
1.8 Cage change and IG-gavage
Laboratory rats may be subjected to a variety
of experimental procedures, e.g. IG
(intragastric)-gavage, which may have a
negative effect on animal welfare. Similar
untoward effects can also result from routine
animal care procedures, such as weekly cage
cleaning. This chapter focuses on these two
common procedures in animal facilities, and
summarizes studies published so far (Table
1.4).
In animal facilities, rats are changed to a
clean cage either once or twice per week
depending on cage density, bedding material
and cage type. This routine procedure has
been shown temporarily to increase blood
pressure, HR, activity (Schnecko et al. 1998,
Duke et al. 2001, Sharp et al. 2002a, Sharp et
al. 2002b, Sharp et al. 2003a, Sharp et al.
2003c, Azar el al. 2005) and behaviour
(Saibaba et al. 1996, Burn et al. 2006a, Burn
et al. 2006b). Moreover, it has been shown
that cage cleaning frequency (Burn et al.
2006a, Burn et al. 2006b), the time of the
cleaning (Schnecko et al. 1998), the type of
the bedding material (Burn et al. 2006a), the
light intensity and the length of the dark
period (Azar et al. 2008) all alter the response
intensity. It is the transfer procedure itself that
is reflected in blood pressure and HR after the
cage change rather than the novelty of the
environment.
In their natural environment, rats have
dominance hierarchies and fighting is related
to their territory, not for any specific object
(Barnett 1958). The study of Burn et al.
(2006a) used the term “skirmishing” to
describe the pattern of behaviours, which were
assumed to be aggressive in rats. The
skirmishing frequency increased significantly
within 15 min after a cage change, but
returned to the normal level within next 15
min (Burn et al. 2006a). Since they are
exploratory animals, rats start to investigate
their new surroundings by ambulating and
rearing (Hughes 1968) and indeed cage
change seems to increase the LA in rats (Burn
et al. 2006b, Saibaba et al. 1996, Schnecko et
al. 1998). The behaviour patterns of rats
change in clean cages; grooming, eating,
drinking, resting, rearing and bedding
manipulation all decrease, while walking and
skirmishing frequencies increase immediately
after the rats are placed in the clean cage
(Burn et al. 2006b).
Rats are known to have a good sense of
smell; the number of identified specific
olfactory receptor genes on the cilia of the
olfactory neurons is high - 1493; in dogs the
corresponding number is 1094; therefore
smell is the rats’ primary sense for monitoring
their environment (Quignon et al. 2005). In
the rat cage, furniture can serve as an odour
cue making the new cage environment more
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
35
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
Table 1.4 Summary of studies on cage change or IG-gavage in rats. Abbreviations: MAP = mean
arterial pressure, SBP = systolic blood pressure, DBP = diastolic blood pressure, PP = pulse pressure,
HR = heart rate, Tb = body temperature.
Reference
Strain/stock
Topic
Parameters
Abou-Ismail et al.
2008
Alban et al. 2001
Wistar
Cage changing
Behaviour, organ weights
Wistar
IG-gavage volumes
Azar et al. 2005
Bonnichsen et al.
2005
Brown et al. 2000
SD
SD
Burn et al. 2006a
Wistar and SD
Cage change
Stress duration and
gavage volume
Different vehicles and
dose volume
Cage changing
frequency
Open-field arena behaviour,
BT
MAP, HR
MAP, HR, Tb
Burn et al. 2006b
Wistar and SD
Burn et al. 2008a
Wistar
Burn et al. 2008b
Lister Hooded
Duke et al. 2001
Honma et al. 1984
Saibaba et al. 1996
Schnecko et al.
1998
Sharp et al. 2002a
Sharp et al. 2002b
Sharp et al. 2003a
Sharp et al. 2003c
Õkva et al. 2006
SD
Wistar
SD
SD
Cage changing
frequency
Cage cleaning
frequency
Preference for clean or
soiled cages
Cage cleaning
Cage cleaning
Cage cleaning time
Cage cleaning time
SD
SD
SD
SD
Wistar
Cage change
Cage change
Cage change
Cage change
IG-gavage
SD
familiar if it is transferred to the clean cage
with the animals; the presence of the old item
in the new cage has been shown to reduce the
aggressive behaviour triggered by regrouping
(Burn et al. 2006b).
The HR of the rats appeared to increase
36
Plasma corticosterone, lung
weight
Behaviour, scoring of
chromodacryorrhoea, wounds,
handleability
Behaviour, scoring of
chromodacryorrhoea
Reproduction, pup mortality
Behaviour, ammonia, food
eaten, amount of faecal pellets
MAP, HR
Plasma corticosterone
Behaviour
SBP, DBP, HR, Activity
MAP, HR
MAP, HR
MAP, HR
HR
SBP, DBP, HR
even by moving the cage to a different place
in the rack (Gärtner et al. 1980), and plasma
corticosterone levels increased significantly
15 min after arrival of rats into a clean cage
(Honma et al. 1984). Schnecko et al. (1998)
have studied the impact of a cage changing
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
General introduction
time on HR and blood pressure and they
reported that if the change took place in the
morning - during the resting period - SBP,
DBP and HR responses ware larger than
following the same procedure during the
active period i.e. the evening. Moreover,
Abou-Ismail et al. (2008) showed that if rat
cages were changed in the light period, they
slept less, had more chromodacryorrhea,
displayed a smaller thymus weight, had higher
levels of aggression and exhibited less
furniture-directed behaviour. Consequently,
the authors suggested that if husbandry
procedures were done during the dark rather
than the lights-on, this might improve rat
welfare. However, this kind of timing is rather
impractical.
The studies into IG-gavaging have
demonstrated that the blood pressure and HR
increase seen in rats immediately after the
procedure and the response can be seen for
30-60 min beyond the procedure (Bonnichsen
et al. 2005, Õkva et al. 2006). Moreover, the
plasma corticosterone levels of rats are known
to increase after an IG-gavage (Brown et al.
2000). The choice of the administration
volume (Alban et al. 2001, Bonnichsen et al.
2005, Brown et al. 2000, Õkva et al. 2006)
and a suitable probe material (Õkva et al.
2006) have both been shown to lead to
refinement of the procedure.
The study of Õkva et al. (2006) showed
essentially the same responses in blood
pressure and HR to cage change and IGgavage in Wistar rats. However, the
corticosterone level seems to increase more
when rats are moved to a novel environment
than to short-term handling (Seggie & Brown
1975). Likewise, plasma corticosterone levels
increase after blood sampling (Sabatino et al.
1991, Haemisch et al. 1999, Márquez et al.
2004), taking a vaginal smear (Honma et al.
1984) or when rats are placed into restrainers
(Sternberg et al. 1992, Dhabhar et al. 1993,
Dhabhar et al. 1995, Sarrieau et al. 1998,
Márquez et al. 2004). Cloutier & Newberry
(2008) have tried to use classical conditioning
to evaluate two stressful procedures, handling
and a saline injection, paired with a rewarding
experience. However, the rewards, food treats
or tickling, did not alleviate the stress
response in SD rats.
The IG-gavage is a short-term procedure,
usually considered more invasive than
handling, and one common feature to both
gavage and handling is that the rats are
returned to the familiar home cage. In the cage
change procedure, the animals are relocated
into a new environment with new odours;
hence it is no surprise that the response to
cage change is more intense.
Only a few of studies have dealt with the
effects of cage change and IG-gavage
procedures with added cage items. Sharp et al.
(2005) studied various potentially stressful
procedures, using both SD and SHR rat
strains, and showed that a multifaceted
enrichment program over a week had no effect
on HR and SBP responses when the rats were
placed in a standard rodent restrainer for 60
min. However, when the rats were removed
from the restrainer, a secondary increase in
HR and SBP occurred, which was
significantly lower in enriched rats compared
to their non-enriched counterparts in both
strains. Moreover, SD and SHR rats housed in
an enriched environment displayed lower HR
and SBP responses to many of the studied
procedures, such as removal of a cage mate,
tail vein injection and exposure to the odour
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
37
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
of urine and faeces of stressed male or female
rats.
1.9. Strain differences in rat
It is well known that different strains and
stocks of laboratory rat are not identical or
even very similar e.g. in their physiology,
behaviour and biochemical characteristics.
Examples of reported differences between
strains and stocks can be seen in Table 1.5. At
least some of these differences can be
attributed to their genetic background. Indeed,
some studies claim that these variables could
be used for the quantitative trait loci (QTL)
analysis of inbred strains (Lipman et al. 1999,
Avsaroglu et al. 2007). QTL analysis is a
statistical method for evaluating the alleles
that occur in a locus and the phenotypes
which they control.
Festing et al. (2002) suggested that by
studying various defined genotypes with a
factorial design for evaluating the strains, then
better precision and applicability within the
species could be achieved. Brown Norway
(BN) and Fisher 344 (F344) strains differ in
various aspects of their physiology,
biochemical characteristics and behaviour
(Table 1.6). These two strains are commonly
used in aging studies (Spangler et al. 1994,
Lipman et al. 1996, Lipman et al. 1999,
Sheridan et al. 2007). Sheridan et al. (2007)
stated the BN and F344 rats are useful models
for investigation of the molecular mechanisms
because they have the same kind of
38
interindividual variation in collateral growth
capacity and a similar impact of age on
compensation as in clinical observations.
Both BN and F344 rats develop
spontaneous lesions at older ages. Lipmann et
al. (1999) detected 80 and 58 different types
of lesions in BN and F344 rats, respectively.
The frequency of lesions was highest in
adrenal glands, kidneys, lungs and pancreas in
BN rats and in eyes, heart, lungs and kidneys
in F344 rats. In addition, the F344 rats had
higher incidence for leukaemia.
Van den Brant et al. (1999) studied blood
pressure, HR and activity in six inbred rat
strains, and they categorised F344 as a
“hypertensive” and the BN as a “hypotensive”
rat strain compared to wild rats. The
difference in day-night activity of BN rats was
also lower compared to F344 and van den
Brant and co-workers concluded that the BN
strain no longer possessed the typical rodent
nocturnal activity. However, in a study of nest
building behaviour (Jegstrup et al. 2005) it
was revealed that there was a closer genetic
relationship between BN and wild rats than
was the case with the two other strains studied
(BDIX and LEW). On the other hand, F344
rats have been considered to be ”stress hyperresponsive”
because
of
their
high
corticosterone responses to restraint and in
behavioural studies (Dhabhar et al. 1995).
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
General introduction
Table 1.5 Summary of differences in laboratory rat strains and stocks. Abbreviations: BP = blood
pressure, HR = heart rate, ACTH = adrenocorticotropic hormone, CBG = corticosteroid-binding
globulin, CRF = corticotropin-releasing factor, GR = glucocorticoid receptor, MR = mineralocorticoid
receptor, HPA = hypothalamic-pituitary-adrenal. Strains and stocks: BN = Brown Norway, DA = Dark
Agouti, F344 = Fischer 344, LE = Long Evans, Lew = Lewis, SD = Sprague Dawley, SHR =
Spontaneously Hypertensive Rat, WIST = Wistar, WKY = Wistar-Kyoto, WF = Wistar-Furth.
Study
Armario et al. 1995
Avsaroglu et al. 2007
Bean et al. 2008
Behmoaras et al. 2005
Dhabhar et al. 1993
Dhabhar et al. 1995
Duclos et al. 2005
Glowa et al. 1992
Gómez et al. 1996
Gómez et al. 1998
Irvine et al. 1997
Jegstrup et al. 2005
Lemaire & Mormède 1995
Lipman et al. 1996
Lipman et al. 1999
Marissal-Arvy et al. 1999
Marissal-Arvy &
Mormède 2004
Nemelka et al. 2008
Ohtsuka et al. 1997
Strains/stocks
BN, F344, Lew, SHR,
WKY
ACI, BN, COP, F344,
Lew, SHR, WAG,
WKY
F344, LE
LE, F344, WF, WAG,
BN, Lew, LOU
F344, Lew, SD
Topic
Behavioural and endocrine response to
forced swimming stress
Response to anaesthetics and analgesics
Housing density
Aortic elastin and collagen
Plasma CBG and corticosterone, and adrenal
receptor activation response to stress
F344, Lew, SD
Plasma ACTH, CBG and corticosterone,
and adrenal receptor activation response to
stress
BN, F344, Lew
HPA-axis activity
F344, Lew, SD
Serum corticosterone response to acoustic
stimuli
BN, F344, Lew, SHR, HPA response to chronic stress
WKY
BN, F344, Lew, SHR, Glucocorticoid feedback on HPA-axis
WKY
SHR, WKY
Influence of restraint on BP
BN, BDIX, Lew
Nest-building behaviour
WIST, LE, BHR
BP and HR during chronic social stress
BN, F1(F344 x BN),
Pathologic characterisation
F1(BN x F344)
BN, F344, F1(BN x
Effect of genotype and diet on age-related
F344)
lesions
BN, F344
Corticosteroid receptor efficiency and
regulation
BN, F344
Excretion of electrolytes
F344, LE
BN, F344
Housing density
Response to formaldehyde inhalation
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
39
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
Study
Strains/stocks
Topic
Prusky et al. 2002
DA, F344, F1(F344 x
Visual acuity
BN), LE, SD, WIST,
wild
Ramos et al. 1997
Rex et al. 1996
Rosenberg et al. 1987
Sarrieau & Mormède
1998
Sarrieau et al. 1998
Sharp et al. 2005b
Sheridan et al. 2007
Spangler et al. 1994
Sternberg et al. 1992
Tordoff et al. 2008
van de Weerd et al. 1996
van den Brant et al. 1999
van den Buuse 1994
van der Staay & Blokland
1996
Webb et al. 2003
40
SHR, WKY, BN, WF,
F344, Lew
WIST, Lew, F344,
BN
Lew, BN, F1(Lew x
BN)
BN, F344
WIST, BN, F344,
F1(BN x F344)
SD, SHR
BN, F344, F1(F344 x
BN)
BN, F344, F1(F344 x
BN)
F344, Lew, SD
14 stocks and strains
WIST, BN
BB, BN, LEW, DA,
F344, WKY, wild
SHR, WKY
WIST, BN, F344,
F1(F344 x BN)
F344, Lew, LE, SD,
WIST
Anxiety-related behaviour
Fear-motivated behaviour
Sleep
HPA-axis activity
Neuroendocrine response to stress
HR, BP and activity
Aging and mesenteric collateral growth
Behavioural assessment of aging
Plasma ACTH and corticosterone and
hypothalamic CRF response to behavioural
and restraint stress
17 taste compounds
Preference for flooring types
HR, BP and activity
HR, BP and activity
Behavioural tests
Morphology, sensorimotor and locomotor
abilities
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
General introduction
Table 1.6 Summary of verified differences between BN and F344 rats. Abbreviations: GR =
glucocorticoid receptor, MR = mineralocorticoid receptor, SBP = systolic blood pressure, DBP =
diastolic blood pressure, HR = heart rate.
Study
Topic
Armario et al. 1995
Forced swimming behaviour
Avsaroglu et al. 2007
Response to anaesthetics and
analgesics
Duclos et al. 2005
HPA-axis activity
Gómez et al. 1996
HPA response to chronic
stress
Glucocorticoid feedback on
HPA-axis
Pathologic characterisation
Effect of genotype and diet
on age-related lesions
Corticosteroid receptor
efficiencies and regulation
Gómez et al. 1998
Lipman et al. 1996
Lipman et al. 1999
Marissal-Arvy et al.
1999
Marissal-Arvy &
Mormède 2004
Ohtsuka et al. 1997
Ramos et al. 1997
Rex et al. 1996
Excretion of electrolytes
Response to formaldehyde
inhalation
Multiple test of anxietyrelated behaviours
Fear-motivated behaviour
Sarrieau & Morméde
1998
Activity of HPA-axis
Sarrieau et al. 1998
Genetic factors involved in
neuroendocrine response to
stress
Sheridan et al. 2007
Mesenteric collateral growth
capacity
Main Results/Differences
BN less struggling and more immobile /
F344 higher corticosterone levels
Both strains resistant to medetomidine /
BN hypersensitive to ketamine /
F344s had longer sleep time with all
anaesthetics studied
F344 corticosterone level higher / BN
more active with running wheel
BN higher increase in ACTH after
immobilization
F344s higher MR and GR levels in
hippocampus and pituitary
BN lower pathology and higher longevity
BN had more kidney related lesions /
F344 liver and eye related lesions
F344s higher MR levels in hippocampus
and pituitary /
BNs higher GR levels in hypothalamus
No differences of urinary of Na+ and K+ /
BN higher preference for saline
BN less sensitive to formaldehyde
BN less anxiety-related behaviours
BN more active and explorative in the
open field
BN adrenals larger / F344 corticosterone
higher / Plasma transcortin level twice that
of F344 / Higher density of MR in F344
BN adrenals larger / F344
corticosterone level higher /
BN ACTH higher / F344
prolactin and renin activity higher
BN collateral growth at a younger age
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
41
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
Study
Topic
Main Results/Differences
Spangler et al. 1994
Behavioural assessment of
aging
Tordoff et al. 2008
Preference for taste
compounds
Blood pressure, HR and LA
F344 more active / F344 worse muscular
strength and coordination / F344 worse
learning / F344 higher number of lesions
(at two years of age)
F344 preferred tasty compounds
Van den Brant et al.
1999
Van der Staay &
Blokland 1996
Behavioural differences
1.10 Aims of the present study
The general aim of this study was to
identify refinement and reduction methods for
rat housing and two commodity procedures.
The specific aims were:
1. To characterize and compare physical
parameters in a common situation, where
IVC-racks are kept in the same room with
open cages (Chapter II).
2. To assess whether a novel system of food
restriction, based on the work for food
principle, would have any effect on weight
gain over a short period, food utilisation and
amount of wood gnawed in adult rats and
whether their time budget would differ from
ad libitum fed rats (Chapter III).
42
F344 higher SBP, DBP, HR and night time
motor activity
F344 less explorative both in the open
field and light-dark box / BN better spatial
discrimination performance /
F344 better shock sensitivity
3. To determine whether isolating walls, cage
tube or restricted feeding change responses to
routine cage change or IG-gavage in both
open and IVCs (Chapter IV).
4. To evaluate the impact of large cage
furniture items on LA, cardiovascular
parameters, and on faecal excretion of
corticosteroid metabolites and IgA, to
determine how applicable the results are, and
two different rat strains would display
variation in their responses, and whether there
would be habituation to the items (Chapters V
and VI).
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
General introduction
1.11 References
Abelson KSP, Fard SS, Nyman J, Goldkuhl R, Hau J.
2009. Distribution of [H-3]-coricosterone in urine,
feces and blood of male Sprague-Dawley rats after tail
vain and jugular vein injections. In Vivo 29:381-386.
Abernathy FW, Flemming CD, Sonntag WB. 1995.
Measurement of cardiovascular response in male
Sprague-Dawley rats using radiotelemetric implants
and tailcuff sphygmomanometry: A comparative study.
Toxicology Methods 5:89-98.
Abou-Ismail UA, Burman OHP, Nicol CJ, Mendl M. 2008.
Let sleeping rat lie: Does the timing of husbandry
procedures affect laboratory rat behaviour, physiology
and welfare? Applied Animal Behaviour Science
111:329-341.
Alban L, Dahl PJ, Hansen AK, Hejgaard KC, Jensen AL,
Kragh M, Thomsen P, Steensgaard P. 2001. The
welfare impact of increased gavaging doses in rats.
Animal Welfare 10:303-314.
Armario A. 2006. The hypothalamic-pituitary-adrenal axis:
What can it tell us about stressors? CNS &
Neurological Disorders – Drug targets 5:485-501.
Armario A, Gavaldá A, Martí J. 1995. Comparison of the
behavioural and endocrine response to forced
swimming stress in five inbred strains of rats.
Psychoneuroendocrinology 20:879-890.
Avsaroglu H, van der Sar AS, van Lith HA, van Zutphen
LFM, Hellebrekers LJ. 2007. Differences in response to
anaesthetics and analgesics between inbred rat strains.
Laboratory Animals 41:337-344.
Azar T, Sharp J, Lawson D. 2005. Stress-like
cardiovascular responses to common procedures in
male versus female spontaneously hypertensive rats.
Contemporary Topics in Laboratory Animal Science
44:25-30.
Azar TA, Sharp JL, Lawson DM. 2008. Effect of housing
rats in dim light or long nights on heart rate. Journal of
the American Association for Laboratory Animal
Science 47:25-34.
Bamberg E, Palme R, Meingassner JG. 2001. Excretion of
corticosteroid metabolites in urine and faeces of rats.
Laboratory Animals 35:307-314.
Barnett SA. 1958. An analysis of social behaviour in wild
rats. Proceedings of the Zoological Society of London
130:107-152.
Batūraitė Z, Voipio HM, Rukšenas O, Luodonpää M,
Leskinen H, Apanavičiene N, Nevalainen T. 2005.
Comparison of and habituation to four common
methods of handling and lifting of rats with
cardiovascular telemetry. Scandinavian Journal of
Laboratory Animal Science 32:137-148.
Baumans V, Schlingmann F, Vonck M, van Lith HA.
2002. Individually ventilated cage: Beneficial for mice
and men? Contemporary Topics in Laboratory Animal
Science 41:13-19.
Bean K, Nemelka K, Canchola P, Hacker S, Sturdivant
RX, Rico PJ. 2008. Effect of housing density on Long
Evans and Fischer 344 rats. Lab Animal Europe 8:3745.
Behmoaras J, Osborne-Pellegrin M, Gauguier D, Jacob MP. 2005. Characteristics of the aortic elastic network
and related phenotypes in seven inbred rat strain.
American Journal of Physiology: Heart and Circulatory
Physiology 288:H769-H777.
Belz EE, Kennell JS, Czambel RK, Rubin RT, Rhodes ME.
2003. Environmental enrichment lowers stressresponsive hormones in singly housed male and female
rats. Pharmacology, Biochemistry, and Behavior
76:481-486.
Beynen AC. 1992. Ad libitum versus restricted feeding:
Pros and cons. Scandinavian Journal of Laboratory
Animal Science 19:19-22.
Birch D, Jacobs GH. 1979. Spatial contrast sensitivity in
albino and pigmented rats. Vision Research 19:933937.
Björk E, Nevalainen T, Hakumäki M, Voipio HM. 2000.
R-weighting provides better estimation for rat hearing
sensitivity. Laboratory Animals 34:136-44.
Boggiano MM, Cavigelli SA, Dorsey JR, Kelley CEP,
Ragan CM, Chandler-Laney PC. 2008. Effect of cage
divider permitting social stimuli on stress and food
intake in rats. Physiology & Behavior 95:222-228.
Bonnichsen M, Dragsted N, Hansen AK. 2005. The
welfare impact of gavaging laboratory rats. Animal
Welfare 14:223-227.
Bradshaw AL, Poling A. 1991. Choice by rats for enriched
versus standard home cages: Plastic pipes, wood
platforms, wood chips, and paper towels as enrichment
items. Journal of the Experimental Analysis of
Behavior 55:245-250.
Brandstetter H, Scheer M, Heinekamp C, Gippner-Stepper
C, Loge O, Ruprecht L, Thull B, Wagner R, Wilhem P,
Scheuber HP. 2005. Performance evaluation of IVC
system. Laboratory Animals 39:40-44.
Brielmeier M, Mahabir E, Needham JR, Lengger C,
Wilhelm P, Schmidt J. 2006. Microbiological
monitoring of laboratory mice and biocontainment in
individually ventilated cages: A field study. Laboratory
Animals 40:247-260.
Broom DM. 1986. Indicators of poor welfare. The British
Veterinary Journal 142:524-526.
Broom DM. 1988. The scientific assessment of animal
welfare. Applied Animal Behaviour Science 20:5-19.
Broom DM. 1991. Animal welfare: Concepts and
measurement. Journal of Animal Science 69:41674175.
Broom DM, Johnson KG. 1993. Stress and Animal
Welfare. Chapman & Hall, London. 211 pp.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
43
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
Brown AP, Dinger N, Levine BS. 2000. Stress produced by
gavage administration in the rat. Contemporary Topics
in Laboratory Animal Science 39:17-21.
Buddaraju AKV, Van Dyke RW. 2003. Effect of animal
bedding on rat liver endosome acidification.
Comparative Medicine, 53:616-621.
Burke DA, Magnuson DSK, Nunn CD, Fentress KG,
Wilson ML, Shum-Siu AH, Moore MC, Turner LE,
King WW, Onifer SM. 2007. Use of environmentally
enriched housing for rats with spinal cord injury: The
need for standardization. Journal of the American
Association for Laboratory Animal Science 46:34-41.
Burn CC, Peters A, Day MJ, Mason GJ. 2006a. Long-term
effects of cage-cleaning frequency and bedding type on
laboratory rat health, welfare, and handleability: A
cross laboratory study. Laboratory Animals 40:353370.
Burn CC, Peters A, Mason GJ. 2006b. Acute effects of
cage cleaning at different frequencies on laboratory rat
behaviour and welfare. Animal Welfare 15:161-171.
Burn CC, Mason GJ. 2008a. Effect of cage-cleaning
frequency on laboratory rat reproduction, cannibalism,
and welfare. Applied Animal Behaviour Science 114:
235-247.
Burn CC, Mason GJ. 2008b. Rats seem indifferent between
their own scent-marked homecages and clean cages.
Applied Animal Behaviour Science 115:201-210.
Carder B, Berkowitz K. 1970. Rats' preference for earned
in
comparison
with
free
food.
Science 167:1273-1274.
Cavigelli SA, Monfort SL, Whitney TK, Mechref YS,
Novotny M, McClintock MK. 2005. Frequent serial
fecal corticoid measures from rats reflect circadian and
ovarian
corticosterone
rhythms.
Journal
of
Endocrinology 184:153-163.
Chamove AS. 1989. Environmental enrichment: A review.
Animal Technology 40:155-178.
Chmiel DJ, Noonan M. 1996. Preference of laboratory rats
for potentially enriching stimulus objects. Laboratory
Animals 30:97-101.
Claassen V. (ed.) 1994. Food restriction. In: Neglected
Factors in Pharmacology and Neuroscience Research:
Biopharmaceutics,
Animal
Characteristics,
Maintenance, Testing Conditions. Techniques in the
Behavioral and Neural Sciences 12:321-342. Elsevier.
Clark JH, Rager DR, Calpin JP. 1997a. Animal well being
I. General Considerations. Laboratory Animal Science
47:564-570.
Clark JH, Rager DR, Calpin JP. 1997b. Animal well being
II. An overview of assessment. Laboratory Animal
Science 47:580-585.
Clough G, Wallace J, Gamble MR, Merryweather ER,
Bailey E. 1995. A positive, individually ventilated
caging system: A local barrier system to protect both
animals and personnel. Laboratory Animals 29:139-51.
44
Cloutier S, Newberry RC. 2008. Use of a conditioning
technique to reduce stress associated with repeated
intra-peritoneal injections in laboratory rats. Applied
Animal Behaviour Science 112:158-173.
Coburn JF, Tarte RD. 1976. The effect of rearing
environment on the contrafreeloading phenomenon in
rats. Journal of the Experimental Analysis of Behavior
26:289-294.
Compton SR, Homberger FR, Paturzo FX, MacArthur
Clark J. 2004. Efficacy of three microbiological
monitoring methods in a ventilated cage rack.
Comparative Medicine 54:382-392.
Council of Europe. 1986. [Internet] European Convention
for the Protection of Vertebrate Animals used for
Experimental and Other Scientific Purposes.
http://conventions.coe.int/Treaty/en/Treaties/Html/123.
htm
Council of Europe. 2006. [Internet] Appendix A of the
European Convention for the Protection of Vertebrate
Animals used for Experimental and Other Scientific
Purposes
(ETS
No.
123).
Guidelines
for
accommodation and care of animals. Strasbourg.
http://conventions.coe.int/Treaty/EN/Treaties/Html/123
-A.htm
Cover CE, Barron MJ. 1998. A new feeder for diet
optimization in rats. Contemporary Topics in
Laboratory Animal Science 37:84-86.
Cruden J. 2007. A comparison of various IVC systems:
The comfort of the mouse. Animal Technology and
Welfare 6:93-115.
Cummins RA, Livesey PJ, Evans JGM. 1977. A
developmental theory of environmental enrichment.
Science 197:692-694.
Cunliffe-Beamer TL, Freeman LC, Myers DD. 1981.
Barbiturate sleeptime in mice exposed to autoclaved or
unautoclaved wood beddings. Laboratory Animal
Science 31:672-675.
Curtis KS, Krause EG, Contreras RJ. 2003. Cardiovascular
function and circadian patterns in rats after area
postrema lesions or prolonged food restriction.
Neuroscience Letters 350:46-50.
Declaration of Bologna. 1999. Reduction, Refinement and
Replacement Alternatives and Laboratory Animal
Procedures. Adopted by the 3rd World Congress on
Alternatives and Animal Use in the Life Sciences, in
Bologna, Italy on 31 August 1999.
Dalhamn T. 1956. Mucous flow and ciliary activity in the
trachea of healthy rats and rats exposed to respiratory
irritant gases (SO2, H3N, HCHO): A functional and
morphologic (light microscopic and electron
microscopic) study with special reference to technique.
Acta Physiologica Scandinavica 36 (Suppl. 123):1-161.
Dhabhar FS, McEwen BS, Spencer RL. 1993. Stress
response, adrenal steroid receptor levels and
corticosteroid-binging globulin levels – A comparison
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
General introduction
between Sprague-Dawley, Fischer 344 and Lewis rats.
Brain Research 616:89-98.
Dhabhar FS, Miller AH, McEwen BS, Spencer RL.1995.
Differential activation of adrenal stereoid receptors in
neural and immune tissues of Sprague Dawley, Fischer
344 and Lewis rats. Journal of Neuroimmunology
56:77-90.
Dirnagl U. 2006. Bench to bedside: The quest for quality in
experimental stroke research. Journal of Cerebral Blood
Flow and Metabolism 26:1465-1478.
Duclos M, Bouchet M, Vettier A, Richard D. 2005.
Genetic differences in hypothalamic-pituitary-adrenal
axis activity and food restriction-induced hyperactivity
in three inbred strains of rats. Journal of
Neuroendocrinology 17:740-752.
Duke JL, Zammit TG, Lawson DM. 2001. The effects of
routine cage-changing on cardiovascular and
behavioural parameters in male Sprague-Dawley rats.
Contemporary Topics in Laboratory Animal Science
40:17-20.
Einhorn D, Young JB, Landsberg L. 1982. Hypotensive
effect of fasting: Possible involvement of the
sympathetic nervous system and endogenous opiates.
Science 217:727-729.
Eller E, Lawson J, Ritskes-Hoitinga M. 2004. The potential
of dietary fiber to achieve dietary restriction. Abstract.
9th Felasa Symposium, Nantes, France
Eriksson E, Royo F, Lyberg K, Carlsson HE, Hau J. 2004.
Effect of metabolic cage housing on immunoglobulin A
and corticosterone excretion in faeces and urine of
young male rats. Experimental Physiology 89:427-433.
Eskola S, Lauhikari M, Voipio HM, Nevalainen T. 1999.
The use of aspen blocks and tubes to enrich the cage
environment of laboratory rats. Scandinavian Journal of
Laboratory Animal Science 26:1-10.
European Commission. 2005. [Internet] Report on the
Statistics on the Number of Animals used for
Experimental and other Scientific Purposes in the
Member States of the European Union in the year 2002.
http://ec.europa.eu/environment/chemicals/lab_animals/
pdf/sec_2005_0045_1.pdf
European Commission. 2006. [Internet] Communication
from the Commission to the European Parliament and
the Council on a Community Action Plan on the
Protection and Welfare of Animals 2006-2010.
http://ec.europa.eu/food/animal/welfare/actionplan/acti
onplan_en.htm
European Commission. 2007a. [Internet] Ethical rules in
FP7. Ethical rules in the EU's Seventh Framework
Programme
for
Research.
http://ec.europa.eu/research/sciencesociety/index.cfm?fuseaction=public.topic&id=129&la
ng=1&CFID=22112&CFTOKEN=47271601
European Commission. 2007b. [Internet] Fifth Report on
the Statistics on the Number of Animals used for
Experimental and other Scientific Purposes in the
Member States of the European Union. http://eurlex.europa.eu/LexUriServ/LexUriServ.do?uri=COM:20
07:0675:FIN:EN:PDF
European Commission. 2008. [Internet] Proposal for a
Directive of the European Parliament and of the
Council on the protection of animals used for scientific
purposes
COM(2008)
543/5.
http://ec.europa.eu/environment/chemicals/lab_animals/
pdf/com_2008_543.pdf
European Union. 1997. [Internet] Treaty of Amsterdam
amending the treaty on European Union, the treaties
establishing the European Communities and related
acts.
http://eurlex.europa.eu/en/treaties/dat/11997D/htm/11997D.html
European Union. 2007a. [Internet] Treaty of Lisbon
amending the Treaty on European Union and the Treaty
establishing the European Community. http://eurlex.europa.eu/JOHtml.do?uri=OJ:C:2007:306:SOM:EN
:HTML
European Union. 2007b. [Internet] Commission
Recommendation on guidelines for the accommodation
and care of animals used for experimental and other
scientific purposes (2007/526/EC). Brussels. http://eurlex.europa.eu/JOHtml.do?uri=OJ:L:2007:197:SOM:EN
:HTML
Ferguson HC. 1966. Effects of red cedar chip bedding on
hexobarbital and pentobarbital sleep time. Journal of
Pharmaceutical Sciences 10:1142-1143.
Ferrari AU, Daffonchio A, Albergati F. 1987. Inverse
relationship between heart rate and blood pressure
variabilities in rats. Hypertension 10:533-537.
Festing M, Overend P, Gaines Das R, Borja MC, Berdoy
M. 2002. The design of animal experiments. Laboratory
Animal Handbooks No. 14. The Royal Society of
Medicine Press Limited, London.
Festing, M. 2004. Is the use of animals in biomedical
research still necessary in 2002? Unfortunately, "yes".
ATLA 32:733-739.
Fraser D, Weary DM, Pajor EA, Milligan BN. 1997. A
Scientific conception of animal welfare that reflects
ethical concerns. Animal Welfare 6:187-205.
Glowa JR, Geyer MA, Gold PW, Sternberg EM. 1992.
Differential startle amplitude and corticosterone
response in rats. Neuroendocrinology 56:719-723.
Gómez F, Lahmame A, De Kloet ER, Armario A. 1996.
Hypothalamic-Pituitary-Adrenal response to chronic
stress in five inbred rat strains: Differential responses
are mainly located at the adrenocortical level.
Neuroendocrinology 63:327-337.
Gómez F, De Kloet ER, Armario A. 1998. Glucocorticoid
negative feedback on the HPA axis in five inbred rat
strains. American Journal of Physiology – Regulatory,
Integrative and Comparative Physiology 274:420-427.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
45
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
Gordon S, Fisher SW, Raymond RH. 2001. Elimination of
mouse allergens in the working environment:
Assessment of individually ventilated cage systems and
ventilated cabinets in containment mouse allergens.
Journal of Allergy and Clinical Immunology 108:288294.
Gorn RA, Kuwabara T. 1967. Retinal damage by visible
light: A physiologic study. Archives of Ophthalmology
77:115-118.
Guhad FA, Hau J. 1996. Salivary IgA as a marker of social
stress in rats. Neuroscience Letters 216:137-140.
Gwosdow AR, Besch EL. 1985. Effect of thermal history
on the rat’s response to varying environmental
temperature. Journal of Applied Physiology 59:413419.
Gärtner K, Büttner D, Döhler K, Friedel R, Lindena J,
Trautschold I. 1980. Stress response of rats to handling
and experimental procedures. Laboratory Animals
14:267-274.
Haemisch A, Guerra G, Furkert J. 1999. Adaptation of
corticosterone – but not β-endorphin – secretion to
repeated blood sampling in rats. Laboratory Animals
33:185-191.
Harkin A, Connor TJ, O’Donnell JM, Kelly JP. 2002.
Physiological and behavioral responses to stress; what
does rat find stressful? Lab Animal Europe 2:32-40.
Hasegawa M, Kurabayashi Y, Ishii T, Yoshida K,
Uebayashi N, Sato N, Kurosawa T. 1997. Intra-cage air
change rate on forced-air-ventilated micro-isolation
system – environment within cages: Carbon dioxide
and oxygen concentration. Experimental Animals
46:251-257.
Hawkins P, Morton DB, Bevan R, Heath K, Kirkwood J,
Pearce P, Scott L, Whelan G, Webb A. 2004.
Husbandry refinements for rats, mice, dogs and nonhuman primates used in telemetry procedures.
Laboratory Animals 38:1-10.
Heffner HE, Heffner RS. 2007. Hearing ranges of
laboratory animals. Journal of the American
Association for Laboratory Animal Science 46:20-22.
Herlihy JT, Stacy C, Bertrand HA. 1992. Long-term
calorie restriction enhances baroreflex responsiveness
in Fischer 344 rats. American Journal of Physiology:
Heart and Circulatory Physiology 263:H1021-H1025.
Ho KJ, Drummond JL. 1975. Circadian rhythm of biliary
excretion and its control mechanisms in rats with
chronic biliary drainage. American Journal of
Physiology 229:1427-1437.
Holm L, Ladewig J. 2007. The effect of housing in a
stimulus rich versus stimulus poor environment on
preference measured by sigmoid double demand
curves. Applied Animal Behaviour Science 107:342354.
Honma KI, Honma S, Hiroshige T. 1984. Feedingassociate corticosterone peak in rats under various
46
feeding cycles. American Journal of Physiology:
Regulatory, Integrative and Comparative Physiology
246:R721-R726.
Howdeshell KL, Peterman PH, Judy BM, Taylor JA,
Orazio CE, Ruhlen RL, vom Saal FS, Welshons WV.
2003. Bisphenol A is released from used polycarbonate
animal cages into water at room temperature.
Environmental Health Perspectives 111:1180-1187.
Hubert MF, P Laroque, JP Gillet, KP Keenan. 2000. The
effects of diet, ad libitum feeding, and moderate and
severe dietary restriction on body weight, survival,
clinical pathology parameters, and cause of death in
control Sprague-Dawley rats. Toxicological Sciences
58:195-207.
Hughes RN. 1968. Behaviour of male and female rats with
free choice of two environments differing in novelty.
Animal Behaviour 16:92-96.
Höglund AU, Renström A. 2001. Evaluation of
individually ventilated cage systems for laboratory
rodents: Cage environment and animal health aspects.
Laboratory Animals 35:51-57.
Ibuka N, Kawamura H. 1975. Loss of circadian rhythm in
sleep-wakefulness cycle in the rat by suprachiasmatic
nucleus lesions. Brain Research 96:76-81.
Inglis IR, Forkman B, Lazarus J. 1997. Free food or earned
food? A review and fuzzy model of contrafreeloading.
Animal Behaviour 53:1171-1191.
Irvine RJ, White J, Chan R 1997: The influence of restraint
on blood pressure in the rat. Journal of Pharmacological
and Toxicological Methods 38: 157 - 162.
Janssen BJA, Tyssen CM, Duindam H, Rietveld WJ. 1994.
Suprachiasmatic lesions eliminate 24-h blood pressure
variability in rats. Physiology & Behavior 55:307-311.
Jegstrup IM, Vestergaard R, Vach W, Ritskes-Hoitinga M.
2005. Nest-building behaviour in male rats from three
inbred strains: BN/HsdCpb, BDIX/OrlIco and
LEW/Mol. Animal Welfare 14:149-156.
Johnson SR, Patterson-Kane EG. 2003. Foraging as
environmental enrichment for laboratory rats: A
theoretical review. Animal Technology and Welfare
2:13-22.
Johnson SR, Patterson-Kane EG, Niel L. 2004. Foraging
enrichment for laboratory rats. Animal Welfare 13:305312.
Keenan KP, Ballam GC, Soper KA, Laroque P, Coleman
JB, Dixit R. 1999. Diet, caloric restriction and the
rodent bioassay. Toxicological Sciences 52(Suppl):2434.
Keller GL, Mattingly SF, Knapke Jr FB. 1983. A forced-air
individually ventilated caging system for rodents.
Laboratory Animal Science 33:580-582.
Kramer K, Voss HP, Grimbergen JA, Mills PA, Huetteman
D, Zwiers L, Brockway B. 2000. Telemetric monitoring
of blood pressure in freely moving mice: A preliminary
study. Laboratory Animals 34:272-280.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
General introduction
Kramer K, Kinter L, Brockway B, Voss HP, Remie R, van
Zutphen BLM. 2001. The use of telemetry in small
laboratory animals: Recent advances. Contemporary
Topics in Laboratory Animal Science 40:8-16.
Krishnan AV, Stathis P, Permuth SF, Tokes L, Feldman D.
1993. Bisphenol-A: An estrogenic substance is released
from polycarbonate flasks during autoclaving.
Endocrinology 132:2277-2278.
Krohn TC, Hansen AK. 2002. Carbon dioxide
concentrations in unventilated IVC cages. Laboratory
Animals 36:209-212.
Krohn TC, Hansen AK, Dragsted N. 2003a. Telemetry as a
method for measuring the impact of housing conditions
on rats’ welfare. Animal Welfare 12:53-62.
Krohn TC, Hansen AK, Dragsted N. 2003b. The impact of
cage ventilation on rats housed in IVC system.
Laboratory Animals 37:85-93.
Krohn TC, Hansen AK, Dragsted N. 2003c. The impact of
low levels of carbon dioxide on rats. Laboratory
Animals 37:94-99.
Kuwabara T, Gorn RA. 1968. Retinal damage by visible
light; an electron microscopic study. Archives of
Ophthalmology 79:69-78.
Lawson DM, Churchill M, Churchill PC. 2000. The effects
of housing enrichment on cardiovascular parameters in
spontaneously hypertensive rats. Contemporary Topics
in Laboratory Animal Science 39:9-13.
Lemaire V, Mormède P. 1995. Telemetered recording of
blood pressure and heart rate in different strains of rats
during chronic social stress. Physiology & Behavior
58:1181-1188.
Lepschy M, Touma C, Hruby R, Palme R. 2007. Noninvasive measurement of adrenocortical activity in male
and female rats. Laboratory Animals 41:372-387.
Lipman NS, Corning BF, Coiro MA. 1992. The effects of
intracage ventilation on microenvironmental conditions
in filter-top cages. Laboratory Animals 26:206-210.
Lipman NS. 1999. Isolator rodent caging systems (state of
the art): A critical view. Contemporary Topics in
Laboratory Animal Science 38:9-17.
Lipman RD, Chrisp CE, Hazzard DG, Bronson RT. 1996.
Pathologic characterization of Brown Norway, Brown
Norway x Fischer 344, and Fischer 344 x Brown
Norway rats with relation to age. Journal of
Gerontology 51:B54-B59.
Lipman RD, Dallal GE, Bronson RT. 1999. Effects of
genotype and diet on age-related lesions in ad libitum
fed and calorie-restricted F344, BN and BNF3F1 rats.
Journal of Gerontology: Biological Sciences
54A:B478-B491.
Manser CE, Broom DM, Overend P, Morris TH. 1998a.
Investigations into the preferences of laboratory rats for
nest-boxes and nesting materials. Laboratory Animals
32:23-35.
Manser CE, Broom DM, Overend P, Morris TH. 1998b.
Operant studies to determine the strength of preference
in laboratory rats for nest-boxes and nesting materials.
Laboratory Animals 32:36-41.
Marissal–Arvy N, Mormède P, Sarrieau A. 1999. Strain
differences in corticosteroid receptor efficiencies and
regulation in Brown Norway and Fischer 344 rats.
Journal of Neuroendocrinology 11:267-273.
Marissal–Arvy N, Mormède P. 2004. Excretion of
electrolytes in Brown Norway and Fischer 344 rats:
Effects of adrenalectomy and mineralocorticoid and
glucocorticoid
receptor
ligands.
Experimental
Physiology 89:753-765.
Markowska AL. 1999. Life-long diet restriction failed to
retard cognitive aging in Fisher-344 rats. Neurobiology
of Aging 20:177-189.
Márquez C, Nadal R, Armario A. 2004. The hypothalamicpituitary-adrenal and glucose responses to daily
repeated immobilisation stress in rats: individual
differences. Neuroscience 123:601-612.
Masoro EJ. 2005. Overview of caloric restriction an
ageing. Mechanisms of Aging and Development
126:913-922.
Matteri RL, Carroll JA, Dyer CJ. 2000. Neuroendocrine
responses to stress. In: Moberg GP, Mench JA (ed.)
The Biology of Animal Stress – Basic Principles and
Implications for Animal Welfare. CABI Publishing,
UK, pp 43-50.
Moberg GP, Mench JA. 2000. The Biology of Animal
Stress – Basic Principles and Implications for Animal
Welfare. CABI Publishing, UK, pp 43-50.
Moncek F, Duncko R, Johansson BB, Jezova D. 2004.
Effect of environmental enrichment on stress related
systems in rats. Journal of Neuroendocrinology 16:423431.
Morimoto A, Nakamori T, Morimoto K, Tan N, Murakami
N. 1993. The central role of corticotrophin-releasing
factor (CRF-41) in physiological stress in rats. Journal
of Physiology 460:221-229.
Morton DB, Hawkins P, Bevan R, Heath K, Kirkwood J,
Pearce P, Scott L, Whelan G, Webb A. 2003.
Refinements in telemetry procedures. Laboratory
Animals 37:261-299.
Myers DD, Smith E, Schweitzer I, Stockwell JD, Paigen
BJ, Bates R, Palmer J, Smith AL. 2003. Assessing the
risk of transmission of three infectious agents among
mice housed in a negatively pressurized caging system.
Contemporary Topics in Laboratory Animal Science
42:16-21.
Möstl E, Palme R. 2002. Hormones as indicators of stress.
Domestic Animal Endocrinology 23:67-74.
Nemelka KW, Bean K, Sturdivant R, Hacker SO, Rico PJ.
2008. Effect of high density housing on behavioral and
physiologic parameters in F344 and Long Evans rats
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
47
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
(Rattus norvegicus). Online Journal of Veterinary
Research 12:28-40.
Neuringer AJ. 1969. Animals respond for food in the
presence of free food. Science 166:399-401.
Nevalainen T, Vartiainen T. 1996. Volatile organic
compounds in commonly used beddings before and
after autoclaving. Scandinavian Journal of Laboratory
Animal Science 23:101-104.
Nevalainen T. 2004. Training for Reduction in Laboratory
Animal Use. ATLA 32 (Supplement 2):55-58.
Newberry RC. 1995. Environmental enrichment;
increasing the biological relevance of captive
environments. Applied Animal Behaviour Science
44:229-243.
Nielsen JB, Andersen O, Svendsen P. 1984. Effect strøelse
på leverens Cytochrom P-450 system i mus. ScansLAS
nyt (Scandinavian Journal of Laboratory Animal
Science) 11:7-13.
Ohtsuka R, Shuto Y, Fujie H, Takeda M, Harada T, Itagaki
S. 1997. Response of respiratory epithelium of BN and
F344 rats to formaldehyde inhalation. Experimental
Animals 46:279-286.
Olsson IAS, Hansen AK, Sandøe P. 2007. Ethics and
refinement in animal research. Science 317:1680.
Orok-Edem E, Key D. 1994. Response of rats (Rattus
norvegicus) to enrichment objects. Animal Technology
45:25-30.
O´Steen WK, Shear CR, Anderson KV. 1972. Retinal
damage after prolonged exposure to visible light: A
light and electron microscope study. The American
Journal of Anatomy 134:5-22.
Paton W. 1984. Man and Mouse: Animals in Medical
Research. Oxford University Press.
Patterson-Kane EG. 2003. Shelter enrichment for rats.
Contemporary Topics in Laboratory Animal Science
42:46-48.
Patterson-Kane EG, Harper DN, Hunt M. 2001. The cage
preferences of laboratory rats. Laboratory Animals
35:74-79.
Pekow C. 2005. Defining, measuring and interpreting
stress in laboratory animals. Contemporary Topics in
Laboratory Animal Science 44:41-45.
Pihl L, Hau J. 2003. Faecal corticosterone and
immunoglobulin A in young adult rats. Laboratory
Animals 37:166-171.
Poole S, Stephenson JD. 1977. Body temperature
regulation and thermoneutrality in rats. Quarterly
Journal of Experimental Physiology 62:143-149.
Pound P, Ebrahim S, Sandercock P, Bracken MB, Roberts
I. 2004. Where is the evidence that animal research
benefits humans? British Medical Journal 328:514-517.
Prusky GT, Harker KT, Douglas RM, Whishaw IQ. 2002.
Variation in visual acuity within pigmented, and
between pigmented and albino rat strains. Behavioural
Brain Research 136:339-348.
48
Purves KE. 1997. Rat and mice enrichment. In: Animal
Alternatives, Welfare and Ethics, ed. LFM van Zutphen
& M Balls. Elsevier Science B.V. pp. 199-207.
Quignon P, Giraud M, Rimbault M, Lavigne P, Tacher S,
Morin E, Retout E, Valin AS, Lindblad-Toh K, Nicolas
J, Galibert F. 2005. The dog and rat olfactory receptor
repertoires. Genome Biology 6:R83.
Rabat A. 2007. Extra-auditory effects of noise in
laboratory animals: The relationship between noise and
sleep. Journal of the American Association for
Laboratory Animal Science 46:35-41.
Ramos A, Berton O, Mormede P, Chalouff F. 1997. A
multiple-test study of anxiety-related behaviours in six
inbred rat strains. Behavioural Brain Research 85:5769.
Reeb CK, Jones RB, Bearg DW, Bedigian H, Paigen B.
1997. Impact of room ventilation rates on mouse cage
ventilation and microenvironment. Contemporary
Topics in Laboratory Animal Science 36:74-79.
Reeb CK, Jones RB, Bearg DW, Bedigian H, Myers DD,
Paigen B. 1998. Microenvironment in ventilated animal
cages with different ventilation rates, mice populations,
and frequency of bedding changes. Contemporary
Topics in Laboratory Animal Science 37:43-49.
Reeb-Whitaker CK, Paigen B, Beamer WG, Bronson RT,
Churchill GA, Schweitger IB, Myers DD. 2001. The
impact of reduced frequency of cage changes on the
health of mice in ventilated cages. Laboratory Animals
35:58-73.
Renström A, Björing G, Höglund U. 2001. Evaluation of
individually ventilated cage systems for laboratory
rodents: Occupational health aspects. Laboratory
Animals 35:42-50.
Rex A, Sondern U, Voigt JP, Franck S, Fink H. 1996.
Strain differences in fear-motivated behavior of rats.
Pharmacology Biochemistry, and Behavior 54:107-111.
Richardson CA, Flecknell PA. 2005. Anaesthesia and postoperative analgesia following experimental surgery in
laboratory rodents: Are we making progress? ATLA
33:119-127.
Ritskes-Hoitinga M, Chwalibog A. 2003. Nutrient
requirements, experimental design, and feeding
schedules in animal experimentation. In: Handbook of
Laboratory Animal Science, second edition. Ed by Hau
J & Hoosier GL, CRC Press, Boca Raton 281-310.
Ritskes-Hoitinga M, Strubbe JH. 2004. Nutrition and
animal welfare. In: The Welfare of Laboratory
Animals. Ed by Kaliste E, Kluwer Academic Publishers
51-80.
Roe FJC. 1994. Historical histopathological control data
for laboratory rodents: Valuable treasure or worthless
trash? Laboratory Animals 28:148-154.
Roe FJC, Lee PN, Conybeare G, Kelly D, Matter B,
Prentice D, Tobin G. 1995. The biosure study:
Influence of composition of diet and food consumption
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
General introduction
on longevity, degenerative diseases and neoplasia in
Wistar rats studied for up to 30 months post weaning.
Food and Chemical Toxicology 33:1S-100S.
Rosenberg RS, Bergmann BM, Son HJ, Arnason BGW,
Rechtschaffen A. 1987. Strain differences in the sleep
of rats. Sleep 10:537-541.
Royo F, Björk N, Carlsson HE, Mayo S, Hau J. 2004.
Impact of chronic catheterization and automated blood
sampling (Accusampler) on serum corticosterone and
fecal immunoreactive corticosterone metabolites and
immunoglobulin A in male rats. Journal of
Endocrinology 180:145-153.
Rubin NH, Alinder G, Riedtveld WJ, Rayford PL,
Thompson JC. 1988. Restricted feeding schedules alter
the circadian rhythms of serum insulin and gastric
inhibitory polypeptide. Regulatory Peptides 23:279288.
Rushen J. 1991. Problems associated with the
interpretation of physiological data in the assessment of
animal welfare. Applied Animal Behaviour Science
28:381-386.
Rushen J, de Passillé AMB. 1992. The scientific
assessment of the impact of housing on animal welfare:
A critical review. Canadian Journal of Animal Science
72:721-743.
Russell WMS, Burch RL. 1959. The principles of humane
experimental technique. Methuen & co Ltd, London,
UK.
Sabatino F, Masoro EJ, McMahan CA, Kuhn RW. 1991.
Assessment of the role of the glucocorticoid system in
aging processes and in the action of food restriction.
Journal of gerontology: Biological Sciences 46:B171B179.
Sabine JR. 1975. Exposure to an environment containing
the aromatic red cedar, Juniperus virginiana:
procarcinogenic, enzyme-including and insecticidal
effects. Toxicology 5:221-235.
Saibaba P, Sales GD, Stodulski G, Hau J. 1996. Behaviour
of rats in their home cages: Daytime variations and
effects on routine husbandry procedures analysed by
time sampling techniques. Laboratory Animals 30:1321.
Saito M, Murakami E, Nishida T, Fujisawa Y, Suda M.
1975. Circadian rhythms in digestive enzymes in the
small intestine of rats. I. Patterns of the rhythms in
various regions of the small intestine. Journal of
Biochemistry 78:475-480.
Saleh MA, Winget CM. 1977. Effect of suprachiasmatic
lesions on diurnal heart rate rhythm in the rat.
Physiology & Behavior 19:561-564.
Sano H, Hayashi H, Makino M, Takezawa H, Hirai M,
Saito H, Ebihara S. 1995. Effects of suprachiasmatic
lesions on circadian rhythms of blood pressure, heart
rate and locomotor activity in the rat. Japanese
Circulation Journal 59:565-573.
Sarrieau A, Mormède P. 1998. Hypothalamic-pituitaryadrenal axis activity in the inbred Brown Norway and
Fischer 344 rat strains. Life Sciences 62:1417-1425.
Sarrieau A, Chaouloff F, Lemaire V, Mormède P. 1998.
Comparison of the neuroendocrine responses to stress
in outbred, inbred and F1 hybrid rats. Life Sciences
63:87-96.
Scheer M, Heinekamp C, Thull B, Wagner R, Scheuber
HP. [Internet, not dated]. Performance evaluation of
IVC systems - Part 1: Test instructions.
www.lal.org.uk/IVC/index.html
Schnecko A, Witte K, Lemmer B. 1998. Effects of routine
procedures on cardiovascular parameters of SpragueDawley rats in periods of activity and rest. Journal of
Experimental Animal Science 38:181-190.
Schreuder MF, Fodor M, van Wijk JAE, Delemarre-van
den Waal HA. 2007. Weekend versus working day:
Differences in telemetric blood pressure in male Wistar
rats. Laboratory Animals 41:86-91.
Schrijver NCA, Bahr NI, Weiss IC, Würbel H. 2002.
Dissociable effects of isolation rearing and
environmental enrichment on exploration, spatial
learning and HPA activity in adult rats. Pharmacology,
Biochemistry, and Behavior 73:209-224.
Seggie JA, Brown GM. 1975. Stress response pattern of
plasma corticosterone, prolactin and growth hormone in
rat, following handling or exposure to novel
environment. Canadian Journal of Physiology &
Pharmacology 53:629-637.
Sei H, Sone M, Kanamori N, Morita Y. 1995. Light-dark
difference in arterial pressure variability during REM
sleep in the rat. Chronobiology International 12:389397.
Sei H, Furuno N, Morita Y. 1997. Diurnal changes of
blood pressure, heart rate and body temperature during
sleep in the rat. Journal of Sleep Research 6:113-119.
Sharp J, Zammit T, Azar T, Lawson D. 2002a. Stress-like
responses to common procedures in male rats housed
alone or with other rats. Contemporary Topics in
Laboratory Animal Science 41:8-14.
Sharp J, Zammit T, Azar T, Lawson D. 2002b. Stress-like
responses to common procedures in rats: Effect of the
estrous cycle. Contemporary Topics in Laboratory
Animal Science 41:15-22.
Sharp J, Zammit T, Azar T, Lawson D. 2002c. Does
witnessing experimental procedures produce stress in
male rats? Contemporary Topics in Laboratory Animal
Science 41:8-12.
Sharp J, Zammit T, Azar T, Lawson D. 2003a. Stress-like
responses to common procedures in individually and
group-housed female rats. Contemporary Topics in
Laboratory Animal Science 42:9-18.
Sharp J, Zammit T, Azar T, Lawson D. 2003b. Are “bystander” female Sprague-Dawley rats affected by
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
49
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
experimental procedures? Contemporary Topics in
Laboratory Animal Science 42:19-27.
Sharp J, Zammit T, Azar T, Lawson D. 2003c. Does cage
size affect heart rate and blood pressure on male rats at
rest or after procedures that induce stress-like
responses? Contemporary Topics in Laboratory Animal
Science 42:8-12.
Sharp J, Azar T, Lawson D. 2005a. Selective adaptation of
male rats to repeated social encounters and
experimental manipulations. Contemporary Topics in
Laboratory Animal Science 44:28-31.
Sharp J, Azar T, Lawson D. 2005b. Effects of a cage
enrichment program on heart rate, blood pressure, and
activity of male Sprague-Dawley and spontaneously
hypertensive rats monitored by radiotelemetry.
Contemporary Topics in Laboratory Animal Science
44:32-40.
Sharp J, Azar T, Lawson D. 2006. Comparison of carbon
dioxide, argon and nitrogen for
inducing
unconsciousness or euthanasia of rats. Journal of the
American Association for Laboratory Animal Science
45:21-25.
Sheridan KM, Ferguson MJ, Distasi MR, Witzmann FA,
Dalsing MC, Miller SJ, Unthank JL. 2007. Impact of
genetic background and aging on mesenteric collateral
growth capacity in Fischer 344 and Brown Norway,
and Fischer 344 x Brown Norway hybrid rats.
American Journal of Physiology: Heart and Circulatory
Physiology 293:H3498-H3505.
Silverman J, Bays DW, Cooper SH, Baker SP. 2008.
Ammonia and carbon dioxide concentrations in
disposable and reusable ventilated mouse cages.
Journal of the American Association for Laboratory
Animal Science 47:57-62.
Siswanto H, Hau J, Carlsson HE, Goldkuhl R, Abelson
KSP. 2008. Corticosterone concentrations in blood and
excretion in faeces after ATCH administration in male
Sprague-Dawley rats. In Vivo 22:435-440.
Spangler EL, Waggie KS, Hengemihle J, Roberts D, Hess
B, Ingram DK. 1994. Behavioral assessment of aging in
male Fischer 344 and brown Norway rat strains and
their F1 hybrid. Neurobiology of Aging 15:319-328.
Spiteri NJ. 1982. Circadian patterning of feeding, drinking
and activity during diurnal food access in rats.
Physiology & Behavior 28:139-147.
Stauffacher M, Peters A, Jennings M, Hubrecht R, Holgate
B, Francis R, Elliot H, Baumans V, Hansen AK. 2002.
[Internet]. Future principles for housing and care of
laboratory rodents and rabbits. Report for the revision
of the Council of Europe Convention ETS 123
Appendix A for rodents and rabbits. Part B. Council of
Europe.
www.coe.int/t/e/legal_affairs/legal_cooperation/biological_safety%2C_use_of_animals/Labor
atory_animals/
50
Stephan FK. 1984. Phase shifts of circadian rhythms in
activity entrained to food access. Physiology &
Behavior 32:663-671.
Sternberg EM, Glowa JR, Smith MA, Calogero AE,
Listwak SJ, Aksentijevich S, Chrousos GP, Wilder RL,
Gold PW. 1992. Corticotropin releasing hormone
related behavioral and neuroendocrine responses to
stress in Lewis and Fischer rats. Brain Research
570:54-60.
Stotzer H, Weisse I, Knappen F, Seitz R. 1970. Die RetinaDegeneration der Ratte. Arzneimittel-Forschung
20:811-17.
Strausbaugh HJ, Green PG, Dallman MF, Levine JD. 2003.
Repeated,
non-habituating
stress
suppresses
inflammatory plasma extravasation by a novel,
sympathoadrenal dependent mechanism. European
Journal of Neuroscience 17:805-812.
Strubbe JH, Keyser J, Dijkstra T, Alingh Prins AJ. 1986a.
Interaction between circadian and caloric control of
feeding behaviour in the rat. Physiology & Behavior
36:489-493.
Strubbe JH, Spiteri NJ, Alingh Prins AJ. 1986b. Effect of
skeleton photoperiod and food availability on the
circadian pattern of feeding and drinking in rats.
Physiology & Behavior 36:647-651.
Strubbe JH, Alingh Prins AJ. 1986. Reduced insulin
secretion after short-term food deprivation in rats plays
a key role in the adaptive interaction of glucose and
fatty free acid utilization. Physiology & Behavior
37:441-445.
Strubbe JH, Alingh Prins AJ, Bruggink J, Steffens AB.
1987. Daily variation of food-induced changes in blood
glucose and insulin in the rat and the control by
suprachiasmatic nucleus and the vagus nerve. Journal
of the Autonomic Nervous System 20:113-119.
Swoap SJ. 2001. Altered leptin signaling is sufficient, but
not required, for hypotension associated with caloric
restriction. American Journal of Physiology. Heart and
Circulatory Physiology 281:H2473-H2479.
Swoap SJ, Overton JM, Garber G. 2004. Effect of ambient
temperature on cardiovascular parameters in rats and
mice: A comparative approach. American Journal of
Physiology. Regulatory, Integrative and Comparative
Physiology 287:R391-R396.
Tamashiro KLK, Nguyen MMN, Fujikawa T, Xu T, Ma
LY, Woods SC, Sakai RR. 2004. Metabolic and
endocrine consequences of social stress in a visible
burrow system. Physiology & Behavior 80:683-693.
Teixeira MA, Chaguri LCAG, Carissimi AS, Souza NL,
Mori CMC, Saldiva PHN, Lemos M, Maccihione M,
Guimarães ET, King M, Merusse JLB. 2006. Effects of
an individually ventilated cage system on the airway
integrity of rats (Rattus norvegicus) in a laboratory in
Brazil. Laboratory Animals 40:419-431.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
General introduction
Tordoff MG, Alarcon LK, Lawler MP. 2008. Preference of
14 rat strains for 17 compounds. Physiology &
Behavior 95:308-332.
Toth LA, Gardiner TW. 2000. Food and water restriction
protocols: Physiological and behavioral considerations.
Contemporary Topics in Laboratory Animal Science
39:9-17.
Townsend P. 1997. Use of in-cage shelters by laboratory
rats. Animal Welfare 6:95-103.
Tsai PP, Oppermann D, Stelzer HD, Mähler M, Hackbarth
H. 2003. The effects of different rack systems on the
breeding performance of DBA/2 mice. Laboratory
Animals 37:44-53.
Tu H, Diberadinis LJ, Lipman NS. 1997. Determination of
air distribution, exchange, velocity and leakage in three
individually ventilated rodent caging systems.
Contemporary Topics in Laboratory Animal Science
36:69-73.
Turner JG, Bauer CA, Rybak LP. 2007. Noise in animal
facilities: Why it matters? Journal of American
Association for Laboratory Animal Science 46:10-13.
d’Uscio LV, Kilo J, Lüscher TF, Gassmann M. 2000.
Circulation. In: Krinke GJ, ed. The Laboratory Rat.
Academic Press, pp. 345-357.
Van de Weerd HA, van den Broek FAR, Baumans V.
1996. Preference for different types of flooring in two
rat strains. Applied Animal Behaviour Science 46:251261.
Van den Brant J, Kovács P, Klöting I. 1999. Blood
pressure, heart rate and motor activity in 6 inbred rat
strains and wild rats (Rattus norvegicus): A
comparative study. Experimental Animals 48:235-240.
Van den Buuse M. 1994. Circadian rhythms of blood
pressure, heart rate, and locomotor activity in
spontaneously hypertensive rats as measured with
radio-telemetry. Physiology & Behavior 55:783-787.
Van den Buuse M. 1999. Circadian rhythms of blood
pressure and heart rate in conscious rats: Effect of light
cycle shift and timed feeding. Physiology & Behavior
68:9-15.
Van den Buuse M, van Acker SABE, Fluttert M, de Kloet
ER. 2001. Blood pressure, heart rate, and behavioral
responses to psychological “novelty” stress in freely
moving rats. Psychophysiology 38:490-499.
Van den Buuse M, van Acker SABE, Fluttert M, de Kloet
ER. 2002. Involvement of corticosterone in
cardiovascular responses to an open-field novelty
stressor in freely moving rats. Physiology & Behavior
75:207-215.
Van den Staay FJ, Blokland A. 1996. Behavioural
differences between outbred Wistar, inbred Fischer
344, Brown Norway and hybrid Fischer 344 x Brown
Norway rats. Physiology & Behavior 60: 97-109.
Van der Harst JE, Fermont PCJ, Bilstra AE, Spruijt BM.
2003. Access to enriched housing is rewarding to rats
as reflected by their anticipatory behaviour. Animal
Behaviour 66:493-504.
Van Ness J, Casto R, Overton J. 1997. Antihypertensive
effects of food-intake restriction in aortic coarctation
hypertension. Journal of Hypertension 15:1253-1262.
Vermeulen JK, de Vries A, Schlingmann F, Remie R.
1997. Food deprivation: Common sense or nonsense?
Animal Technology 48:45-54.
Vesell ES. 1968. Genetic and environmental factors
affecting hexobarbital metabolism in mice. Annals of
the New York Academy of Sciences 151:900-912.
Voipio HM. How do rats react to sound? 1997.
Scandinavian Journal of Laboratory Animal Science 24
(Suppl 1):1-80.
Voipio HM, Nevalainen T, Halonen P, Hakumäki M, Björk
E. 2006. Role of cage material, working style and
hearing sensitivity in perception of animal care noise.
Laboratory Animals 40:400-409.
Voipio HM, Korhonen T, Koistinen T, Kuronen H, Mering
S, Nevalainen T. 2008. Durability and hygiene of aspen
tubes used for providing environmental complexity for
laboratory rats. Scandinavian Journal of Laboratory
Animal Science 35:107-115.
Wade AE, Holl JE, Hillard CC, Molton E, Greene FE.
1968. Alteration of drug metabolism in rats and mice
by an environment of cedarwood. Pharmacology 1:317328.
Webb AA, Gowribai K, Muir G. 2003. Fischer (F-344) rats
have different morphology, sensorimotor and
locomotor abilities compared to Lewis, Long-Evans,
Sprague-Dawley and Wistar rats. Behavioural Brain
Research 144:143-156.
Webster J. 1995. Animal Welfare. A Cool Eye Towards
Eden. Blackwell Science, Oxford. 273 pp.
Webster AJF. 2003. Assessment of animal welfare at farm
and group level: Introduction and overview. Animal
Welfare 12:429-431.
Weichbrod RH, Cicar CF, Miller JG, Simmonds RC,
Alvares AP, Ueng TH. 1988. Effects of cage bedding
on microsomal oxidative enzymes in rat liver.
Laboratory Animal Science 38:96-298.
Weisse I, Stotzer H, Seitz R. 1974. Age and lightdependent changes in the rat eye. Virchows Archiv für
pathologische Anatomie und Physiologie und für
klinische Medizin 362:145-56.
Williams CM, Riddell PM, Scott LA. 2008. Comparison of
preferences for object properties in the rats using
paired- and free-choice paradigms. Applied Animal
Behaviour Science 112:146-157.
Witte K, Grebmer W, Scalbert E, Delagrange, P,
Guardiola-Lemaître B, Lemmer B. 1998. Effects of
melatoninergic agonists on light-suppressed circadian
rhythms in rats. Physiology & Behavior 65:219-224.
Yamauchi C, Fujita S, Obara T, Ueda T. 1981. Effects of
room temperature on reproduction, body and organ
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
51
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
weights, food and water intake, and hematology in rats.
Laboratory Animal Science 31:251-258.
Young JB, Mullen D, Landsberg L. 1978. Caloric
restriction lowers blood pressure in the spontaneously
hypertensive rat. Metabolism 27:1711-1714.
Yu BP, Masoro EJ, Murata I, Bertrand HA, Lynd FT.
1982. Life span study of SPF Fisher 344 male rats fed
ad libitum or restricted diets: Longevity, growth, lean
body mass and disease. Journal of Gerontology 37:130141.
Yu BP, Masoro EJ, McMahan CA. 1985. Nutritional
influences on aging of Fisher 344 rats: Physical,
metabolic, and longevity characteristics. Journal of
Gerontology 40:657-670.
Zaias J, Queeney TJ, Kelley JB, Zakharova ES, Izenwasser
S. 2008. Social and physical environmental enrichment
differentially affect growth and activity of
preadolescent and adolescent male rats. Journal of the
American Association for Laboratory Animal Science
47:30-34.
Zhang B, Sannajust F. 2000. Diurnal rhythms of blood
pressure, heart rate, and locomotor activity in adult and
old male Wistar rats. Physiology & Behavior 70:375380.
Zucker I. 1971. Light-dark rhythms in rat eating and
drinking behavior. Physiology& Behavior 6:115-126.
Õkva K, Tamoševičiūtė E, Čižiūtė A, Pokk P, Rukšėnas O,
Nevalainen T. 2006. Refinements for intragastric
gavage in rats. Scandinavian Journal of Laboratory
Animal Science 33:243-252.
52
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 258:1-52 (2009)
CHAPTER II
Exposure in the shoebox: Comparison of physical environment of IVCs and open rat
cages.
Niina Kemppinen, Anna Meller, Erkki Björk, Tarja Kohila and Timo Nevalainen. 2008.
Scandinavian Journal of Laboratory Animal Science 35:97-103.
Reprinted with permission from Scandinavian Journal of Laboratory Animal Science. Copyright
(2008) the Scandinavian Journal of Laboratory Animal Science.
Scand. J. Lab. Anim. Sci. 2008 Vol. 35 No. 2
Exposure in the Shoebox:
Comparison of Physical Environment of IVCs
and Open Rat Cages
by Niina Kemppinen1,*, Anna Meller1, Erkki Björk2, Tarja Kohila1 & Timo Nevalainen3,4
1
Laboratory Animal Centre, 4Department of Basic Veterinary Sciences, University of Helsinki, Helsinki, Finland
2
Department of Environmental Sciences, 3National Laboratory Animal Center, University of Kuopio, Finland
Summary
New caging and innovative items for more structured environment within the cage have been introduced.
Many of these innovations cannot be seen as 'pure' or individual procedures, but rather they represent a
mixed exposure with a multitude of operant factors, some possibly having an impact on animals and
research. One kind of new caging system is the individually ventilated cage (IVC), where each cage
receives its own non-contaminated airflow, primarily designed for health status maintenance and occupational safety. Even though those cages may be the same as those used in open cage systems, the physical
environment inside the cage may not identical. Comparison between cage types is difficult without characterization of the physical environment, because the change may involve alterations in several parameters
in the environment. The aim of this study is to characterize and compare common physical parameters in
the ordinary situation, where IVC-racks are kept in the same room with open cages. The cage type used
was a polysulfone solid bottom cage. The parameters measured in this study were: illumination, temperature, relative humidity (RH) and acoustic level in both IVCs and open top cages. No animals were in the
cages during light intensity, but there was bedding in the cage during acoustic measurements and both bedding as well as a half-full food hopper during the illumination measurements. The temperature and (RH)
measurements were carried out with three male rats in each cage. There were differences between IVCs
and open top cages in all measured parameters. The light intensity was lower in IVCs, most likely due to
more compact cage placement in the rack and the additional plastic cover lid of the cage. Both maximum
and minimum temperatures were 1-4 oC higher in IVCs; which suggests that their ventilation is incapable
of taking away heat, produced inside the cage. Similarly, the relative humidity was higher in the IVCs. The
sound level adjusted to rat's hearing with R-weighting was higher in IVCs when compared to open cages.
Furthermore, the sound level was highest in the corners next to the ventilation valves. In conclusion, there
may be differences between open cages with IVCs involving several physical parameters of cage environment and this may confound comparisons between results obtained in these cage systems.
Introduction
New caging and innovative items are being introduced to provide a more structured environment
*Correspondence: Niina Kemppinen
Laboratory Animal Centre, P.O. Box 56 FIN - 00014
University of Helsinki, Finland
Tel.
+358 9 191 59070
Fax
+358 9 191 59480
E-mail [email protected]
within the cage. Many of these innovations cannot
be seen as 'pure' or single procedures, but rather as
a mixed exposure with a multitude of operant factors, possibly having an impact on animals and
research.
One of those new kinds of caging systems is the
individually ventilated cage (IVC), where each cage
receives its own filtered air flow, primarily designed
for health status maintenance and occupational
97
Published in the Scandinavian Journal of Laboratory Animal Science - an international journal of laboratory animal science
Scand. J. Lab. Anim. Sci. 2008 Vol. 35 No. 2
safety. Other potential benefits include: protection
of small groups of animals against infections, protection of the environment from the animals and
compensation for poor air change rates in the room
(Brandstetter et al., 2004). Even though the cages
may be the same as those used in open cage systems, the physical environment inside the cage may
not be identical.
Reports on IVC-systems in the scientific journals
can be divided into those concerned with the design
and recommendations for (Höglund & Renström et
al., 2001; Renström et al., 2001; Hawkins et al.,
2003; Brandstetter et al., 2005) and characterization of the IVC environment (Krohn et al., 2003;
Clough et al., 1995).
The move from traditional open cages to IVCs is
bound to change the physical environment of the
animals living in; what we have called, the ”shoebox”. It could be anticipated that at least temperature, relative humidity (RH), acoustic environment
and light intensity may change in this transition.
Traditionally in biomedical research, attempts are
made to assess the individual effects of compounds
and procedures and, usually, evaluation of many
simultaneous events and their combinations are
avoided. The term used here is standardization, and
emphasis is on the fact that all other items are
exactly the same between study and control groups.
Comparison of open-top cages and IVCs without
characterization of the physical environment may not
reveal a single causative feature, because the change
inevitably involves a mixed exposure. The aim of this
study is to characterize and compare physical parameters in a common situation, where IVC-racks are
housed in the same room with open cages.
Materials and Methods
Animal room
All the cages were kept in the same room (length x
width x height; 5.5 x 3.5 x 3.0 m) along opposite
walls. The locations of cage racks, room furniture,
fluorescent tubes, air inlet and outlet are illustrated
in Figure 1. The IVC-rack (Figure 2) included 20,
while the open cages (Figure 3) were in two racks,
ten cages each. The height of the open cage rack
was 176 cm and that of IVC-rack was 186 cm.
Open top cage racks
Stainless steel table
IVC ventilation unit
IVC rack
Rack for empty cages
Computer cabinet
Inlet air diffuser below ceiling
Air outlet above the door
Fluorescent tubes, height 244 cm from floor
Figure 1. Layout of the animal room.
98
Scand. J. Lab. Anim. Sci. 2008 Vol. 35 No. 2
V
e
n
t
i
l
a
t
i
o
n
u
n
i
t
16.4
26.0
29.2
28.9
8.5
7.2
° 6.2
8.5
3.8
4.4
4.9
Sound measurements
° Temperature and RH measurements
Illumination measurements
Figure 2. Frontal view of cage and ventilation unit
location in IVC-cage rack with cage-specific illumination values (lux) and cages where acoustic, temperature and humidity measurements were done.
Cage types
The measurements were done from two different
caging systems: open top cages and individually
ventilated cages (IVC). Cages made of polysulfone
(Tecniplast, Buguggiate, Italy, type1500U, dimensions 48.0 x 37.5 x 21.0 cm) with a solid bottom
were used. Both cage types had a stainless steel
wire lid, while in the IVCs there was an additional
polysulfone cover, which contained the air supply
and the exhaust air valves and a passive filter at the
top of the cover. This filter allows gas exchange for
a short period, when the cage is not docked to the
IVC-system.
The IVC-system consisted of a ventilation module,
which had both supply and exhaust units (Slim
LineTM, Tecniplast, Buguggiate, Italy) and the individually ventilated cage rack (SealsafeTM,
Tecniplast, Buguggiate, Italy). The supply unit
delivered HEPA (High Efficiency Particulate Air)
filtered air into the cage, taken from the room itself,
separately through each cage’s supply valve, at the
cage cover end. The exhaust air was taken out from
the cages, also at the same end, through the exhaust
valve and voided back to the room through a three
filter set down to HEPA level.
Illumination
Artificial lights with two fluorescent tubes (light
color warm white) were on from 06.00 to 18.00;
their location in the room (106 cm below the ceiling) is illustrated in Figure 1. The IVC-rack had a
stainless steel shield on top, and the open cages
were covered with black plastic sheeting to prevent
direct light entering the cages. No animals were in
the cages during the light intensity measurements,
but there was bedding in the cage and the food hopper was half full. The illuminometer (Roline digital
lux meter RO 1332, Rotronic AG, Bassersdorf,
Switzerland) was placed at the center of the cage
floor on the bedding layer and single measurements
were taken from both IVCs (Figure 2) and open
cages (Figure 3).
Acoustic environment
A sound analyzer (Norsonic 121, Norsonic AS,
Lierskogen, Norway.) was used for noise measurement. The measurement system was calibrated
using a sound level calibrator (Wärtsilä model
5274, MIP Electronics Oy, Kerava, Finland). Four
cages from both cage types were measured four
times, once from each corner. The cages had bedding, but no animals were inside the cages during
the measurements. Measurements were taken from
cages marked with
as shown in Figures 2 and 3.
Equal sound pressure levels for one minute (Leq,
1min) in third-octave bands between 31.5 Hz and
20 000 Hz were measured with 1/2 an inch condenser microphone (Norsonic 1225, Norsonic AS,
Lierskogen, Norway.). The microphone was placed
about 5 cm from the walls and the bottom of the
cage, and directed towards the corner of the cage.
In the weighted equal sound pressure level calculations, R-weighting and A-weighting were used. The
99
Scand. J. Lab. Anim. Sci. 2008 Vol. 35 No. 2
46.4º
91.0
53.8
40.9
27.2
17.5
17.3
14.2
17.6
Table plate
16.5
26.7
25.1
16.2
28.8
25.3
Sound measurements
° Temperature and RH measurements
Illumination measurements
Figure 3. Frontal view of cages in open cage racks with cage-specific illumination values (lux) and cages
where acoustic, temperature and humidity measurements were done.
basis of the R-weighting for rat hearing sensitivity
and the numerical values are described in detail by
Björk et al., (2000) and Voipio (1997). The Aweighting is commonly used in human sound
experiments. The total weighted equal sound pressure levels were computed summing the weighted
third-octave band levels on the energy bases. The
weighted equal sound pressure level in each cage
was computed as the mean value in the four corners
on the energy bases.
Temperature and RH
Temperature and (RH) measurements were carried
out with animals in the cages. Fischer344
(F344/NHsd, Harlan, Horst, The Netherlands) male
rats were used in this study. The rats were 40 weeks
old and weighed 380 - 400 g, three animals per
cage. The cage floors were covered with 3.0 l aspen
100
chip bedding (size 4 x 4 x 1 mm, 4HP, Tapvei Oy,
Kaavi, Finland).
Municipal tap water was provided in polycarbonate
bottles with stainless steel drinking nipples,
changed once a week and refilled once in between.
Irradiated pelleted (25 kGy) feed (2016 Global
Rodent Maintenance, Teklad, Bicester, UK) was
given ad libitum, added once a week.
Temperature and RH were measured with Besser®
7009032 Wireless Weather Station and with two
Techno line TX4 433 MHz sensors (Besser, Borken,
Germany). Measurements were taken simultaneously from both cage types (IVC and open top) and
from the room for 7 days. The sensors were placed
inside the feed hopper next to the pellets. Readings
were taken once a day to provide minimum and
maximum values over the previous 24 h period for
Scand. J. Lab. Anim. Sci. 2008 Vol. 35 No. 2
B
A
4
C
o
3
2
Room
1
IVC-min
0
IVC-max
Temperature
o
Temperature C
4
-1
3
2
1
Room
Open-min
0
Open-max
-1
1
2
3
4
5
6
7
1
2
3
Day
4
6
7
D
C
8
6
4
2
Room
0
IVC-min
-2
IVC-max
Relative humidity %
8
Relative humidity %
5
Day
6
4
2
Room
0
Open-min
Open-max
-2
-4
-4
1
2
3
4
5
6
7
Day
1
2
3
4
5
6
7
Day
Figure 4. Deviation in the 24 hourly single cage maximum and minimum values inside the cage for temperature and relative humidity from the corresponding room values during the week and between cage changes.
A and C are values for IVC-cages and B and D for open cages. Cage changes are marked with an arrow.
temperature and RH. Measurements were taken
from cages as marked in Figures 2 and 3.
Results
Illumination
The light intensity at 1 m above floor in the open
cages was 16-18 lux compared to 6–9 lux in the
IVC´s, with upper cage rows showing considerably
higher values. The light intensities of IVC-racks
were lower than those measured in the open cages
at corresponding levels. More detailed, cage specific, values are shown in Figures 2 and 3.
Acoustic environment
The sound level adjusted with R-weighting in
empty IVC cages was 20-25 dB(R) compared to 1218 dB(R) in the empty open cages, with the corresponding adjusted A-weighting being 45-47 dB(A)
and 46-49 dB(A), respectively. The sound level was
less in the open cages in the lower shelves of the
rack, while in the IVCs the front of the cages
showed higher sound levels compared to the back
corners in the vicinity of the air valves. The sound
frequency in both cage types was 16 – 16000 Hz.
The mean sound pressure levels in the third-octave
bands between 31.5 Hz and 20000 Hz on the energy bases of both cage types with R- and A-weighting and un-weighted (lin) are shown in Figures 5a
and 5b.
Temperature and RH
There was a marked difference in temperature and
RH between inside the IVC’s and open top cages
when compared to both open top cage and room
values. In the IVCs, the maximum and minimum
temperature values in the IVCs were 1-4 °C higher
than the room temperature. In the open top cages
the temperatures were at the same level as the room
temperatures. There was a similar tendency noted in
RH as shown in Figure 4.
101
Scand. J. Lab. Anim. Sci. 2008 Vol. 35 No. 2
Figure 5 a. Sound spectra in open cages.
Figure 5 b. Sound spectra in IVC cages.
Discussion
IVCs are a new isolation system being installed in a
large number of laboratory animal facilities. The
system provides protection to the animals against
infections, and has clear occupational benefits to
personnel, especially in leading to a reduction in the
levels of airborne allergens (Renström et al., 2001).
It is often assumed that the physical environment is
the same in open cages and IVCs, when they are
kept in the same room. Surprisingly it was found
that this is not the case, and furthermore that the
magnitude of the changes in physical environment
is great enough that it could have an impact on the
animals housed in these cages. Therefore it was
decided to assess a set of easily measurable physical parameters in both caging systems.
The light intensity in IVCs was 10-60 lux lower
than the corresponding values in open top cages. In
the IVCs, the illumination varied between 3.8-28.9
lux and in the open top cages between 14.2-91.0
lux. The brightest cages were on the top row of both
racks and the dimmest cages on the bottom row.
102
Clough et al., (1995) has shown similar results in
the transparent, polycarbonate positive individually
ventilated (PIV)-cages but in the translucent,
polypropylene control cages the illumination was
much brighter than used in our experiment. This
difference in results may be due to the black plastic
sheeting that was placed on top of the open top cage
racks to equalize the lighting in both cage types. It
appears that in some open top cages at the highest
level, in contrast to the situation in the IVCs, the
lighting was too bright, and even exceeded values
shown to cause retinal damage in albino rats
(Stotzer et al., 1970; Weisse et al., 1974).
Sound spectra (Figures 5a and 5b) show decreasing
sound levels when approaching 16000 Hz and consequently it can be assumed that no ultrasound
exists in either cage type. Accordingly the measured
R-weighted sound pressure levels depict the correct
sound level which would be heard by the rats. In
IVCs, the R-weighted sound levels were about 7
dB(R) higher than in open top cages; energywise
the difference was five fold and in terms of loudness almost twice as great. Although the difference
in sound levels audible to the rat (R-weighted) is
large, it remains to be determined whether the levels measured (< 25 dB(R)) have any major impact
on the animals.
Scheer et al., state the obvious: The climatic conditions in the cage are dependent on those of the surrounding room as well as the air supply of the cagerack. In this study the temperature and RH in IVCs
were the same as in the animal room when measured without animals, but placement of animals into
the IVCs increased the temperature by 3-4 °C and
RH by about 6 % in these cages.
In the open top cages, the temperature and RH were
at the same level as in the animal room. This is in
agreement with the results of Clough et al. (1995)
who have shown similar results in PIV-cages. It
appears that IVC-ventilation is unable to remove all
the heat produced in the cage. Potential sources are
heat from the animals as well as heat generated
from urine-feces-bedding fermentation. Heat emission for three animals in a cage is estimated to be
Scand. J. Lab. Anim. Sci. 2008 Vol. 35 No. 2
about 12 W (Heine, 1998). However, fermentation
reactions are unlikely to occur because ventilation
tends to keep bedding too dry.
It appears that the transition from traditional open
top cages to IVCs may lead to changes in the physical environment. This makes any comparisons of
these caging systems problematic without characterization of the physical parameters e.g. lighting intensity, sounds, temperature and RH. In conclusion,
comparison of open cages with IVCs involves several physical parameters in the cage environment,
which may confound straightforward comparisons.
Acknowledgements
This study has received financial support from the
Finnish Ministry of Education, the Academy of
Finland, the ECLAM and ESLAV Foundation, the
North-Savo Cultural Foundation, the Finnish
Research School for Animal Welfare, the Oskar
Öflund Foundation and the University of Kuopio.
References
Björk E, T Nevalainen, M Hakumäki M & HM
Voipio. R-weighting provides better estimation
for rat hearing sensitivity. Lab Anim, 2000, 34,
136-144.
Brandstetter H, M Scheer, C Heinekamp, C
Gippner-Stepper, O Loge, L Ruprecht, B Thull,
R Wagner, P Wilhem & HP Scheuber.
Performance evaluation of IVC system. Lab
Anim, 2005, 39, 40-44.
Clough G., J Wallace, MR Gamble, ER
Merryweather & E Bailey. A positive, individually ventilated caging system: a local barrier
system to protect both animals and personnel.
Lab Anim, 1995, 29, 139-151.
Hawkins P, D Anderson, K Applebee, D Key, J
Wallace, G Milite, J MacArthur Clark, R
Hubrecht & M Jennings. Individually ventilated
cages and rodent welfare: Report of the 2002
RSPCA/UFAW rodent welfare group meeting.
Anim Technol Welf, 2003, 2, 23-34.
Heine WOP. Environmental management in laboratory animal units. Basic Technology and
Hygiene Methods and Practice. Pabst Science
Publishers, Berlin, 1998.
Höglund AU & A Renström. Evaluation of individually ventilated cage systems for laboratory
rodents: cage environment and animal health
aspects. Lab Anim, 2001, 35, 51-57.
Krohn TC, AK Hansen & N Dragsted. The impact of
cage ventilation on rats housed in IVC system.
Lab Anim, 2003, 37, 85-93.
Renström A, G Björing & U Höglund. Evaluation of
individually ventilated cage systems for laboratory rodents: occupational health aspects. Lab
Anim, 2001, 35, 42-50.
Scheer M, C Heinekamp, B Thull, R Wagner, & HP
Scheuber. Performance evaluation of IVC systems - Part 1: Test instructions. Available:
www.lal.org.uk/IVC/index.html
Stotzer H, I Weisse, F Knappen & R Seitz. Die
Retina-Degeneration
der
Ratte.
Arzneimittelforschung, 1970, 20, 811-817.
Voipio HM. How do rats react to sound? Scand J
Lab Anim Sci, 1997, 24 (Suppl 1), 1-80.
Weisse I, H Stotzer & R Seitz. Age and light-dependent changes in the rat eye. Virchows Arch
Pathol Anat Physiol Klin Med, 1974, 362, 145156.
103
CHAPTER III
Work for food – A solution to restricted food intake in group housed rats?
Niina Kemppinen, Anna Meller, Kari Mauranen, Tarja Kohila and Timo Nevalainen. 2008.
Scandinavian Journal of Laboratory Animal Science 35:81-90.
Reprinted with permission from Scandinavian Journal of Laboratory Animal Science. Copyright
(2008) the Scandinavian Journal of Laboratory Animal Science.
Scand. J. Lab. Anim. Sci. 2008 Vol. 35 No. 2
Work for Food – A Solution to Restricting Food Intake in
Group Housed Rats?
by Niina Kemppinen1,*, Anna Meller1, Kari Mauranen2, Tarja Kohila1 & Timo Nevalainen3, 4
Laboratory Animal Centre and 4Department of Basic Veterinary Sciences, University of Helsinki, Finland
2
Department of Mathematics and Statistics and 3National Laboratory Animal Center, University of Kuopio, Finland
1
Summary
Rodents spend a great proportion of their time searching for food. The foraging drive in rats is so strong
that the animals readily work for food even when food is freely available. Commonly used ad libitum feeding is associated with a reduced life span, increased incidence of tumours and risk of liver and kidney diseases. It is also considered to be the most poorly controlled variable in rodent bioassays. The aim of this
study was to assess whether rats will gnaw wood in order to obtain food hidden in wooden walls, whether
this activity has a beneficial effect on controlling weight gain, and whether a typical diurnal activity rhythm
is maintained. A total of 18 BN/RijHsd and 18 F344/NHsd male rats were housed in either open or individually ventilated cages (IVC), three rats in each cage. 10 of 36 were fitted with a telemetric transponder.
Four groups were used: two groups (diet board and plain board) with a maze made of two crossed aspen
boards, the third having a rectangular aspen tube. One maze was of plainboard, but the other included
drilled holes snugly loaded with food pellets, the “diet board”, such that the rats had to gnaw wood to reach
the food. The other two groups – and the controls – were fed ad libitum. The study used a crossover design
and the added item was changed every two weeks. Rats, added items, and amount of food left at the end of
the two week period were weighed. The statistical assessment showed that in terms of weight gain there
was a significant interaction both in IVC- (p = 0.005) and in open cages (p < 0.001) between the strains
and the group. In the F344 rats the diet board was more effective in controlling weight, but when combining the strains, all comparisons with diet board were significant (p < 0.05). Use of strain and added item
as main effects, and age as covariate, showed that in the IVC-system there was a significant (p < 0.001)
interaction between the strain and the group, this effect being rather clear in the F344 rats in terms of
amount of food disappearing. In the open cage system, both strain and group were significant (p < 0.001)
factors; all three comparisons with diet board were significant (p < 0.001) in the amount of food disappearing. In conclusion, the work-for-food approach appears to be a promising way of avoiding obesity
without causing untoward effects on diurnal activity in rats. Hence, the approach may have considerable
refinement and reduction potential.
Introduction
Obesity resulting from overeating is a universal
problem; and restricted feeding is the best remedy
to cure obesity-associated problems. This is also
*Correspondence: Niina Kemppinen
Laboratory Animal Centre, P.O. Box 56 FIN - 00014
University of Helsinki, Finland
Tel.
+358 9 191 59070
Fax
+358 9 191 59480
E-mail [email protected]
true in laboratory animals. Laboratory rodents are
commonly fed ad libitum, e.g. food is available all
the time. However, there is ample evidence that ad
libitum feeding increases the incidence of kidney,
heart diseases, and neoplasias and shortens lifespan
in rats (Roe, 1994; Roe et al., 1995; Hubert et al.,
2000).
Keenan et al. (1999) has stated that ad libitum feeding of rodents is the most poorly controlled experimental factor in animal-based research. In the long-
81
Published in the Scandinavian Journal of Laboratory Animal Science - an international journal of laboratory animal science
Scand. J. Lab. Anim. Sci. 2008 Vol. 35 No. 2
term, studies rats die prematurely due to malignancies and degenerative diseases, and this impairs the
statistical sensitivity of the study and leads to more
animals being needed.
Group housing is the preferred method, and indeed
this is a regulatory requirement in Europe (Council
of Europe 2007; European Union 2007). However,
when animals are group housed, there is no practical or effective way to restrict evenly the food intake
of all individuals within the group. Food consumption within the group may also vary, with the dominant animal eating more than the others. When animals are housed individually, restricted feeding is
technically possible, but it may, depending how and
when food is offered, change the diurnal rhythm.
Furthermore, solitary housing is not practical
because it requires more cages, and hence is costly.
Rats are nocturnal animals and in their natural environment they forage for food and eat mainly during
the dark phase because there is less risk posed by
predators. In animal facilities, rats also eat predominantly during the dark period when the food is
available ad libitum (Spiteri, 1982; Strubbe et al.,
1986b; Strubbe & Alingh Prins, 1986), in fact eating during the dark is probably genetically determined (Ritskes-Hoitinga & Strubbe, 2004). It has
been shown that when ad libitum feeding was reinstated after a restricted feeding schedule, the rats
will immediately revert to their original feeding
pattern (Spiteri, 1982; Strubbe et al., 1986b).
Locomotion behaviour also increases if the food
deprivation period is longer than six hours
(Vermeulen et al., 1997); a probable consequence of
food searching behaviour.
Daily feeding activity and other diurnal rhythms are
controlled by the circadian oscillator, which is
located in the suprachiasmatic nuclei in the hypothalamus (Stephan, 1984; Strubbe, et al., 1987;
Ritskes-Hoitinga & Chwalibog, 2003). When rats
are fed with restricted feeding they have access to
food for a few hours, and in most cases this coincides with the housing facility's working hours. In
this kind of situation, they eat all the food immediately, which will impair both natural feeding pat-
82
terns and gastrointestinal physiology. This can lead
to a phase-shift of many biochemical and physiological functions in the gastrointestinal tract of nocturnally active rodents and further changes in serum
insulin and glucose (Strubbe & Alingh Prins, 1986;
Strubbe, 1987; Rubin et al., 1988), mucosal
enzymes of small intestine (Saito et al., 1975) and
bile flow (Ho & Drummond, 1975) in rats.
Moreover, it has also been shown that an altered
feeding schedule results in changes of blood pressure, heart rate and behavioural activity of rats (van
den Buuse, 1999).
A decrease in rat food intake in the early studies
was achieved with meal feeding; i.e. rats had access
to food for only couple of hours a day (Saito et al.,
1975; Stephan, 1984; Strubbe, & Alingh Prins,
1986; Roe et al., 1995; van den Buuse, 1999), or
simply offering them a certain amount of food
(Vermeulen et al., 1997; Markowska, 1999; Hubert
et al,. 2000). However, these methods necessitate
solitary housing of rats.
There are studies trying to combine group housing
and restricted feeding. Johnson et al. (2004) covered the feeding area except for a one cm wide slot,
where the food was available to the rats. In the same
study they also had a “foraging device”, where rats
had to work, i.e. to move gravel for access to food.
With the slot approach the rats spent more time
feeding but consumed less food and with no effect
on body weight. The rats preferred eating from the
“foraging device”, and though they had to work for
food, the body weights of these rats were even significantly higher than in ad libitum fed controls. A
third approach that had been tried is the addition of
largely indigestible sugar beet pulp fibre to the
chow; there were reduced weight gain benefits, but
also enlarged GI-track - especially caecum - in the
increased fibre-fed group (Eller et al., 2004).
We hypothesized that rats will only work - in this
case gnaw wood - for food they necessarily need,
provided that the work intensity is correctly set. The
aim of this study was to assess whether a novel system of food restriction would have any effect on
weight gain over a short period, food utilisation and
Scand. J. Lab. Anim. Sci. 2008 Vol. 35 No. 2
amount of wood gnawed in adult rats and whether
their time budget differs from ad libitum fed rats.
Materials and Methods
Animals
A total of 18 BN (BN/RijHsd) and 18 Fischer344
(F344/NHsd) male rats, all supplied from Harlan,
(Horst, The Netherlands), were used in this study.
10 of which were fitted with a telemetric transponder (details below). The rats were 25 weeks old and
weighed 280 - 370 g (BN) or 350 - 460 g (F344),
respectively, at the beginning of the experiment.
Animal housing and care
Rats were housed in the same room either in open top
polysulfone cages (Tecniplast, Buguggiate, Italy) or
polysulfone individually ventilated cages (IVC)
(Tecniplast, Buguggiate, Italy) (3 rats / cage). The
cage type used was 1500U ***Eurostandard IV S
(48.0 x 37.5 x 21.0 cm – floor area 1500 cm2) with a
solid bottom and stainless steel wire lid; IVC cages
had their own double lids. The cage floor was covered
with 3.0 l aspen chip bedding (of size 4 x 4 x 1 mm,
4HP, Tapvei Oy, Kaavi, Finland). The cages were
changed weekly. The room temperature was 21.2 ±
0.3°C and relative humidity (RH) 53.5 ± 7.7 %, but
the temperature was 1 – 4 °C and RH 2 – 3 % higher
in the IVCs than both in open cages and in the room.
Artificial lighting with fluorescent tubes (light colour
warm white) were on from 06.00 to 18.00 and the
light intensity at 1 m above floor in the open cages
was 16-18 lx compared to 6–9 lx in the IVC´s. The
sound level adjusted with R-weighting in empty IVC
cages was 20-25 dB(R) compared to 12-18 dB(R) in
the empty open cages, with the corresponding adjusted A-weighting being 45-47 dB(A) and 46-49 dB(A),
respectively. Tap water was provided in polycarbonate bottles and changed once a week and refilled once
in between. For a more thorough description, see
Kemppinen et al. (2008) preceding paper.
Experimental procedure
Animals were housed three animals per cage, one of
them with telemetric transponder. The experiment
utilized a crossover design with two week rounds
and a rotational order. Within both strains there
were two different kinds of mazes (diet board and
plain board) made of two crossed aspen boards
(34.0 x 14.7 x 3.2 cm; 21.1 x 14.7 x 3.2 cm), a rectangular aspen tube (20.0 x 12.0 x 12.0 cm), or controls without any addition (Figure 1). One maze
included holes for food pellets, the diet board,
where rats had to gnaw for food, the other was of
plain board. The items were made out of aspen
because this was the same material as the bedding
presumably with the same emissions.
Figure 1. Illustration of the study groups: A: diet
board, B: plain board, C: tube, D: control. Both
strains had one of each added item for two weeks in
both the IVCs and open top cages.
Irradiated (25 kGy) pelleted feed (2016 Global
Rodent Maintenance, Harlan Teklad, Bicester, UK)
was offered to three groups (plain board, tube and
control groups) ad libitum, while the diet board
group had the food pellets embedded snugly in
drilled holes (12 mm) of the aspen board. The feed
was added once a week and weighed. The aspen
boards were weighed before and after the food pellets were placed into the holes. These diet boards
were changed once a week. After the change, the
remaining food pellets were removed from the dietboards and weighed. Rats were weighed before and
83
Scand. J. Lab. Anim. Sci. 2008 Vol. 35 No. 2
after every study round. All the aspen items were
weighed before use and at cage change.
In addition, to assess the effect of the various feeding regimens on the rats’ physiological activity and
heart rate, ten rats had been implanted with a radio
telemetry transmitter (model TA11PA-C40; Data
Sciences International, St.Paul, MN, USA). The
cylinder shape transmitter body (3.0 cm long, Ø 1.5
cm) monitored pressure and activity via a fluid
filled catheter (8 cm long) for sending the signals to
an electronics module. The electronics module
translated the signals into digitized form and transmitted them to the receiver plate located under the
cage. The receiver detected the transmitted signal
and converted it to a form readable by the computer.
The rats were anesthetized with the combination of
fentanyl/fluanisone
(Hypnorm®,
Janssen
Pharmaceutica, Beerse, Belgium) + midazolam
(Dormicum®, Hoffmann - La Roche AG, GrenzachWyhlen, Germany)(0.15 - 0.20 ml/100g SC). The
abdominal area was clipped and then scrubbed with
MediScrub®, 1 % triclosan solution (Medichem
International, Sevenoaks, UK) solution and disinfected with chlorhexidine solution (Klorohexol® 5
mg/ml, Leiras, Turku, Finland), and an ocular lubricant
(Viscotears®,
Novartis
Healthcare,
Copenhagen, Denmark) was applied on both
corneas. A sterile drape was placed over the surgical area and a small area cut away to enable a 3 cm
incision to be made through the skin along the
abdominal midline. The sterile transmitter was presoaked in sterile saline for at least 20 min before the
surgery and then placed into the abdominal cavity,
and the catheter into the abdominal aorta. The transmitter was sutured into the abdominal wall with 40 Ethicon® Ethilon®II (Johnson & Johnson Intl, StStevens-Woluwe, Belgium) and the abdominal and
skin incisions were closed with 5.-0 Ethicon®
Vicryl® (Johnson & Johnson Intl, St-StevensWoluwe, Belgium). After the surgery, the animals
were given twice a day 0.01 – 0.05 mg/kg SC
buprenorphine (Temgesic®; Schering-Plough
Europe, Brussels, Belgium) and once a day a dose
84
of 5 mg/kg SC carprofen (Rimadyl®; Vericore Ltd.,
Dundee, UK) and parenteral fluids for three days.
The pain medication for each rat was titrated with
individual response. All rats were given initially
buprenorphine at the highest dose; this was continued for at least two days; and carprofen medication
for at least three days. The animals were allowed to
recover for ten days before the experiment was
started.
Data processing and statistical analysis
Activity and heart rate were processed for time
budget graphs from the telemetric signals for ten
min periods on the first, third, seventh and 13th night
and the following light period for each night for all
instrumented rats. The number of ten minute periods without activity (activity = 0) were calculated
from the graphs, and comparisons made between
the groups during the 13th night, and between the
days processed in the diet board and plain board
group.
All data was assessed with Kolmogorov-Smirnov
for normality of distribution. Mixed-model repeated
measures ANOVA using strain and group as main
effects and age as covariate was applied to weight,
disappearance of food, wood gnawed and activity
during the dark. Significance was set at p < 0.05.
Results
Calculation on a rat basis showed that with respect
to the weight gain, there was a significant interaction both in IVC (p = 0.005) and in open cages (p <
0.001) between strain and group (Figures 2A &
2B). In F344 rats, the diet board was more effective
in controlling weight, but when combining the
strains, all comparisons with diet board were significant (p < 0.05). When the calculation was done on
a cage basis, then it seemed that only the rats with
the open-cage type diet board displayed any significantly (p = 0.008) reduced weight gain as compared to the plain board group.
In terms of food consumption and in the IVC-system, there was a significant (p < 0.001) interaction
between strain and group, with the effect being
Scand. J. Lab. Anim. Sci. 2008 Vol. 35 No. 2
Weight gain in IVC
A.
BN
A.
BN
Food consumption in IVC
F344
20
10
0
-10
-20
Diet board
Plain board
Tube
Control
F344
Food consumption (g) / 2 weeks
Weight gain (g) / 2 weeks
30
1200
1000
800
600
400
200
0
Diet board
Weight gain in open cage
B.
Plain board
Tube
Control
BN
B.
F344
Food consumption in open cage
BN
F344
20
10
0
-10
-20
Diet board
Plain board
Tube
Control
Figure 2. The weight gain (mean ± SD) of BN and
F344 rats in IVC (A) and open (B) top cages. There
was a significant interaction both in IVCs (p =
0.005) and in open cages (p < 0.001) between strain
and group. In F344 diet board was more effective in
controlling weight, but when combining the strains,
all comparisons with diet board were significant (p
< 0.05).
clear in F344 rats (Figures 3A and 3B). In the open
cage system, both strain and group were significant
(p < 0.001) factors; all three comparisons with diet
board were significant (p < 0.001). When the strains
were pooled, the difference was between 12 - 18 %
less food eaten as compared to the respective controls.
The amount of wood gnawed differed significantly
from normal distribution; hence a mixed model was
applied to the ranks. In terms of the amount of
wood gnawed, there was a significant (p = 0.001 –
0.005) interaction between strain and group in both
cage types. The rats gnawed more wood with diet
Food consumption (g) / 2 weeks
Weight gain (g) / 2 weeks
30
1200
1000
800
600
400
200
0
Diet board
Plain board
Tube
Control
Figure 3. Food consumption (mean ± SD) of BN
and F334 rats in IVC (A) and open top cages (B).
In IVC-system there was a significant (p < 0.001)
interaction between strain and group, and the effect
was quite clear in F344 rats. In the open cage system, both strain and group were significant (p <
0.001) factors; in all three comparisons differences
with diet board were significant (p < 0.001). When
the strains were pooled, the difference was between
12 - 18 % less as compared to respective controls.
board as compared to the plain board and tube
groups in both caging systems. Furthermore, F344
rats gnawed wood more than BN rats (Figures 4A
and 4B).
Typical activity and heart rate recordings for the last
light and dark period of the two week round for
both BN and F344 rats are shown in Figures 5A 5D. Calculation from all diet board and plain board
activities shows that in both cage types there was a
significant interaction (p < 0.001) between the
85
Scand. J. Lab. Anim. Sci. 2008 Vol. 35 No. 2
A.
B.
Strain
BN
F344
10
1
Strain
BN
F344
100
Wood gnawed (g) / 2 weeks
Wood gnawed (g) / 2 weeks
100
10
1
0,1
0,1
-0,1
-0,1
Diet board
Plain board
Tube
Group
Diet board
Plain board
Tube
Group
Figure 4. Amount of wood gnawed shown as box plots in both the IVC (A) and open top cages (B). There
was a significant (p = 0.001 – 0.005) interaction between strain and group in both cage types. The rats
gnawed more wood with diet board as compared to plain board and tube groups in both caging systems.
Furthermore, F344 rats gnawed more wood than the BN rats.
strain and light, with both of the strains being more
active during the dark. F344 rats were significantly
(p < 0.05) more active in the dark phase than BN
rats in both groups. There were no differences in the
activity of the rats between the diet board and plain
board groups.
Discussion
It has been demonstrated that rats prefer to work for
food. Carder & Berkowitz (1970) and Neuringer
(1969) reported that even if the rats had free access
to food they would rather earn their food as long as
the work demands were low. In a preference test,
rats preferred to eat mostly from the foraging device
which required digging gravel to achieve access
(Johnson et al., 2004). This preference of the rats
may reflect their need to perform foraging behaviour as they would in their natural environment.
All the rats with the diet board grew less than other
groups in both cage types; especially in the F344
rats the diet board was effective in controlling
86
weight. The F344 rats lost weight in the diet board
group especially in the open cages, when the rats
were older, but the magnitude of loss was marginal
– only a few grams over two weeks, most likely fat
tissue (Figure 2). The working hypothesis has been
that rats should grow less on the restricted feeding
(Roe et al., 1995; Hubert et al., 2000), but this has
not been observed in all studies. With the “foraging
device”, the weight gain of the rats was higher than
in ad libitum fed controls; and when the rats had
limited access to food, their body weights remained
unchanged, both being indications that the approach
had been unsuccessful (Johnson et al., 2004).
Eller et al. (2004) have tried to determine whether
consuming sugar beet pulp fibre made from watersoluble polysaccharides would have any effect on
the weight gain of rats. The rats indeed grew less
with the fibre diet, but autopsy after the study
revealed an enlarged digestive system in the rats
that had received the fibre enriched diet – especially the caecum was enlarged. This may be attributa-
Scand. J. Lab. Anim. Sci. 2008 Vol. 35 No. 2
BN16 Diet Board - IVC - dark
A.
600
60
40
Activity
Heart rate (bpm)
500
400
20
300
200
6:00
5:00
4:00
3:00
2:00
1:00
0:00
23:00
22:00
21:00
20:00
19:00
18:00
0
HR
Activity
Time (h)
BN16 Diet Board - IVC- light
B.
60
600
40
Activity
Heart rate (bpm)
500
400
20
300
0
18:00
17:00
16:00
15:00
14:00
13:00
12:00
11:00
10:00
9:00
8:00
7:00
6:00
200
HR
Activity
Time (h)
F5 Diet Board - IVC - dark
C.
60
600
40
400
Activit y
Heart rate (bpm)
500
20
300
200
6:00
5:00
4:00
3:00
2:00
1:00
0:00
23:00
22:00
21:00
20:00
19:00
18:00
0
HR
Activity
Time (h)
F5 Diet board - IVC - light
D.
600
60
40
400
Activity
Heart rate (bpm)
500
20
300
200
Time (h)
18:00
17:00
16:00
15:00
14:00
13:00
12:00
11:00
10:00
9:00
8:00
7:00
6:00
0
HR
Activity
Figure 5. Typical single rat (A = BN – dark, B = BN – light, C = F344 – dark, D = F344 - light) activity
and heart rate (HR) recording for the last 24 h of the two week round. There was a significant interaction
(p = 0.000) between the strain and light, and both strains were more active during the dark. F344 rats were
significantly (p < 0.05) more active in the dark phase than BN rats.
87
Scand. J. Lab. Anim. Sci. 2008 Vol. 35 No. 2
ble to the hygroscopic effect of the fibre.
The F344 rats ate more than the BN rats in all of the
groups. In the open cage system rats ate significantly less in the diet board group compared to the
other three study groups. When the strain specific
data was pooled the difference was between 12 - 18
% less as compared to the respective controls. The
rats in the open cages ate more in the plain board
group than in the two other control groups – apparently because the plain board round followed the
diet board round, and the animals regained their
weight loss in that round (Figure 3). In the study of
Johnson et al. (2004) the rats consumed less food
when they had limited access to food, while the
contrary was true with the “foraging device”, both
as compared to controls.
The rats gnawed the wood most with the diet board
as compared to plain board and tube groups in both
cage types. This was unavoidable task if they
wished to eat the food pellets. The F344 rats
gnawed wood significantly more than the BN rats this may relate to a difference in the natural behaviour of these two rat strains (Figure 4). Eskola et al.
(1999) have shown that rats would spontaneously
gnaw aspen blocks and tubes but this opportunity
for gnawing combined with ad libitum feeding had
no effect on the growth of Wistar rats, a situation
similar to F344 rats in plain board and tube groups.
The F344 rats were significantly more active during
the dark than the BN rats in both cage systems.
There were no differences in the activity between
the plain board and diet board groups suggesting
that working for food was not overly strenuous to
the rats. Furthermore, the activity of the rats at that
time did not differ from their activity during ad libitum feeding. It has been shown that when rats have
limited access to food they spend more time feeding, but with the ”foraging device” the time spent
feeding was markedly decreased. There were only
negligible changes between the study groups in
their relative total activity levels (Hawkins et al.,
1999; Johnson et al., 2004). There were no changes
in the social hierarchy of the rats and no increased
fighting or stereotype behaviour when rats had lim-
88
ited access to food (Hawkins et al., 1998).
The rats eat most of their food in the dark. In the
study of the Spiteri (1982) the rats consumed 94 %
of their food intake during the dark. The normal
feeding activity of rats consists of two peaks during
the dark, the first one at the beginning of the dark
phase and the other at the end (Spiteri, 1982;
Strubbe et al., 1986a). Light is a strong ‘Zeitgeber’
because it shifts the clock in a circadian timedependent way ensuring synchrony with the external light-dark cycle. The feeding activity and other
diurnal rhythms are controlled by the circadian
oscillator of the suprachiasmatic nuclei in the hypothalamus. It has been claimed that there are more
oscillators involved in the circadian system and this
provides the flexibility needed for adaptation to different external and internal stimuli (AnglésPujolrás et al., 2006).
When the rats are given access to meals at set times
for a few hours each day, they eat all the food
almost instantaneously and spend the rest of the day
without food; this impairs their natural feeding
activity and associated gastrointestinal physiology.
This study used the diet board for food restriction
allowing rats to enjoy a natural feeding pattern and
indeed feeding activity was similar to the plain
board group (Figure 5).
The diet-board has clear advantages over previous
methods of restricted feeding. The rats can eat at
any time and in addition is unlikely to alter biochemical and physiological phenomena timed by
circadian rhythm, as opposed to set meal times and
most other, if not all, restricted feeding methods
(Ho & Drummond, 1975; Saito et al., 1975;
Stephan, 1984; Strubbe et al., 1986b; Strubbe et al.,
1987; Rubin et al., 1988; van den Buuse, 1999). We
conclude that the diet board seems to be a promising way to control obesity and health problems in
laboratory rats. Challenging questions still need to
be answered to determine whether this approach has
refinement and reduction potential.
Scand. J. Lab. Anim. Sci. 2008 Vol. 35 No. 2
Acknowledgements
This study has received financial support from the
Finnish Ministry of Education, the Academy of
Finland, the ECLAM and ESLAV Foundation, the
North-Savo Cultural Foundation, the Finnish
Research School for Animal Welfare, the Oskar
Öflund Foundation and the University of Kuopio..
We acknowledge Mr Heikki Pekonen for surgical
procedures.
References
Anglés-Pujolrás M, JJ Chiesa, A Díez-Noguera & T
Cambras. Motor rhythms of forced desynchronized rats subjected to restricted feeding.
Physiol Behav, 2006, 88, 30 – 38.
Buuse van den M. Circadian rhythms of blood pressure and heart rate in conscious rats: effect of
light cycle shift and timed feeding. Physiol
Behav, 1999, 68, 9 – 15.
Carder B & K Berkowitz. Rats' preference for earned in comparison with free food.
Science, 1970, 167, 1273 - 1274.
Council of Europe. Appendix A of the European
Convention for the Protection of Vertebrate
Animals used for Experimental and Other
Scientific Purposes (ETS No. 123). Guidelines
for Accommodation and Care of Animals.
Strasbourg 2007.
Eller E, J Lawson & M Ritskes-Hoitinga. The
potential of dietary fiber to achieve dietary
restriction. Abstract. Internationalisation and
Harmonisation of Laboratory Animal Care and
Use Issues. 9th FELASA symposium, Nantes,
France 2004.
Eskola S, M Lauhikari, H-M Voipio & T
Nevalainen. The use of aspen blocks and tubes
to enrich the cage environment of laboratory
rats. Scan J Lab Anim Sci, 1999, 26, 1 – 10.
European Union. Commission Recommendation
on Guidelines for the Accommodation and Care
of Animals Used for Experimental and Other
Scientific Purposes (2007/526/EC). Brussels
2007.
Hawkings P, M Heath, C Dickson, D Wrightson, P
Brain, G Grant & R Hubrecht. Report of the
1998 RSPCA/UFAW rodent welfare group
meeting. Anim Technol, 1999, 50, 41 – 49.
Ho KJ & JL Drummond. Circadian rhythm of biliary excretion and its control mechanisms in rats
with chronic biliary drainage. Am J Physiol,
1975, 229, 1427 – 1437.
Hubert M-F, P Laroque, J-P Gillet & KP Keenan.
The effects of diet, ad libitum feeding, and
moderate and severe dietary restriction on body
weight, survival, clinical pathology parameters,
and cause of death in control Sprague-Dawley
rats. Toxicol Sci, 2000, 58, 195 - 207.
Johnson SR, EG Patterson-Kane & L Niel. Foraging
enrichment for laboratory rats. Anim Welf,
2004, 13, 305 - 312.
Keenan KP, GC Ballam, KA Soper, P Laroque, JB
Coleman & R Dixit. Diet, caloric restriction,
and the rodent bioassay. Toxicol Sci, 1999, 52
(Suppl), 24 - 34.
Kemppinen N, A Meller, E Björk, T Kohila & T
Nevalainen. Exposure in the shoebox: comparison of physical environment of IVC- and open rat
cages. Scan J Lab Anim Sci, 2008, 35, 97-103.
Markowska AL. Life-long diet restriction failed to
retard cognitive aging in Fisher-344 rats.
Neurobiol Aging, 1999, 20, 177 - 189.
Neuringer AJ. Animals respond for food in the presence of free food. Science, 1969, 166, 399 –
401.
Ritskes-Hoitinga M & A Chwalibog. Nutrient requirements, experimental design, and feeding
schedules in animal experimentation. In:
Handbook of laboratory animal science, second
edition. Ed by Hau J & Hoosier GL, CRC Press,
Boca Raton, 2003, 281 - 310.
Ritskes-Hoitinga M & JH Strubbe. Nutrition and
animal welfare. In: The welfare of laboratory
animals. Ed by Kaliste E, Kluwer Academic
Publishers, 2004, 51 – 80.
Roe FJC. Historical histopathological control data
for laboratory rodents: valuable treasure or
worthless trash? Lab Anim, 1994, 28, 148 - 154.
Roe FJC, PN Lee, G Conybeare, D Kelly, B Matter,
89
Scand. J. Lab. Anim. Sci. 2008 Vol. 35 No. 2
D Prentice & G Tobin. The biosure study: influence of composition of diet and food consumption on longevity, degenerative diseases and
neoplasia in Wistar rats studied for up to 30
months post weaning. Food Chem Toxic, 1995,
33, 1S - 100S.
Rubin NH, G Alinder, WJ Riedtveld, PL Rayford &
JC Thompson. Restricted feeding schedules
alter the circadian rhythms of serum insulin and
gastric inhibitory polypeptide. Regul Pept,
1988, 23, 279 – 288.
Saito M, E Murakami, T Nishida, Y Fujisawa & M
Suda. Circadian rhythms in digestive enzymes
in the small intestine of rats. I. Patterns of the
rhythms in various regions of the small intestine. J Biochem, 1975, 78, 475 – 480.
Spiteri NJ. Circadian patterning of feeding, drinking and activity during diurnal food access in
rats. Physiol Behav, 1982, 28, 139 - 147.
Stephan FK. Phase shifts of circadian rhythms in
activity entrained to food access. Physiol Behav,
1984, 32, 663 - 671.
90
Strubbe JH, J Keyser, T Dijkstra & AJ Alingh Prins.
Interaction between circadian and caloric control of feeding behaviour in the rat. Physiol
Behav, 1986a, 36, 489 - 493.
Strubbe JH, NJ Spiteri & AJ Alingh Prins. Effect of
skeleton photoperiod and food availability on
the circadian pattern of feeding and drinking in
rats. Physiol Behav, 1986b, 36, 647 - 651.
Strubbe JH & AJ Alingh Prins. Reduced insulin
secretion after short-term food deprivation in
rats plays a key role in the adaptive interaction
of glucose and fatty free acid utilization.
Physiol Behav, 1986, 37, 441 - 445.
Strubbe JH, AJ Alingh Prins, J Bruggink & AB
Steffens. Daily variation of food-induced changes in blood glucose and insulin in the rat and
the control by suprachiasmatic nucleus and the
vagus nerve. J Auton Nerv Syst, 1987, 20, 113
- 119.
Vermeulen JK, A de Vries, F Schlingmann & R
Remie: Food deprivation: common sense or
nonsense? Anim Technol, 1997, 48, 45 - 54.
CHAPTER IV
The effect of dividing walls, a tunnel and restricted feeding on cardiovascular
responses to cage change and gavage in rats (Rattus norvegicus).
Niina Kemppinen, Anna Meller, Kari Mauranen, Tarja Kohila & Timo Nevalainen. 2009. Journal of
the American Association for Laboratory Animal Science 48:157-165.
Reprinted with permission from Journal of the American Association for Laboratory Animal
Science. Copyright (2009) the American Association for Laboratory Animal Science.
Journal of the American Association for Laboratory Animal Science
Copyright 2009
by the American Association for Laboratory Animal Science
Vol 48, No 2
March 2009
Pages 157–165
The Effect of Dividing Walls, a Tunnel,
and Restricted Feeding on Cardiovascular
Responses to Cage Change and Gavage
in Rats (Rattus norvegicus)
Niina M Kemppinen,1,* Anna S Meller,1 Kari O Mauranen,2 Tarja T Kohila,1 and Timo O Nevalainen3, 4
Cage change and gavage are routine procedures in animal facilities, yet little is known about whether housing modifications
change responses to these procedures. Telemetric activity and cardiovascular parameters were assessed in this experiment.
BN and F344 male rats were housed in open or individually ventilated cages, each containing 3 rats, 1 of which had a transponder. A crossover design was used, in which 2 groups were given dividers made of 2 intersecting boards (1 form contained
holes loaded with food pellets; the other did not) and 1 group was given a rectangular tunnel. On day 8 of each 2-wk period,
the cages were changed; on day 11, rats were gavaged. The parameters were evaluated for the first hour and for the following
17 h. Baseline values for each rat were subtracted from the corresponding response values. The presence of objects did not
affect the responses of F344 rats to cage changing or gavage. In BN rats with IVCs, the presence of the plain divider modified
the response to both procedures. Responses to procedures appeared to be dependent on both the strain and the cage object,
thus complicating the establishment of valid general recommendations.
Abbreviations: bpm, beats per minute; HR, heart rate; IVC, individually ventilated cage; MAP, mean arterial pressure.
The new European regulations on laboratory rodents12,15
mandate the provision of sufficient nest material to build a
complete, covered nest or, if doing so is not possible, providing
a nest box. Because rats are poor nest-builders,19 they must be
provided with objects for this purpose. Moreover, objects that
provide cover or divide the cage area may allow the rats to
initiate or avoid contact with cagemates.35
All these regulations are rather specific, but generally the
same for all laboratory rodents, that is, rats, mice, gerbils, hamsters, and guinea pigs. However, all rodent species and even
strains and stocks within a species may have different needs.
These differences raise the question of whether general guidelines, which may be valid for 1 species, may have a negative
effect on welfare in other species and strains.
Cage change is a frequent routine procedure in animal
facilities that induces temporary, but significant, cardiovascular and behavioral changes in rats.9,10,13,27,29,31-33 Similarly
the frequency9,10 and time29 of changing, type of the bedding
material,9 light intensity, and length of the dark period3 all
modify the intensity of the response to cage changing. The
effects on physiologic parameters, such as blood pressure and
heart rate, after the cage change seem to be a consequence of the
transfer procedure itself and of the novel environment.
Two features of rats suggest potential advantages of placing
objects in the cage. First, rats are known to have a good sense
of smell—1493 specific olfactory receptor genes have been
identified on the cilia of the olfactory neurons—and smell is
Received: 02 Apr 2008. Revision requested: 12 May 2008. Accepted: 11 Aug 2008.
1Laboratory Animal Centre and 4Department of Basic Veterinary Sciences, University of
Helsinki, Finland; 2Department of Mathematics and Statistics and 3National Laboratory
Animal Center, University of Kuopio, Finland.
* Corresponding author. Email: [email protected]
their primary sense for monitoring their environment.24 Second,
rats have dominance hierarchies in which fighting is essentially
territorial, rather than for any specific object.4 The term ‘skirmishing’ has been used to describe a pattern of behaviors often
assumed to be aggressive in rats; in 1 study,10 the frequency of
skirmishing was increased during the first 15 min after a cage
change. Consequently, cage objects may retain a familiar odor
cue during cage change; the presence of the old item in the new
cage reduces the aggressive behavior of rats that is triggered
by regrouping.10
Gavage is a method widely used to administer test compounds into the stomach of laboratory rats. Rats display
increased blood pressure and heart rate (HR) immediately after
gavage, and these increases may persist for 30 to 60 min after the
procedure.7,40 Furthermore, elevations in plasma corticosterone
levels have been measured in rats after gavage.8 The selection
of the correct administration volume2,7,8,40 and a suitable probe
material40 are important to performing this procedure properly,
but whether housing can be advantageous is unknown.
Housing refinements have not been assessed in regard to
their effect on refining the performance of procedures in rats,
although housing modifications can alter their physiology and
behavior. Rats have lower blood pressure and HR when housed
on bedding compared with a grid or plastic floor.22 Rats also
prefer a cage with shelter to a barren environment,36 perhaps
because they prefer to spend most of the light phase inside the
shelter.14 Rats with a furnished environment are more active
than those which lack such objects,38 and the presence of a shelter in the cage decreases fearfulness.36 Finally, the availability
of cage objects may allow the rats to exhibit species-specific
behaviors.11
157
Vol 48, No 2
Journal of the American Association for Laboratory Animal Science
March 2009
In many previous studies of the effects of cage changing or
gavage,7,8,13,27,29,31-33 the presence of objects in the cages was
not described. However, 1 study of both Sprague–Dawley and
spontaneously hypertensive male rats showed that providing a
multifaceted enrichment program over a week did not affect HR
or systolic blood pressure responses to placement in a standard
rodent restrainer for 60 min.34 However, after removal from the
restrainer, the rats showed a secondary increase in HR and systolic blood pressure that was significantly attenuated in enriched
compared with nonenriched rats of both strains. Moreover,
enriched rats of both strains had lower HR and systolic blood
pressure responses to a variety of procedures, including removal
of a cagemate, tail-vein injection, and exposure to the odor of
urine and feces of stressed male or female rats.34
We hypothesized that cage objects would alter the effect of
cage change and gavage on telemetrically recorded cardiovascular parameters and locomotor activity. This study was designed
to evaluate the effect of an aspen wall divider with or without
restricted feeding and of the presence of an aspen tunnel in the
cage on these measures after routine cage changing and gavage
of laboratory rats in both open-top cages and IVCs.
Materials and Methods
The study was done in the Laboratory Animal Centre,
University of Helsinki. The study protocol was reviewed and
approved by the Animal Ethics Committee of the University
of Helsinki.
Animals. A total of 12 BN (BN/RijHsd) and 12 Fischer 344
(F344/NHsd) male rats, all supplied from Harlan (Horst, The
Netherlands) were used in this study. The rats were 25 wk old
and weighed 280 to 370 g (BN) or 350 to 460 g (F344) at the
beginning of the experiment.
Animal housing and care. For the first 8 wk, all rats were
housed (3 rats per cage) in the same room in polysulfone IVCs
(Tecniplast, Buguggiate, Italy) and then for 8 wk in open-top
polysulfone cages (Tecniplast). The caging (48.0 × 37.5 × 21.0
cm; floor area, 1500 cm2) had a solid bottom and stainless steel
wire lid; each IVC had its own double lid. The cage floor was
covered with 3.0 L aspen chip bedding (4 × 4 × 1 mm; 4HP,
Tapvei Oy, Kaavi, Finland). The cages were changed weekly.
The room temperature was 21.2 ± 0.3 °C and relative humidity
was 53.5% ± 7.7%, but the temperature was 1 to 4 °C higher and
relative humidity was 2% to 3% higher in IVCs than in open
cages and the room. Artificial lighting with fluorescent tubes
(light color, warm white) was on from 0600 to 1800, and the light
intensity at 1 m above floor in the open cages was 16 to 18 lx
compared with 6 to 9 lx in the IVCs. The sound level adjusted
with R-weighting (adjusted for the hearing sensitivity of rats6)
in empty IVC cages was 20 to 25 dB(R) compared with 12 to 18
dB(R) in empty open cages, with the corresponding adjusted
A-weighting (adjusted for the hearing sensitivity of humans)
being 45 to 47 dB(A) and 46 to 49 dB(A), respectively. Tap water
was provided in polycarbonate bottles, changed once weekly,
and refilled once between the cage changes. A more thorough
description of husbandry has been published previously.20
Experimental procedure. The experiment used a crossover
design with 2-wk periods and a rotational order; the first item
provided to each group was allocated randomly (Figure 2).
Within both strains, variables included 2 kinds of dividers
made of 2 intersecting aspen boards (34.0 × 14.7 × 3.2 cm; 21.1
× 14.7 × 3.2 cm) and a rectangular aspen tunnel (20.0 × 12.0 ×
12.0 cm); control groups lacked these cage additions (Figure 1).
One divider included round holes for food pellets, from which
the rats had to gnaw wood to obtain food (diet board, Figure 1
158
A), whereas the other had no holes or food (plain board, Figure
1 B). The feed was added once weekly and weighed. Data on
food consumption are presented elsewhere.21
Irradiated (25 kGy) pelleted feed (2016 Global Rodent Maintenance, Harlan Teklad, Bicester, UK) was offered to 3 groups
(plain board, tunnel, and control groups) ad libitum, whereas
the diet board group had the food pellets embedded snugly
into drilled holes (12 mm) in the aspen board. The diet board
reduces food consumption by 12% to 18% in both F344 and BN
rats.21 The transition to the diet board was carried out without
acclimation, as for the other items.
Eight rats, 4 BN and 4 F344, were implanted with radiotelemetry transmitters (model TA11PA-C40, Data Sciences
International, St Paul, MN). The cylinder-shaped transmitter
body (length, 3.0 cm; diameter, 1.5 cm) monitors blood pressure and locomotor activity by means of a fluid-filled catheter
(length, 8 cm), which transmits signals to the electronic control
module. The electronic module translates the signals into a
digitized form and transmits them to the receiver plate located
under the cage. The receiver detects the transmitted signal and
converts it to a computer-readable form. For locomotor activity
measurements, the telemetric receiver has 2 antennas, located
at 90º to one another, and the receiver records the difference in
signal strength when an animal is moving in relation to these
antennas.
For implantation of the transmitters, the rats were anesthetized with the combination of fentanyl–fluanisone [Hypnorm
(fentanyl citrate, 0.315 mg/mL; fluanisone, 10 mg/mL), Janssen
Pharmaceutica, Beerse, Belgium] and midazolam [Dormicum
(midazolam, 5 mg/mL), Hoffmann–La Roche AG, GrenzachWyhlen, Germany] at a dose of 0.15 to 0.20 mL per 100 g SC
(1 part Hypnorm, 1 part Dormicum, and 2 parts sterile water).
The abdominal area was clipped, scrubbed with 1% triclosan
solution (MediScrub, Medichem International, Sevenoaks, UK)
solution, and disinfected with chlorhexidine solution (5 mg/
mL; Klorohexol, Leiras, Turku, Finland), and an ocular lubricant (Viscotears, Novartis Healthcare, Copenhagen, Denmark)
was applied on both corneas. A sterile drape was placed over
the surgical area, and a small area was cut away to enable a 3
cm incision to be made through the skin along the abdominal
midline. The sterile transmitter was presoaked in sterile saline
for at least 20 min before surgery and then placed into the abdominal cavity; the catheter was inserted into the abdominal
aorta. The transmitter was sutured onto the abdominal wall with
4-0 absorbable suture (Ethilon II, Johnson and Johnson International, St Stevens Woluwe, Belgium) and the abdominal and
skin incisions were closed with 5-0 suture (Vicryl, Johnson and
Johnson International). After the surgery, the animals were given
buprenorphine twice daily (0.01 to 0.05 mg/kg SC; Temgesic,
Schering–Plough Europe, Brussels, Belgium), carprofen daily
(5 mg/kg SC; Rimadyl, Vericore, Dundee, UK), and parenteral
fluids for 3 d. The pain medication for each rat was titrated
according to individual response. All rats initially were given
buprenorphine at the highest dose for at least 2 d and carprofen
medication for at least 3 d. The animals were allowed to recover
for 10 d before the experiment was started.
After 1 wk of each 2-wk period, the rats were transferred to
a clean cage between 11:00 and 13:00 by lifting the rats by the
body with encircled fingers. Cage cleaning involved replacing
the cage, all of the bedding and the water bottle; the cage lid
and feed were retained. The tunnel and plain board were moved
to the new cage, the diet board was changed. In the ad libitum
feeding groups, more feed was added after weighing.
Cardiovascular effects of cage objects and procedures in rats
Figure 1. Study groups. (A) Diet board. (B) Plain board. (C) Tunnel. (D) Control. Both rat strains (BN and F344) in both IVCs and open cages had
an additional object for 2 wk.
The implanted rats were gavaged 3 d after cage change between 09:00 and 10:00 h; they were lifted from the cage gently
by grasping the chest. The grip was changed to the scruff of the
neck when the rats were in the holder’s lap.5 The rat was gently
stroked twice from chin to the base of the tail, and a stainless
steel gavage tube (length, 84 mm; shaft diameter, 1.2 mm) was
passed through the esophagus into the stomach and maintained
in that position for 3 s before being retracted slowly; nothing
was administered. The time line of the study is illustrated in
Figure 2.
Data processing and statistical analysis. Means of locomotor
activity, mean arterial pressure (MAP), and HR were processed
at 5-min periods for the first hour after the procedure and for 17
h thereafter at 30-min periods, separately for the light and dark
periods and for the 2 cage types. Mean baseline values of MAP
and HR for each rat for the dark and light periods and both cage
types were calculated from recordings obtained on day 7, the
day before the procedures; these values were subtracted from
the corresponding response values. Mixed-model repeated
measures ANOVA (SPSS Windows, version 14.0, SPSS, Chicago,
IL, USA) was used to assess means and parameter responses,
and Bonferroni correction was used for posthoc comparisons.
For activity calculation, group was used as a main effect and age
as the covariate. For the MAP and HR calculation, activity and
parameter baseline values were added into the covariates.
159
Vol 48, No 2
Journal of the American Association for Laboratory Animal Science
March 2009
Figure 2. Cage item order in F344 and BN rats in 1 cage type (the order
was the same in both IVCs and the open cages) and 1 crossover round,
with the timing of the procedures performed within each round.
Response duration for both MAP and HR was calculated as
described previously.5 The resulting durations were compared
by mixed-model repeated measures ANOVA using group,
strain, and cage type as main effects, and age as a covariate.
Individual mean values of control group on day 7 were calculated separately for light and dark periods; these values were
processed to cage type- and strain-specific average night–day
difference values.
Results
Cage change. The locomotor activity response during the first
hour after IVC cage change was higher (P < 0.001) in the tunnel
group of F344 rats than in the control and in the plain board
groups (Figure 3A). In the open-top cages during the first dark
period, the F344 rats in the plain board group were significantly
less active as compared with both control (P < 0.01) and tunnel
(P < 0.05) groups (Figure 3C). Overall, the locomotor activity
response decreased (P < 0.001) in both strains after the first hour
(Figure 3). MAP and HR response durations exhibited no differences between the cage items. The night–day difference values
for the MAP and HR in the control group before the procedures
are illustrated in Table 1.
F344 rats and MAP response. In the IVCs, the F344 rats displayed a significant (P < 0.001) difference between item MAP
responses only during the subsequent dark period, during
which the MAP of the diet board group was higher than that
of the controls and the other 2 groups (Figure 4C). In the open
cages during the first hour, the MAP responses experienced by
the plain board F344 rats were significantly higher than those
of the control group (P < 0.05) and the tunnel group (P < 0.01;
Figure 4 A). Later, during the dark period, the MAP response
was lower in the diet board group compared with the control (P
< 0.05) and plain board (P < 0.01) F344 groups (Figure 4 C).
BN rats and MAP response. No differences between groups
of BN rats were detected in either cage type during the first
60 min after cage change (Figure 4 A). During the remaining
light period in the IVCs, the BN tunnel group displayed a significantly larger MAP response than did the control (P < 0.001)
and diet board (P < 0.05) groups. In the open-top cages, the diet
board BN animals exhibited a significantly (P < 0.01) higher
MAP response compared with controls (Figure 4 B). During the
160
Figure 3. Activity (mean ± SEM) of rats after cage change in IVCs and
open-top cages. (A) First hour after cage change. (B) From 1 h after
cage change to start of dark period. (C) First dark period after cage
change. Overall there are 16 experimental units, 4 animals in 4 rounds.
*, P < 0.05; #, P < 0.01; §, P < 0.001.
subsequent dark phase in the IVCs, the plain board BN group
expressed a significantly (P < 0.01) smaller MAP response than
did the control group, and in the open-top cages, the tunnel
group exhibited a significantly (P < 0.05) lower MAP response
than did control animals (Figure 4 C).
F344 rats and HR response. In both cage types of F344 rats,
no differences HR response were seen before the dark period
(Figure 5 A, B). The HR response of IVC F344 rats during the
first dark phase was significantly (P < 0.05) lower in those exposed to the plain board as compared with the diet board. In
open-top cages, the HR response was significantly decreased
in F344 rats with the diet board in comparison with control rats
Cardiovascular effects of cage objects and procedures in rats
Table 1. The night and day baseline values and night–day differences of MAP (mmHg) and HR (bpm) of control F344 and BN rats in 2 different
cage types (IVC and open top).
IVC
Open top
F344
BN
F344
BN
MAP
HR
MAP
HR
MAP
HR
MAP
HR
Night
115.3
376.4
93.8
309.0
113.1
395.1
92.4
301.3
Day
108.9
320.9
92.0
277.6
105.2
327.1
91.1
282.8
6.4
55.5
1.8
31.4
7.9
68.0
1.3
18.5
Night – day
Figure 4. MAP response (change from baseline ± SEM) of rats to cage
change in IVCs and open-top cages. (A) First hour after cage change.
(B) From 1 h after cage change to start of dark period. (C) First dark
period after cage change. The y axis scale is the same in all figures.
Overall there are 16 experimental units, 4 animals in 4 rounds. *, P <
0.05; #, P < 0.01; §, P < 0.001.
Figure 5. HR response (change from baseline ± SEM) of rats to cage
change in IVCs and open-top cages. (A) First hour after cage change.
(B) From 1 h after cage change to start of dark period. (C) First dark
period after cage change. The y axis scale is the same in all figures.
Overall there are 16 experimental units, 4 animals in 4 rounds. *, P <
0.05; #, P < 0.01; §, P < 0.001.
(P < 0.05) and the plain board (P < 0.001) and tunnel (P < 0.05)
groups (Figure 5 C).
BN rats and HR response. Regardless of cage type, no differences in HR response between groups of BN rats were detected
during the first hour or the first dark period after the cage change
161
Vol 48, No 2
Journal of the American Association for Laboratory Animal Science
March 2009
(Figure 5 A, C). During the period after the first hour until the
start of the dark period in IVCs, there was a significantly (P <
0.05) higher HR response in BN rats with the tunnel compared
with the diet board group (Figure 5B).
IG-gavage
Locomotor activity response to gavage did not differ between
groups of both strains in both cage types until the first dark
period, when the F344 rats in IVCs with plain boards were
significantly more active than control rats (P < 0.01) and those
with diet boards (P < 0.05; Figure 6 A). The locomotor activity
response in both strains and cage types diminished (P < 0.001)
after the first hour (Figure 6). There were no significant differences in the durations of the MAP and HR responses.
F344 rats and MAP response. The F344 rats living in open-top
cages and with access to the tunnels had a significantly smaller
MAP response during the first hour after gavage than did control (P < 0.01) and plain board (P < 0.001) groups, whereas from
that point until dark, the plain board group exhibited a higher
(P < 0.001) MAP response compared with those of all other
groups, including the controls (Figure 7 A, B). During the subsequent dark phase, F344 rats housed in IVCs with diet boards
or plain boards showed smaller (P < 0.001 for both groups) MAP
responses than that of the control group, but in open-top cages,
the F344 rats in the tunnel group had a lower (P < 0.05) response
than did control and plain board rats (Figure 7 C).
BN rats and MAP response. BN rats in IVCs experienced a
lower (P < 0.01) MAP response when the plain board was in the
cage during the first hour after gavage, compared with controls.
During the same period, the BN rats living in open-top cages
with tunnels had a significantly lower MAP response than
did control rats (P < 0.05) or those with diet boards (P < 0.01;
Figure 7 A). During the subsequent light period (before dark),
the MAP response in the IVC plain board group was decreased
compared with those of control (P < 0.05), diet board (P < 0.01)
and tunnel (P < 0.001) groups of BN rats. BN rats in open-top
cages with plain boards displayed significantly (P < 0.01) greater
MAP responses than did those with tunnels (Figure 7 B). During
the dark in IVCs, the response differences disappeared, but in
open-top cages, the MAP responses of the plain board group
of BN rats were still significantly greater than those in control
(P < 0.05), tunnel (P = 0.01), and diet board (P < 0.001) groups
(Figure 7 C).
F344 rats and HR response. Significant response differences
during the first hour after gavage were seen only in rats in IVCs;
that is, HR response was significantly (P < 0.01) lower in the
tunnel group compared with controls (Figure 8 A). During the
remainder of the light period in open-top cages, the plain board
group displayed a significantly (P < 0.001 for all comparisons)
larger HR response compared with that of controls, rats with
diet boards, and with a rats with tunnels (Figure 8 B). During
the subsequent dark phase, groups exposed to the diet board
(P < 0.01) and plain board (P < 0.001) in IVCs had decreased
HR responses when compared with those of controls, and in
open-top cages, the plain board group of F344 rats showed a
significantly higher HR response than did the diet board (P <
0.05) and tunnel (P < 0.01) groups (Figure 8 C).
BN rats and HR response. During the first hour after gavage,
the BN rats in IVCs with tunnels or plain boards had smaller
(P < 0.01 for both comparisons) HR response than did controls,
whereas in open-top cages, the diet board group had higher HR
responses than did controls (P < 0.05) and the tunnel group (P <
0.01; Figure 8 A). During the remaining light period, the plain
board group of BN rats in IVCs displayed a significantly (P <
0.05) lower HR response than did controls, whereas in open-top
162
Figure 6. Activity (mean ± SEM) of rats after gavage in IVCs and opentop cages. (A) First hour after cage change. (B) From 1 h after cage
change to start of dark period. (C) First dark period after cage change.
Overall there are 16 experimental units, 4 animals in 4 rounds. *, P <
0.05; #, P < 0.01.
cages, the BN rats in the tunnel group exhibited a significantly
(P < 0.01 to 0.05) lower response compared with those of the
rats with the 2 types of board (Figure 8 B). During the subsequent dark phase, the rats in the plain board group exhibited a
significantly (P < 0.05) higher HR response compared with the
diet board group in the IVCs and those provided with a tunnel
and living in open-top cages (Figure 8 C).
Discussion
Virtually nothing is known about whether cage objects alter
the responses of rats to routine care and research procedures (for
example, cage change and gavage). Most published studies on
cage change or gavage7,8,13,27,29,31-33,40 either lacked cage items
or details regarding the objects provided or provided the same
item for all groups. Only 1 study34 has addressed the effect of
Cardiovascular effects of cage objects and procedures in rats
Figure 7. MAP response (change from baseline ± SEM) of rats to gavage in IVCs and open-top cages. (A) First hour after cage change. (B)
From 1 h after cage change to start of dark period. (C) First dark period
after cage change. The y axis scale is the same in all figures. Overall
there are 16 experimental units, 4 animals in 4 rounds. *, P < 0.05; #, P
< 0.01; §, P < 0.001.
Figure 8. HR response (change from baseline ± SEM) of rats to gavage
in IVCs and open-top cages. (A) First hour after cage change. (B) From
1 h after cage change to start of dark period. (C) First dark period after
cage change. The y axis scale is the same in all figures. Overall there
are 16 experimental units, 4 animals in 4 rounds. *, P < 0.05; #, P < 0.01;
§, P < 0.001.
a multifaceted enrichment program on procedures in rats. The
present study evaluates the effect of 2 different objects (a tunnel
and dividing boards), 1 of which was used with and without
restricted feeding. To achieve better representation of the rat as
a species, 2 inbred strains of rats (F344 and BN) were used.16
The current study demonstrates that the cardiovascular response to both cage change and gavage in F344 and BN rats is
modified by the cage item added and the strain of rat evaluated.
These intraspecies differences in cardiovascular responses are
not surprising, given that these rat strains differ extensively in
their baseline systolic and diastolic blood pressures and HR,37
plasma corticosterone,28 and brain and pituitary mineralocorticoid receptor levels.23 These strains also differ in their motor
activity level, diurnal rhythm21,37 and behavior.25,26,39 Therefore,
the effect of cage objects on the procedures appears to include
a genetic component. A previous study34 also showed a difference between SHR and SD rats in responses to procedures, and
the authors concluded that making generalized recommendations to the animal care community regarding rat enrichment
programs is difficult.
Cardiovascular telemetry allows continuous recording. The
items themselves resulted in a variable baseline (Table 1), but
this variability was accommodated through adding item-specific
individual baseline values as covariates for both day and night
responses. Consequently, the statistically significant differences
obtained can be considered as true differences between groups.
Overall, even small differences may be important because these
procedures are done in many animals.
163
Vol 48, No 2
Journal of the American Association for Laboratory Animal Science
March 2009
The current study assessed responses to cage change during
3 subsequent time windows. The response during the first hour
after the procedure is considered to result from the combined
effects of lifting and transferring the rat to a new cage and its
exposure to the new environment. The period between the first
hour after a procedure until the start of the dark phase is considered to reflect the rat’s reaction to the new environment during
the inactive (that is, lights-on) period. Finally, the subsequent
12-h active period should reveal any long-lasting consequences
of the cage items.
The immediate MAP and HR responses to gavage appear to
be smaller in magnitude than those associated with cage change
(Figure 4 A and 7 A). In 1 study,40 the immediate responses in
blood pressure and HR to cage change and gavage in an outbred
Wistar stock were essentially the same as ours. Another study30
found a larger increase in the corticosterone level when rats were
moved to a novel environment compared with that associated
with short-term handling. Gavage is a short-term procedure
that is usually considered more invasive than handling. A
feature common to both gavage and handling is that the rats
are returned back to the familiar home cage. In the cage change
procedure, the animals are relocated to a new environment with
new odors; therefore the more intense response to cage change
is not surprising.
The locomotor activity of the rats increased immediately after
placement into the clean cages (Figure 3). This finding agrees
with several studies10,27,29 in which cage change has been shown
to increase the motor activity of rats. Clean cages also change the
behaviors displayed by rats: grooming, eating, drinking, resting, rearing, and bedding manipulation all decrease, whereas
the walking and skirmishing increase immediately after cage
change.10
Skirmishing or fighting of rats after cage changing may be
attributable to the establishment of the dominance hierarchies
within the group and is related to territory4 (for example, the
new cage). Rats investigate their surroundings by ambulating
and rearing.18 As a result of the increased exploratory behavior, cardiovascular parameters and activity increase after cage
changes. Cage objects can provide an odor cue that makes the
new cage more familiar to rats.
The cage change procedure increased MAP, HR, and locomotor activity in all groups, but the values returned to near baseline
within 1 h (Figures 3 to 5). These findings are consistent with
other studies,13,31-33 that also found increased blood pressure
and HR after cage changes. However, in the cited studies, both
parameters returned to baseline within 60 to 180 min.
Seemingly minor differences in the cage changing procedure
can have cardiovascular effects in rats. The HR of the rats is
increased even by moving the cage to a different location in
the cage rack.17 In another study,29 if cage changes took place
in the morning, during the resting period, the systolic and
diastolic blood pressures and HR responses were larger than
those when cage changes occurred during the active period in
the evening.29 Another study1 reported that if rats experienced
a cage change during the light period, they slept less and had
more chromodacryorrhea, reduced thymus weight, increased
aggression, and less object-directed behavior. Consequently, the
authors suggested that performing husbandry procedures during the dark period rather than the light period might improve
the wellbeing of rats. This timing may be impractical, however.
In our current study, cages were changed at about noon—clearly
within working hours.
We used strain-specific reference values (that is, night–day
differences in MAP and HR calculated for the control group) to
164
assess whether statistically significant differences detected in the
current study have biologic or welfare relevance. Based on this
comparison, the statistically significant MAP responses to both
cage change and gavage for F344 rats in IVCs were not greater
than the night–day MAP difference for this strain (that is, 6.4
mm Hg) or the corresponding HR value [that is, 55.5 beats per
minute (bpm)]. Therefore, a biologically valid effect attributable
to cage objects is not apparent in the current study.
The presence of a plain board in IVC-housing BN rats influenced the MAP response to gavage until dark (Figure 7 A and
B). This significant difference exceeded the BN-specific MAP
night–day difference (1.8 mm Hg). However, the corresponding
HR differences (Figure 8 A through C) were less than the HR
night–day difference (31.4 bpm).
The MAP response to cage change of BN rats in the tunnel
group, as compared with both control and diet board groups,
was greater during the second sampling window (Figure 4 B),
and the magnitude of the difference exceeded the corresponding night–day difference. This effect appeared to be related to
the light phase, because during the subsequent dark period, the
presence of the plain dividing board did not change the MAP as
compared with the controls (Figure 4 C). The between-groups
HR differences were not close to the corresponding night-day
difference. BN rats generally rested on top of rather than inside
of the tunnel and tend to be aggressive toward their cagemates.
A single tunnel may not have provided sufficient numbers of
compartments to function as safe havens compared with those
created by inclusion of the plain board. This explanation could
account for the MAP changes that occurred when a tunnel was
present in the cage.
In open-top cages, the F344 night–day difference of the control
group was 7.9 mm Hg for MAP and 68.0 bpm for HR. Although
many statistically significant differences were detected in the
responses to both procedures (Figures 4, 5, 7, and 8), none of
them were large enough to achieve biologic significance.
Compared with the F344 strain, BN rats in open-top cages
showed lower night–day differences than they had shown 8
wk earlier when they were in IVCs (Table 1). In BN rats, the
presence of the tunnel changed responses to gavage during
the first hour in terms of the MAP response, as compared with
the diet board and control groups (Figure 7 A), although with
respect to HR, the tunnel-associated difference was significant
only in comparison to the diet board (Figure 8 A). Thereafter
the presence of the plain board lessened the response, especially
in MAP during the subsequent dark period (Figure 7 B, C). A
similar trend occurred for HR, but a biologically meaningful effect occurred only for comparison of the plain board and tunnel
during the 5 h before the dark period (Figure 8 B).
After the cage change, the BN rats showed no significant
differences in HR. The change in MAP in BN rats supplied
with a diet board, as compared with the controls, reduced the
response during the period from 1 h after cage change until dark
(Figure 4 B). Perhaps the new diet board allowed easier access
to food than did the old, worn-out board, thereby leading to
the changes in MAP.
When living in open-top cages, rats can smell other rats in the
room. This situation may influence postprocedural responses.
Comparison of the cage types used in the present study is difficult due to the 8-wk age difference in the rats in the 2 cage
types and the different physical environments.20
Overall, cardiovascular telemetry allowed us to assess the
impact of cage objects on responses to procedures. However, the
importance of statistically significant effects must be interpreted
with regard to biologic significance. In this regard, we detected
Cardiovascular effects of cage objects and procedures in rats
no meaningful effect of cage objects on the responses of F344
rats to either cage change or gavage. In BN rats, the presence of
the plain board in the IVC modified the responses to both procedures. The only effect observed in open-top cages during the
first 60 min was associated with gavage and the presence of the
tunnel in BN rats. In conclusion, the response of rats to various
husbandry procedures appears to be specific to strain and cage
objects, and perhaps also to age and cage type, complicating the
establishment of valid general recommendations.
Acknowledgments
This study was supported by the Academy of Finland, the Finnish
Ministry of Education, ECLAM and ESLAV Foundation, the North-Savo
Cultural Foundation, the Finnish Research School for Animal Welfare,
the Oskar Öflund Foundation, and the University of Kuopio. We acknowledge Mr Heikki Pekonen for help with the surgical procedures
and Ms Virpi Nousiainen for carrying out the gavage procedure.
References
1. Abou-Ismail UA, Burman OHP, Nicol CJ, Mendl M. 2008. Let
sleeping rats lie: does the timing of husbandry procedures affect
laboratory rat behavior, physiology, and welfare? Appl Anim
Behav Sci 111:329–341.
2. Alban L, Dahl PJ, Hansen AK, Hejgaard KC, Jensen AL, Kragh M,
Thomsen P, Steensgaard P. 2001. The welfare impact of increased
gavaging doses in rats. Anim Welf 10:303–314.
3. Azar TA, Sharp JL, Lawson DM. 2008. Effect of housing rats in
dim light or long nights on heart rate. J Am Assoc Lab Anim Sci
47:25–34.
4. Barnett SA. 1958. An analysis of social behaviour in wild rats. Proc
Zool Soc Lond 130:107–152.
5. Batūraitė Z, Voipio HM, Rukšenas O, Luodonpää M, Leskinen
H, Apanavičiene N, Nevalainen T. 2005. Comparison of and habituation to four common methods of handling and lifting of rats
with cardiovascular telemetry. Scand J Lab Anim Sci 32:137–148.
6. Björk E, Nevalainen T, Hakumäki M, Voipio HM. 2000. Rweighting provides better estimation for rat hearing sensitivity.
Lab Anim 34:136–144.
7. Bonnichsen M, Dragsted N, Hansen AK. 2005. The welfare impact
of gavaging laboratory rats. Anim Welf 14:223–227.
8. Brown AP, Dinger N, Levine BS. 2000. Stress produced by gavage
administration in the rat. Contemp Top Lab Anim Sci 39:17–21.
9. Burn CC, Peters A, Day MJ, Mason GJ. 2006. Long-term effects
of cage-cleaning frequency and bedding type on laboratory rat
health, welfare, and handleability: a cross laboratory study. Lab
Anim 40:353–370.
10. Burn CC, Peters A, Mason GJ. 2006. Acute effects of cage cleaning
at different frequencies on laboratory rat behavior and welfare.
Anim Welf 15:161–171.
11. Chamove AS. 1989. Environmental enrichment: a review. Anim
Technol 40:155–178.
12. Council of Europe. 2007. [Internet] Appendix A of the European
Convention for the Protection of Vertebrate Animals Used for
Experimental and Other Scientific Purposes (ETS No. 123). Guidelines for accommodation and care of animals. Available at http://
conventions.coe.int/Treaty/en/Treaties/Html/123.htm.
13. Duke JL, Zammit TG, Lawson DM. 2001. The effects of routine
cage-changing on cardiovascular and behavioural parameters in
male Sprague–Dawley rats. Contemp Top Lab Anim Sci 40:17–
20.
14. Eskola S, Lauhikari M, Voipio HM, Nevalainen T. 1999. The
use of aspen blocks and tubes to enrich the cage environment of
laboratory rats. Scand J Lab Anim Sci 26:1–10.
15. European Union. 2007. [Internet] Commission recommendation
on guidelines for the accommodation and care of animals used
for experimental and other scientific purposes (2007/526/EC).
Available at: http://eur-lex.europa.eu/JOHtml.do?uri=OJ:L:200
7:197:SOM:EN:HTML
16. Festing M, Overend P, Gaines Das R, Borja MC, Berdoy M. 2004.
The design of animal experiments. Laboratory animal handbooks,
no. 14. London: The Royal Society of Medicine Press.
17. Gärtner K, Büttner D, Döhler K, Friedel R, Lindena J, Trautschold
I. 1980. Stress response of rats to handling and experimental procedures. Lab Anim 14:267–274.
18. Hughes RN. 1968. Behaviour of male and female rats with free
choice of two environments differing in novelty. Anim Behav
16:92–96.
19. Jegstrup IM, Vestergaard R, Vach W, Ritskes-Hoitinga M. 2005.
Nest-building behaviour in male rats from three inbred strains:
BN/HsdCpb, BDIX/OrlIco and LEW/Mol. Anim Welf 14:149156.
20. Kemppinen N, Meller A, Björk E, Kohila T, Nevalainen T. 2008.
Exposure in the shoebox; comparison of physical environment of
IVC- and open rat cages. Scand J Lab Anim Sci 35:97–103.
21. Kemppinen N, Meller A, Mauranen K, Kohila T, Nevalainen T.
2008. Work for food – a solution for restricted feeding in group
housed rats? Scand J Lab Anim Sci 35:81–90.
22. Krohn TC, Hansen AK, Dragsted N. 2003. Telemetry as a method
for measuring the impact of housing conditions on rats’ welfare.
Anim Welf 12:53–62.
23. Marissal–Arvy N, Mormède P, Sarrieau A. 1999. Strain differences
in corticosteroid receptor efficiencies and regulation in Brown
Norway and Fischer 344 rats. J Neuroendocrinol 11:267–273.
24. Quignon P, Giraud M, Rimbault M, Lavigne P, Tacher S, Morin
E, Retout E, Valin AS, Lindblad-Toh K, Nicolas J, Galibert F.
2005. The dog and rat olfactory receptor repertoires. Genome Biol
6:R83.
25. Ramos A, Berton O, Mormede P, Chalouff F. 1997. A multiple-test
study of anxiety-related behaviours in six inbred rat strains. Behav
Brain Res 85:57–69.
26. Rex A, Sondern U, Voigt JP, Franck S, Fink H. 1996. Strain differences in fear-motivated behavior of rats. Pharmacol Biochem
Behav 54:107–111.
27. Saibaba P, Sales GD, Stodulski G, Hau J. 1996. Behaviour of rats
in their home cages: daytime variations and effects on routine
husbandry procedures analysed by time sampling techniques.
Lab Anim 30:13–21.
28. Sarrieau A, Mormède P. 1998. Hypothalamic–pituitary–adrenal
axis activity in the inbred Brown Norway and Fischer 344 rat
strains. Life Sci 62:1417–1425.
29. Schnecko A, Witte K, Lemmer B. 1998. Effects of routine procedures on cardiovascular parameters of Sprague–Dawley rats in
periods if activity and rest. J Exp Anim Sci 38:181–190.
30. Seggie JA, Brown GM. 1975. Stress response pattern of plasma
corticosterone, prolactin, and growth hormone in rat, following
handling or exposure to novel environment. Can J Physiol Pharmacol 53:629–637.
31. Sharp J, Zammit T, Azar T, Lawson D. 2002. Stress-like responses
to common procedures in male rats housed alone or with other
rats. Contemp Top Lab Anim Sci 41:8–14.
32. Sharp JL, Zammit TG, Lawson DM. 2002. Stress-like responses to
common procedures in rats: effect of the estrous cycle. Contemp
Top Lab Anim Sci 41:15–22.
33. Sharp J, Zammit T, Azar T, Lawson D. 2003. Stress-like responses
to common procedures in individually and group-housed female
rats. Contemp Top Lab Anim Sci 42:9–18.
34. Sharp J, Azar T, Lawson D. 2005. Effects of a cage enrichment
program on heart rate, blood pressure, and activity of male
Sprague–Dawley and spontaneously hypertensive rats monitored
by radiotelemetry. Contemp Top Lab Anim Sci 44:32–40.
35. Stauffacher M, Peters A, Jennings M, Hubrecht R, Holgate B,
Francis R, Elliot H, Baumans V, Hansen AK. 2002. [Internet].
Future principles for housing and care of laboratory rodents and
rabbits. Report for the revision of the Council of Europe Convention ETS 123 Appendix A for rodents and rabbits. Part B. Available
at www.coe.int/t/e/legal_affairs/legal_co-operation/biological_safety%2C_use_of_animals/Laboratory_animals/
36. Townsend P. 1997. Use of in-cage shelters by laboratory rats. Anim
Welf 6:95–103.
37. Van Den Brandt J, Kovács P, Klöting I. 1999. Blood pressure,
heart rate, and motor activity in 6 inbred rat strains and wild rats
(Rattus norvegicus): a comparative study. Exp Anim 48:235–240.
38. van der Harst JE, Fermont PCJ, Bilstra AE, Spruijt BM. 2003. Access to enriched housing is rewarding to rats as reflected by their
anticipatory behaviour. Anim Behav 66:493–504.
39. van der Staay FJ, Blokland A. 1996. Behavioral differences between
outbred Wistar, inbred Fischer 344, Brown Norway, and hybrid
Fischer 344 × Brown Norway rats. Physiol Behav 60:97–109.
40. Okva K, Tamoseviciute E, Ciziute A, Pokk P, Ruksenas O,
Nevalainen T. 2006. Refinements for intragastric gavage in rats.
Scand J Lab Anim Sci 33:243–252.
165
CHAPTER V
Impact of aspen furniture and restricted feeding on activity, blood pressure, heart
rate, and faecal corticosterone and immunoglobulin A excretion in rats (Rattus
norvegicus) housed in individually ventilated cages.
Niina Kemppinen, Jan Hau, Anna Meller, Kari Mauranen, Tarja Kohila and Timo Nevalainen. 2010.
Published online Laboratory Animals, doi: 10.1258/la.2009.009058.
Reprinted with the permission from the Laboratory Animals. Copyright (2009) Laboratory Animals
Limited.
Published online on 23 October 2009 Lab Anim, doi: 10.1258/la.2009.009058
Original Article
Impact of aspen furniture and restricted feeding on activity,
blood pressure, heart rate and faecal corticosterone and
immunoglobulin A excretion in rats (Rattus norvegicus)
housed in individually ventilated cages
N Kemppinen1, J Hau2, A Meller1, K Mauranen3, T Kohila1 and T Nevalainen4,5
1
Laboratory Animal Centre, University of Helsinki, Finland; 2Department of Experimental Medicine, University and University Hospital
of Copenhagen, 3 Blegdamsvej, 2200 Copehagen N, Denmark; 3Department of Mathematics and Statistics; 4National Laboratory Animal
Center, University of Kuopio, PO Box 1627, 70211 Kuopio, Finland; 5Department of Basic Veterinary Sciences, PO Box 66, 00014
University of Helsinki, Finland
Corresponding author: N Kemppinen, Laboratory Animal Centre, PO Box 56, FIN - 00014 University of Helsinki, Finland.
Email: [email protected]
Abstract
This study aims to evaluate the impact of adding different items in individually ventilated rat cages on the animal’s activity,
cardiovascular parameters and faecal stress indicators. The following three cage items made of aspen were compared: a
cross made of two intersecting boards, a similar cross where drilled holes were loaded with food pellets (restricted feeding)
and a rectangular tube. Male rats of the strains BN and F344 (n ¼ 12) were housed in groups of three; one rat in each group
was implanted with a telemetric transponder to measure mean arterial pressure (MAP) and heart rate (HR). In a crossover
design, each group spent 14 days with each type of cage furniture, thereafter faecal pellets were collected for faecal analyses.
The means of activity and means and coefficient of variation for MAP and HR were calculated for days 2, 6, 10 and 14.
As a way of determining which of the statistically significant MAP and HR mean changes were biologically meaningful, the
night –day differences of the controls on day 14 were used. Both board types lowered MAP of F344 rats; hence dividing walls
seem beneficial for F344 welfare. None of the MAP or HR differences in BN rats were biologically significant. No statistically
significant differences in faecal corticosterone or IgA excretion were detected. In conclusion, provision of general
recommendations with respect to cage furniture for rat cages is complicated because there is a clear genetic component
involved in how animals respond to these structures.
Keywords: Refinement, reduction, rodents, environmental enrichment, telemetry
Laboratory Animals 2010: 1 –9. DOI: 10.1258/la.2009.009058
Compliance with the new European regulations mandates
the use of nesting material or nest boxes in rodent
cages.1,2 These regulations are based on clear scientific, primarily ethological, rationale.3,4 Regulatory compliance is
easily achieved by adding structures into the cage, but not
all of these may function as housing refinements, some
may have no value and some may even have a negative
refinement or reduction impact.5 – 9 The potential value of
structures to be placed into rodent cages requires systematic
studies to assess and compare the value of any new item or
combination of items.
Regulatory texts have a tendency to generalize research
findings. Indeed, the current European guidelines on
laboratory animal housing and care are identical for
all rodent species; and within a species they have to be
implemented irrespective of animal strain, and caging
system in use.1,2 One widely used type of housing is the
individually ventilated caging (IVC) system, which has
been shown to change the physical environment inside the
cages compared with that of open cages in terms of illumination, acoustic environment, temperature and relative
humidity (RH).10
When animals are stressed, their attempts to cope
with their situation may be evaluated by the activity of
the sympathetic nervous system, the hypothalamic –
pituitary– adrenal (HPA) axis activity and their behaviour.11
Common feature of all stress assessment methods is the
requirement to sample by using non-disturbing and
Laboratory Animals 2010: 1– 9
Copyright 2009 by the Laboratory Animals Limited
2
Laboratory Animals
................................................................................................................................................
non-invasive methods. Moreover, single indicators may be
misleading, and an evaluation of improvements in cage
environment often requires an assessment of a combination
of indicators.12,13
Radiotelemetry is a method for measuring physiological
parameters, such as blood pressure and heart rate (HR), in
laboratory rodents which allows the animals to move
freely during recording, making any restraint unnecessary.
Indeed, the values of HR and blood pressure are considerably lower in animals implanted with a radiotelemetry
transmitter than the corresponding values obtained with
other methods.14 The effect of the different combinations
of added cage items in rats is an active study area, but
telemetry assessments have rarely been incorporated
into these experiments. The study of Sharp et al. 15 using
Sprague-Dawley (SD) and spontaneously hypertensive
(SH) male rats showed that their multifaceted enrichment
programme reduced HR in both dark and light periods
and increased activity during the afternoons for SH rats.
Corticosteroids and their metabolites are excreted into
urine and faeces, both of which can be regarded as timeintegrated indices of stress. Unlike serum sampling,
samples of both urine and faeces can be obtained noninvasively though the changes in the concentrations in the
circulation occur with a delay of approximately 10 h in
urine and 16 h in faeces.16 About 20% of the recovered
metabolites in rats appear in urine and 80% in faeces.16,17
In a similar manner to serum corticosterone,18 concentrations of its metabolites in faeces follow a diurnal
rhythm; but conversely to the serum corticosterone, the
highest concentrations of the faecal corticosterone metabolites appear to be present in the morning samples.16,19 If
stress persists for a longer time, it may also cause immunosuppression. This can be assessed by quantification of
secretory immunoglobulin A (IgA) in faeces. Faecal IgA
excretion also exhibits a diurnal variation, but the values
detected in morning and evening samples have been somewhat controversial.19 – 21 Faecal samples for both assays are
easy to collect from the cage without disturbing the
animals, enabling long-term, longitudinal studies.22
Rats are crepuscular and nocturnal animals and their blood
pressure and HR are higher during the night than during the
day, but there are major differences between different stocks
and strains.15,23 – 26 In the present study, two rat strains, F344
and BN, were studied in order to elucidate whether
there would be any genetic contribution to the variation in
the results, and to achieve better applicability within the
species.27 These strains were chosen because they differ in
various aspects of their physiology, including locomotor
activity level, diurnal rhythm of activity,25,28 blood pressure,
HR25 and levels of circulating corticosterone.29,30
Laboratory rodents are commonly fed ad libitum, e.g. food
is available all the time, a feeding regime known to cause
obesity, increase the incidence of diseases and shorten lifespan in rats.31 – 33 On the other hand, restrictive feeding
may be difficult to combine with the recommended group
housing of rats,1,2 as there is no practical or effective way to
evenly restrict the food intake of all individuals within the
group. Rats eat predominantly during the dark period
when the food is available ad libitum 34 – 36 and when the
restricted feeding is timed in the light phase in rats, it may
impair both natural feeding patterns and gastrointestinal
physiology. The diet board, where the access to food requires
rats to gnaw wood, has been shown to significantly reduce
the weight gain in rats with 15–18% less food being eaten.28
We hypothesized that a refinement value could be
detected via changes in mean arterial pressure (MAP),
HR, faecal corticosteroid metabolites and faecal IgA, and a
reduction value by changes in the coefficients of variation
(CV) of these parameters, this being attributable to various
cage items and restricted feeding. This study was designed
to evaluate the impact of dividing aspen walls with or
without restricted feeding, as well as an aspen tube on laboratory rats using activity, circulatory parameters and faecal
stress indicators to determine whether two different rat
strains would display variation in their responses, and
whether there would be habituation to the items.
Materials and methods
The study was carried out in the Laboratory Animal Centre,
University of Helsinki. The protocol of the study was
reviewed and approved by the Animal Ethics Committee
of the University of Helsinki.
Animals
A total of 12 BN (BN/RijHsd) and 12 Fischer344 (F344/
NHsd) male rats (Harlan, Horst, The Netherlands) were
used in this study. The rats were 25 weeks old and
weighed 280– 370 g (BN) or 350 –460 g (F344) at the beginning of the study. Upon arrival from the commercial
breeder at the age of 10 weeks, the animals were housed
in IVCs with the same bedding material and the same
social groups as during the study.
Animal housing and care
All rats were housed in the same room in groups of three rats
per cage in polysulphone IVCs (Tecniplast, Buguggiate,
Italy). The cage type used was 1500U Eurostandard IV S
(44.5 33.5 21.0 cm – floor area 1500 cm2) with a solid
bottom and IVC-double lids. The cage floor was covered
with 3.0 L aspen chip bedding (size 4 4 1 mm, 4HP,
Tapvei Oy, Kaavi, Finland). The cages were changed once
weekly. The room temperature was 21.2 + 0.38C (mean +
SD) and the RH was 53.5 + 7.7%, but actual values inside
the IVCs were 1–48C and up to 6% RH higher. Artificial
lighting with fluorescent tubes (light colour warm white)
was on from 06:00 to 18:00 and the light intensity in cages
1 m above the floor was 6–9 lx. The sound level with
R-weighting (adjusted for the hearing sensitivity of rats), in
empty IVC cages, was 20–25 dB(R), with the corresponding
A-weighting (adjusted for the hearing sensitivity of
humans) being 45–47 dB(A).37 Tap water was provided in
polycarbonate bottles, changed once a week and refilled
once in between. Feeding was restricted or ad libitum depending on treatment as outlined below. For a more thorough
description, see Kemppinen et al. 10
Kemppinen et al. Cage furniture, restricted feeding and rat welfare
3
................................................................................................................................................
Cage furniture and study design
Animals were housed in permanent groups of three. The
experiment utilized a crossover design with two-week
periods and a rotational order between control and the
following three cage furniture items (Figure 1):
(1) Cage without furniture (control);
(2) Cage equipped with a cross made of intersecting two
aspen boards (34.0 14.7 3.2 and 21.1 14.7 3.2 cm, plain board);
(3) The same as (2) but holes (12 mm) were drilled into the
boards and then were loaded snugly with food pellets;
rats had to gnaw wood to gain access to the food, no
other food source was available (diet board);
(4) Cage provided with a rectangular aspen tube (20.0 12.0 12.0 cm, external dimensions).
The order of the cage items was arranged at random, and
the first item in each group was randomly allocated. The
illustration of the item order can be seen elsewhere.38
The items were made of aspen because this was the same
material as the bedding and they can be assumed to have
the same volatile compound emissions, in this case
especially the absence of a- and b-pinenes,39 but also to
be able to endure several bouts of sanitation.40 The day
when the rats were introduced to the new cage furniture
was designated day 1 in all study periods. Rats were
changed to clean cages on day 8 at noon during every
period.
Irradiated (25 kGy) pelleted feed (2016 Global Rodent
Maintenance, Harlan Teklad, Bicester, UK) was available
to the three groups (control, plain board, tube) ad libitum,
while the diet board group had the food pellets in holes
drilled into the aspen board. When fed with the diet
board, rats have been shown to eat 12 – 18% less and gain
weight slower.28
Surgical procedure
Eight rats were implanted with a radiotelemetry transmitter
(TA11PA-C40; Data Sciences International, St Paul, MN,
USA). Anaesthesia was induced with a mixture of fentanyl/fluanisone (Hypnormw, Janssen Pharmaceutica,
Beerse, Belgium; one part), midazolam (Dormicumw,
Hoffmann-La Roche AG, Grenzach-Wyhlen, Germany; one
part and two parts of sterile water) at the doses of 0.15 –
0.20 mL/100 g subcutaneously. The ventral abdomen area
was shaved and scrubbed with MediScrubw, 1% triclosan
solution (Medichem International, Sevenoaks, UK)
and disinfected with chlorhexidine solution (Klorohexolw
5 mg/mL, Leiras, Turku, Finland). Ocular lubricant
(Viscotearsw, Novartis Healthcare, Copenhagen, Denmark)
was applied to both eyes. After a small incision on the
ventral abdominal midline had been made, the presoaked
transmitter was placed into the abdominal cavity and
the catheter into the abdominal aorta. The transmitter was
secured into the abdominal wall with 4 –0 Ethiconw
Ethilonw II sutures (Johnson & Johnson Intl, St
Stevens-Woluwe, Belgium) and the incision was closed
with 5 – 0 Ethiconw Vicrylw (Johnson & Johnson Intl).
The surgery procedure lasted about 20–30 min.
Postoperative pain alleviation was carried out with 0.01 –
0.05 mg/kg subcutaneous buprenorphine (Temgesicw;
Schering-Plough Europe, Brussels, Belgium) twice a day
and once a day a dose of 5 mg/kg subcutaneous carprofen
(Rimadylw; Vericore Ltd, Dundee, UK) for at least three
days, and parenteral fluids were given for three days. On
these three days, the implanted rats were housed alone
and then placed back into their home cages. The pain medication for each rat was titrated against the individual
response. The animals were allowed to recover for 10 days
before the experiment was started.
Sampling
Figure 1 Pictures of the cage items used: the diet board above, the plain
board on the left below and the tube on the right below. Both rat strains
(BN and F344) had an added furniture item for two weeks
Mean blood pressure, HR and locomotor activities were
recorded telemetrically every 75 s for 24 h on days 2, 6, 10
and 14. In order to conduct the locomotor activity measurements, the telemetric receiver had two antennae, located at
two sides, like an x- and y-axis, and the receiver detected
the difference in signal strength when the animal moved
in relation to these antennae.
At the end of each period, the rats were housed singly for
6 h (06:00 –12:00) and all faecal pellets voided from each
4
Laboratory Animals
................................................................................................................................................
individual were collected and frozen (2188C). All samples
were analysed individually.
Faecal corticosterone and faecal IgA quantification
The extraction of both corticosterone and IgA was performed
as described by Pihl and Hau.19 The corticosterone enzymelinked immunosorbent assay was performed using a commercial corticosterone kit (DRG Diagnostics, Marburg,
Germany) using the manufacturer’s instruction manual.
The quantification of IgA was performed using the assay
also described by Pihl and Hau19 and reagents were obtained
from AbD Serotec (Kidlington, Oxfordshire, UK; purified rat
IgA standard, PRP01), concentrations 0–1000 ng/mL; coating
antibody (mouse anti rat IgA heavy chain, MCA191); and
detection antibody (mouse anti rat kappa/lambda light
chain: HRP, MCA1296P; diluted 1:1000).
Data processing and statistical analysis
The number of animals needed in the study was estimated
with the resource equation method.27 The cage was used
as an experimental unit, and with the crossover design
used, this resulted in 12 degrees of freedom for error per
strain, well within the optimum range, i.e. 10– 20. Means
and CV for MAP, HR and mean for locomotor activity
were calculated in the light and dark periods for days 2, 6,
10 and 14 in each period for 30 min intervals from the raw
data. The 12 h mean values for MAP and HR of the controls
were subtracted from those of plain board, diet board and
tube groups for the same days, and CV values of four furniture items were used as such. Mixed-model repeated
measures analysis of variance (ANOVA) on SPSS for
Windows (version 14.0) was used, followed by Bonferroni
correction for post hoc comparisons. For locomotor activity,
furniture item was used as the main effect and age as covariate. For MAP, HR and all CVs, furniture item was used
as the main effect and age and activity as covariates.
Analogous calculations were carried out to compare data
within a furniture item between the days; the day was
used as the main effect instead of furniture item. We
assumed that for a cardiovascular effect of housing to be
biologically meaningful, it would have to be at least as
large as the difference between night and day values.
Therefore, mean night – day difference was calculated from
day 14 data for the control group of both rat strains.
Significant CV differences between groups were processed
further to point estimates [¼(CV1/CV2)2]. Significance was
set at P , 0.05 for mean value calculations, but at P , 0.01
for CV statistics to increase the reliability of the findings.
The faecal corticosteroids and faecal IgA results of three
rats from the same cage were averaged and calculated
with repeated measures mixed-model ANOVA using the
age as a covariate.
Results
F344 rats were far more active than the BN rats during the
dark phase, whereas the activity during the light phase
was similar in these strains (Figure 2). In the dark phase,
Figure 2 Activity (SEM) of F344 (a) and BN (b) rats with different cage items
and controls during the dark and light periods in individually ventilated cages
(IVC). P , 0.05; #P , 0.01. Number of experimental units within a strain ¼ 16
F344 rats were significantly (P , 0.01) more active in cages
with diet boards than in the control cage on day 6, while
on day 10, they exhibited significantly (P , 0.05) higher
activity with the tube compared with those living with the
diet board and controls (Figure 2a), but these differences
disappeared by the end of the two-week period. The BN
rats exhibited no differences in activity between the cage
furniture items (Figure 2b).
When calculated from data of both strains and all the
groups, F344 rats displayed higher values of MAP and HR
than BN rats throughout the study. In both dark and light
phases, MAP exhibited a significant (P , 0.001) group strain interaction on all observation days. HR showed a significant (P , 0.05) group strain interaction during the
dark phase from day 6 onwards. Due to the multiple interactions encountered, the following results will be presented
separately for both strains and for each light phase.
Effect of housing item
F344 rats
MAP and HR night – day differences for F344 controls were
5 (+2) mmHg and 52 (+13) BPM at day 14. Throughout the
two-week periods, both during the dark and light phases,
F344 rats living with the tube exhibited increased MAP,
while the other two items decreased MAP compared with
the baseline of the controls; all these comparisons being
statistically significant (P , 0.001). When housed with the
plain board, rats had lower MAP on day 6 light phase
(P , 0.001), on day 10, dark phase (P , 0.01) and on day
14 all throughout the 24 h (P , 0.01 – 0.05). In most cases,
these differences were also biologically significant, i.e.
Kemppinen et al. Cage furniture, restricted feeding and rat welfare
5
................................................................................................................................................
Figure 3 The mean arterial pressure (MAP) and heart rate (HR) differences (SEM) of F344 (a and b) and BN (c and d) rats to controls with three cage items during
the dark and light periods in individually ventilated cages (IVC). BPM: beats per min; P , 0.05; #P , 0.01; §P , 0.001. Number of experimental units ¼ 12
exceeded 5 mmHg (Figure 3a). HR changes are shown in
Figure 3b with statistical significances, but none of the
differences came even close to the HR night – day difference
of the F344 control group, i.e. 52 BPM.
In F344 rats on day 2, the CV of MAP was significantly
higher when the animals were housed with the plain
board than with the two other items and the control cages
during both the dark (all P , 0.01) and the light (all P ,
0.001) phases. On day 10 in the light, the CV of HR was
significantly lower in the diet board animals than in the
control cages (P , 0.01). On day 14, the CV of MAP in
the light phase was significantly (P , 0.01) higher with the
plain board compared with the tube (P , 0.01), while CV
of HR in the dark phase was significantly lower with the
plain board cages than in the tube cages (P , 0.01). All
these results are shown graphically in Figures 4a and b
and in table format with the corresponding point estimates
in Table 1.
rather similar (Figure 3d), but in analogy to F344 rats,
none of the differences reached a biologically significant
value. In BN rats on day 14 in the light phase, the CVs of
the MAP and HR were significantly lower in the diet
board cages than in the control cages (P , 0.01). All possible
comparisons are illustrated graphically in Figures 4c and d
and those statistically significant in table format with the
corresponding point estimates in Table 1.
BN rats
The night –day differences in MAP and HR for BN controls
were 3 (+1) mmHg and 25 (+7) BPM at day 14. In BN rats,
there were few statistically significant changes in MAP and
HR between the different cage items. Overall MAP was significantly (P , 0.001 – 0.05) lower in the diet board cages
than in the plain board cages early in the period; this
became less and finally it stabilized as the lowest of all the
groups at the end of the two-week period (Figure 3c).
Nonetheless, it was only by day 14 that the MAP decrement
of the diet board, as compared with the plain board,
exceeded the limiting night – day difference. The relationship of the HR between the cage furniture items was
Faecal assays
Between days comparison
When the days were compared within the furniture item
during the two-week period in both strains, there was a
decreasing trend both in MAP and HR compared with the
control baseline, and although between-day differences
were small, they were in most cases statistically significant
(P , 0.001 – 0.05), particularly in the F344 rats (Figure 5).
There was a large variation in the number of faecal pellets
voided, from none up to more than 10 pellets from one
animal. No group strain interaction was seen, and none
of the two strains exhibited any between-furniture item
differences in the faecal corticosterone or in faecal IgA
(results only in F344 rats) excreted (Figure 6).
Discussion
The F344 rats consistently had higher MAP and HR than BN
rats, and the dark activity levels of the F344 rats were also
6
Laboratory Animals
................................................................................................................................................
Figure 4 Coefficient of variation (CV) of the mean arterial pressure (MAP) and heart rate (HR) for F344 (a and b) and BN (c and d) rats with three cage items and
control groups during the dark and light periods in individually ventilated cages (IVC). P , 0.05; #P , 0.01; §P , 0.001. Number of experimental units ¼ 16
higher than those of BN rats (Figure 2). This is well in line
with the results of van den Brant et al. 25 who concluded
that the BN strain no longer possesses the typical rodent
nocturnal activity. The present study showed that in comparable environments, F344 rats display larger night –day
differences both in MAP and HR values compared with
the BN rats. This agrees with the results of van den Brant
et al. 25 who categorized the F344 as a ‘hypertensive’ and
the BN as a ‘hypotensive’ rat strain. We compared all the
MAP and HR level differences to the corresponding
night – day difference of the controls, and propose that statistically significant differences in means should exceed the
normal night – day difference, in order to be considered
to be biologically meaningful. The results of this study
suggest that a fixed percentage, e.g. 6 –7% as suggested by
Krohn et al. 41 may not be applicable for all strains, and
that a strain-specific measure, such as night –day difference,
may be more valid for this purpose.
This study shows that cardiovascular responses of F344
rats and BN rats to the added furniture items differed. The
F344 rats had the highest MAP in the tube cages all the
time throughout the two-week period (Figure 3a). It has
been shown that albino Wistar rats prefer a cage with a
shelter rather than a barren environment42,43 and that they
tend to stay inside the tube during the light phase.44 The
elevated MAP, as seen in this study, indicates that this
type of covered structure does not serve the purpose for
which it was intended; a result that contradicts earlier findings.44,45 However, it is possible that the tubes used in the
present study may have been too small to comfortably
accommodate three large rats, and that the finding is applicable to the F344 strain only.
Table 1 P values for significant mean arterial pressure (MAP) and heart rate (HR) coefficient of variation (CV) comparisons between the groups and
corresponding point estimates (PE) for both F344 and BN rats and both light phases on the observation days
F344 rats, P < /PE
Day 2
Plain board/diet board
Plain board/tube
Plain board/control
Day 10
Control/diet board
Day 14
Plain board/tube
Control/diet board
BN rats, P < /PE
MAP dark
HR dark
MAP light
HR light
MAP dark
HR dark
MAP light
HR light
0.01/1.36
NS
0.01/1.56
NS
0.01/1.41
NS
NS
NS
NS
0.01/0.70
NS
NS
0.001/1.42
NS
0.001/1.81
NS
0.001/1.52
NS
NS
0.01/1.75
0.01/1.45
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
0.01/1.45
0.01/1.68
Non-significant comparisons are not shown in the table. NS ¼ not significant
Kemppinen et al. Cage furniture, restricted feeding and rat welfare
7
................................................................................................................................................
Figure 5 The mean arterial pressure (MAP) and heart rate (HR) differences (SEM) to controls of F344 (a and b) and BN (c and d) rats with three
cage items during the dark and light periods in individually ventilated cages (IVC). BPM: beats per min; P , 0.05; #P , 0.01; §P , 0.001. Number of experimental
units ¼ 16
Figure 6 Corticosterone (a) and immunoglobulin A (IgA) (b) excreted (SEM)
via faeces for F344 and BN rats with three cage items during the dark and
light periods in individually ventilated cages (IVC). Number of experimental
units within a strain ¼ 16
Both board structures lowered the MAP values of F344
rats throughout the two-week period and towards the
end, the periods with the plain board seemed to be most
effective in this respect (Figure 3a). Based on this observation, it is likely that the dividing walls represent a suitable
cage item for F344 rats, but also the diet board appears
better than the tube, and certainly also better than no furniture at all. It is interesting to note that both boards, unlike
the tube, could not be used to provide cover, a feature
mandated by the European guidelines.1,2 The diet board
involves the concept of work for food, a feature that
would be expected to raise at least the active period MAP,
but nonetheless the difference was minor compared with
the plain board. For routine animal observation purposes,
dividing walls are clearly better than covered structures
that obscure the animals. This seems to challenge
the concept that all albino rats need to be provided with
structures for hiding places and privacy.44,45 The BN rats
exhibited no biologically significant MAP or HR betweenfurniture item differences.
Only a handful of earlier studies on the effect of new cage
items have utilized telemetry. Sharp et al. 15 evaluated their
enrichment programme in rats with telemetrically recorded
HR, systolic blood pressure and activity. They showed that
HR of SH rats was significantly reduced by their combination of cage items, but for the SD rats they could detect
no effect. Their programme consisted of several smaller
items added to the cage between three and seven days.
8
Laboratory Animals
................................................................................................................................................
There were some similarities between these items and those
used in our study, such as the simulated burrow, which is
comparable to the tube used in the present study, and a
gnawing and food foraging item, which is comparable to
the diet board of our study. A detailed comparison
between the two studies is not relevant because we used
one item at a time for 14 days, as opposed to their combination of items and much shorter exposure time.
When the processed days were compared within the furniture item during the two-week periods, there was a
decreasing trend both in MAP and HR levels in both
strains (Figure 5), but statistical and biological significant
findings were only found for MAP of F344 plain board
rats when comparing days 2 and 14. We interpret this to
mean that the F344 rats had habituated to the plain board,
but not to the other items. Conversely, no such effects
were detectable in BN rats.
The CV of MAP in F344 rats was highest in the plain
board groups on day 2, both in the dark and light phases.
This may simply be a novelty effect of this cage item
which was introduced into the cage one day before
(Figure 4a). The corresponding point estimates showed
that with the plain board and MAP as the result parameters,
the number of animals needed would be 1.36– 1.81 times
greater than those with other items including controls.
Similar results were found between plain board and the
tube on day 14 dark (Table 1). The high MAP seen in the
F344 rats living with the tube was not associated with excessive variation but rather the opposite. In the BN rats, the CV
values of both MAP and HR were higher in the control
groups compared with the diet board groups (Figure 6)
and the corresponding point estimates were 1.45 and 1.68,
respectively (Table 1). Cage furniture may thus have
marked, strain specific, effects on the within-group variation
and this could be anticipated to influence the number of
animals needed in blood pressure studies.
In the present study, the cage furniture items used exhibited no effect on faecal corticosterone or IgA excretion and
there were no differences compared with the rats housed
in control cages. Sarrieau and Mormède30 have shown significantly higher plasma corticosterone levels in F344 rats
compared with BN rats, but we could not detect any significant differences in faecal corticoids between the strains
(Figure 6a). A recent study by Siswanto et al. 46 showed
that injection of at least of 100 mg/kg adrenocorticotropin
hormone is required to detect a subsequent change in the
faecal corticosteroid level in rats. Hence, it was not surprising that the HPA axis was not sufficiently stimulated by the
changes in cage environment for this to be detectable as
changes in faecal corticosteroid excretion. Faecal IgA concentrations in rats have also been measured in response to
housing in metabolic cages20 but not to cage furniture.
Royo et al. 21 stated that stress-induced changes in excreted
IgA concentrations are slower and perhaps less pronounced
than those of corticosteroids; hence, IgA may be useful only
for assessing long-term animal wellbeing. Obviously, the
lack of significant changes in this study suggests that two
weeks may not have been long enough or alternatively the
response was simply too small to be detected with this
assay.
In conclusion, cardiovascular parameters can be used
to assess the welfare value of cage furniture, whereas
changes in faecal corticosterone and faecal IgA excretion
would appear to be too small to be quantifiable.
Furthermore, covered structures may not be any better
than no structure at all in the cage, and the establishment
of general recommendations may be difficult because
there is a clear genetic component involved, resulting in
major between-strain differences.
ACKNOWLEDGEMENTS
This study was supported by the Academy of Finland,
the Finnish Ministry of Education, ECLAM and ESLAV
Foundation, the North-Savo Cultural Foundation, the
Finnish Research School for Animal Welfare, the Oskar
Öflund Foundation and the University of Kuopio. We are
thankful to Mr Heikki Pekonen for surgical procedures
and to Ms Virpi Nousiainen for daily care of the rats.
REFERENCES
1 Council of Europe. Appendix A of the European Convention for the
Protection of Vertebrate Animals Used for Experimental and Other
Scientific Purposes (ETS No. 123). Guidelines for Accommodation and
Care of Animals. Strasbourg, 2006. See http://conventions.coe.int/
Treaty/EN/Treaties/Html/123-A.htm
2 European Union. Commission Recommendation on 18 June 2007 on
Guidelines for the Accommodation and Care of Animals Used for
Experimental and Other Scientific Purposes (2007/526/EC). Brussels,
2007. See http://eur-lex.europa.eu/JOHtml.do?uri=OJ:L:2007:197:SOM:
EN:HTML
3 Council of Europe. Species-specific Provisions for Rodents and Rabbits.
Background Information for the Proposals presented by the Group
of Experts on Rodents and Rabbits. Part B. Strasbourg, 2002. See
http://www.coe.int/t/e/legal_affairs/legal_co-operation/
biological_safety%2C_use_of_animals/laboratory_animals/
GT123(2001)4%20Final%20PART%20B%20Rodents.pdf
4 Olsson A, Dahlborn K. Improving housing conditions for laboratory
mice: a review of environmental enrichment. Lab Anim 2002;36:243 – 70
5 Kaliste EK, Mering SM, Huuskonen HK. Environmental modification
and agonistic behavior in NIH/S male mice: nesting material enhances
fighting but shelters prevent it. Comp Med 2006;56:202 –8
6 Moncek F, Duncko R, Johansson BB, Jezova D. Effect of environmental
enrichment on stress related systems in rats. J Neuroendocrinol
2004;16:423 –31
7 Tsai PP, Pachowsky U, Stelzer HD, Hackbarth H. Impact of
environmental enrichment in mice. 1: effect of housing conditions on
body weight, organ weights and haematology in different strains. Lab
Anim 2002;36:411 – 19
8 Tsai PP, Stelzer HD, Hedrich HJ, Hackbarth H. Are the effects of
different enrichment designs on the physiology and behaviour of DBA/2
mice consistent? Lab Anim 2003;37:314 – 27
9 Tsai PP, Stelzer HD, Schraepler A, Hackbarth H. Importance and effects
of enrichment on physiology, behaviour and breeding performance in
mice. ALTEX 2006;23(Suppl):96– 8
10 Kemppinen N, Meller A, Björk E, Kohila T, Nevalainen T. Exposure in
the shoebox: comparison of physical environment of IVC- and open rat
cages. Scand J Lab Anim Sci 2008;35:97– 103. See http://biomedicum.ut.
ee/sjlas/35_2_97–103.pdf
11 Broom DM. Indicators of poor welfare. Br Vet J 1986;142:524 – 6
12 Duncan IJH, Filshie JH. The use of telemetry devices to measure
temperature and heart rate in domestic fowl. In: Amlaner CJ, MacDonald
DW, eds. Handbook on Biotelemetry and Radio Tracking. Oxford: Pergamon,
1979:579
Kemppinen et al. Cage furniture, restricted feeding and rat welfare
9
................................................................................................................................................
13 Duncan IJH. In: Wegner RM, ed. Proceedings of the Second European
Congress on Poultry Welfare. Celle: World Poultry Science Association,
1985:96
14 Kramer K, Kinter L, Brockway B, Voss H-P, Remie R, van Zutphen BLM.
The use of telemetry in small laboratory animals: recent advances.
Contemp Top Lab Anim Sci 2001;40:8 –16
15 Sharp J, Azar T, Lawson D. Effects of a cage enrichment program on
heart rate, blood pressure, and activity of male Sprague – Dawley and
spontaneously hypertensive rats monitored by radiotelemetry. Contemp
Top Lab Anim Sci 2005;44:32 – 40
16 Bamberg E, Palme R, Meingassner JG. Excretion of corticosteroid
metabolites in urine and faeces of rats. Lab Anim 2001;35:307 – 14
17 Lepschy M, Touma C, Hruby R, Palme R. Non-invasive measurement of
adrenocortical activity in male and female rats. Lab Anim 2007;41:372 – 87
18 Dhabhar FS, McEwen BS, Spencer RL. Stress response, adrenal steroid
receptor levels and corticosteroid-binding globulin levels – a comparison
between Sprague – Dawley, Fischer 344 and Lewis rats. Brain Res
1993;616:89 –98
19 Pihl L, Hau J. Faecal corticosterone and immunoglobulin A in young
adult rats. Lab Anim 2003;37:166 –71
20 Eriksson E, Royo F, Lyberg K, Carlsson H-E, Hau J. Effect of metabolic
cage housing on immunoglobulin A and corticosterone excretion in
faeces and urine of young male rats. Exp Physiol 2004;89:427 –33
21 Royo F, Björk N, Carlsson H-E, Mayo S, Hau J. Impact of chronic
catheterization and automated blood sampling (Accusampler) on serum
corticosterone and fecal immunoreactive corticosterone metabolites and
immunoglobulin A in male rats. J Endocrinol 2004;180:45 –153
22 Möstl E, Palme R. Hormones as indicators of stress. Domest Anim
Endocrinol 2002;23:67 –74
23 Van den Buuse M. Circadian rhythms of blood pressure, heart rate, and
locomotor activity in spontaneously hypertensive rats as measured with
radiotelemetry. Physiol Behav 1994;55:783 –7
24 Lemaire V, Mormède P. Telemetered recording of blood pressure and
heart rate in different strains of rats during chronic social stress. Physiol
Behav 1995;58:1181 –8
25 Van den Brant J, Kovács P, Klöting I. Blood pressure, heart rate and
motor activity in 6 inbred rat strains and wild rats (Rattus norvegicus): a
comparative study. Exp Anim 1999;48:235 –40
26 Zhang B, Sannajust F. Diurnal rhythms of blood pressure, heart rate, and
locomotor activity in adult and old male Wistar rats. Physiol Behav
2000;70:375 –80
27 Festing M, Overend P, Gaines Das R, Cortina Borja M, Berdoy M. The
design of animal experiments. In: Laboratory Animal Handbooks. No. 14.
London: RSM Press, 2002
28 Kemppinen N, Meller A, Mauranen K, Kohila T, Nevalainen T. Work
for food – a solution for restricted feeding in group housed rats? Scand
J Lab Anim Sci 2008;35:81 –90. See http://biomedicum.ut.ee/sjlas/35_2_
81– 90.pdf
29 Armario A, Gavaldá A, Martı́ J. Comparison of the behavioural and
endocrine response to forced swimming stress in five inbred strains of
rats. Psychoneuroendocrinology 1995;20:879 –90
30 Sarrieau A, Mormède P. Hypothalamic– pituitary – adrenal axis activity
in the inbred Brown Norway and Fischer 344 rat strains. Life Sci
1998;62:1417 – 25
31 Hubert M-F, Laroque P, Gillet J-P, Keenan KP. The effects of diet, ad
libitum feeding, and moderate and severe dietary restriction on body
weight, survival, clinical pathology parameters, and cause of death in
control Sprague – Dawley rats. Toxicol Sci 2000;58:195 – 207
32 Roe FJC. Historical histopathological control data for laboratory rodents:
valuable treasure or worthless trash? Lab Anim 1994;28:148 –54
33 Roe FJC, Lee PN, Conybeare G, et al. The biosure study: influence of
composition of diet and food consumption on longevity, degenerative
diseases and neoplasia in Wistar rats studied for up to 30 months post
weaning. Food Chem Toxicol 1995;33:1S –100S
34 Spiteri NJ. Circadian patterning of feeding, drinking and activity during
diurnal food access in rats. Physiol Behav 1982;28:139 –47
35 Strubbe JH, Alingh Prins AJ. Reduced insulin secretion after
short-term food deprivation in rats plays a key role in the adaptive
interaction of glucose and fatty free acid utilization. Physiol Behav
1986;37:441 –5
36 Strubbe JH, Spiteri NJ, Alingh Prins AJ. Effect of skeleton photoperiod
and food availability on the circadian pattern of feeding and drinking in
rats. Physiol Behav 1986;36:647 –51
37 Björk E, Nevalainen T, Hakumäki M, Voipio H-M. R-weighting
provides better estimation for rat hearing sensitivity. Lab Anim
2000;34:136 –44
38 Kemppinen N, Meller A, Mauranen K, Kohila T, Nevalainen T. The effect
of dividing walls, a tunnel and restricted feeding on cardiovascular
responses to cage change and gavage in rats (Rattus norvegicus). J Am
Assoc Lab Anim Sci 2009;48:157 –65
39 Nevalainen T, Vartiainen T. Volatile organic compounds in commonly
used beddings before and after autoclaving. Scand J Lab Anim Sci
1996;23:101 –4
40 Voipio H-M, Korhonen T, Koistinen T, Kuronen H, Mering S, Nevalainen
T. Durability and hygiene of aspen tubes used for providing
environmental complexity for laboratory rats. Scand J Lab Anim Sci
2008;35:107 –115. See http://biomedicum.ut.ee/sjlas/35_2_107 –115.pdf
41 Krohn TC, Hansen AK, Dragsted N. Telemetry as a method for
measuring the impact of housing condition on rats’ welfare. Anim Welf
2003;12:53 –62
42 Patterson-Kane EG. Shelter enrichment for rats. Contemp Top Lab Anim
Sci 2003;42:46 –8
43 Townsend P. Use of in-cage shelters by laboratory rats. Anim Welf
1997;6:95 –103
44 Eskola S, Lauhikari M, Voipio H-M, Nevalainen T. The use of aspen
blocks and tubes to enrich the cage environment of laboratory rats. Scand
J Lab Anim Sci 1999;26:1– 10
45 Batchelor GR. The rest/activity rhythm of the laboratory rat housed
under different systems. Anim Technol 1994;45:181 –7
46 Siswanto H, Hau J, Carlsson H-E, Goldkuhl R, Abelson KSP.
Corticosterone concentrations in blood and excretion in faeces after
ATCH administration in male Sprague –Dawley rats. In Vivo
2008;22:435 –40
(Accepted 21 July 2009)
CHAPTER VI
Refinement and reduction value of aspen furniture and restricted feeding of rats in
conventional cages.
Niina Kemppinen, Anna Meller, Jann Hau, Kari Mauranen, Tarja Kohila and Timo Nevalainen.
Submitted to Scandinavian Journal of Laboratory Animal Science.
access to cages furnished with a box or
nesting material (Manser et al., 1998). New
European regulations state that if there is not
enough nesting material available to build a
complete, covered nest, a nest box should be
provided (Council of Europe, 2006; European
Union, 2007). Dividing structures and shelters
in the cage may offer rats opportunities to
seek or avoid contact with other group
members and are regarded as beneficial to
animal welfare (Stauffacher et al., 2002).
The benefits and problems of housing
refinement - also called environmental
enrichment - achieved by inclusion of
different items or materials in the cage, need
to be evaluated because the putative positive
effect on the welfare of the animals may
depend on various characteristics of the
animals, e.g. strain, stock, sex, and age. Some
structures may even have a negative impact on
animal welfare (Kaliste et al., 2006; Moncek
et al., 2004; Tsai et al., 2002; Tsai et al.,
2003). A true value assessment requires
systematic evaluation of the individual items
and the relevant combinations of items.
A stressful environment activates the
autonomic nervous system (ANS) causing
persistent elevations of both heart rate (HR)
and blood pressure. A lowering of HR and
blood pressure may thus be considered to
reflect an increase in the welfare of the
animals. Radiotelemetry as a method to record
physiological parameters, such as HR and
blood pressure, allows animals to move freely
while recording i.e. restraint is unnecessary.
The values of HR and blood pressure are thus
considerably lower in animals implanted with
a telemetry transmitter than those obtained
with other methods (Kramer et al., 2001).
Summary
This study evaluated the impact of aspen
furniture on cardiovascular parameters,
locomotor activity (LA) and faecal welfare
indicators in rats. A total of 12 BN and 12
F344 male rats were group housed (n=3) in
conventional cages. Two groups received a
simple maze, one group received a rectangular
tube and the controls had no furniture. In one
of the two maze groups, the rats had to gnaw
through wood in order to obtain food. The
mean values of the LA in all groups and
differences in mean arterial pressure (MAP)
and heart rate of the rats housed in the various
item groups were compared to the values of
the rats housed in control cages with no
furniture, on days two, six, ten and 14 in each
period (both light and dark phases). The F344
rats were generally more active than the BN
rats during the dark phase, but not during the
light phase. Based on the MAP results, the
tube appeared to be a poor choice for F344
rats, while for BN rats all furniture items
seemed beneficial, with both board types
apparently superior to the tube. In general,
F344 rats had higher faecal corticosterone
levels than BN rats with the reverse being true
for secretory IgA values. In conclusion, LA
and cardiovascular parameters seemed
appropriate ways to evaluate the impact of
cage furniture on physiological parameters,
and covered structures such as tubes do not
seem to provide any enrichment value in these
two rat strains.
Introduction
Rats prefer cages containing a nest box
(Patterson-Kane, 2003; Townsend, 1997) and
they are willing to work in order to gain
101
Telemetry has been used to analyze the impact
of different cage flooring in rats (Krohn et al.,
2003a), and Sharp et al. (2005) studied a
multifaceted enrichment program’s effect in
Sprague-Dawley (SD) and spontaneously
hypertensive (SHR) male rats. The cage
enrichments had no significant effects on
basal or undisturbed HR, systolic blood
pressure (SBP) or activity in SD rats or on
SBP in SH rats. However, in SHR rats with
enrichment, the HR was reduced in both dark
and light phases and the SHR rats’ activity
increased during the afternoons. In our
previous study, cage furniture had item- and
strain-specific effects on blood pressure and
HR in BN and F344 rats housed in an IVCsystem, and the rats exhibited habituation to
some of the items (Kemppinen et al., in press).
Increases in the serum corticosterone level
are attributable to activation of the
hypothalamic-pituitary-adrenal (HPA) axis in
response to stressful stimulation in rats; but
this response is not as rapid as that of ANS.
Corticosteroid metabolites are excreted both
into urine and faeces, but unlike the situation
in serum, they are not detectable until 6-10ten
hours later in urine and 4-1216 hours later in
faeces after stressful event (Bamberg et al.,
2001, Royo et al. 2004, Lepschy et al. 2006,
Siswanto et al. 2008, Abelson et al. 2009 ).
The major pathway of excretion in rats is via
faeces; this accounts for 80 % of the
recovered metabolites in rats (Bamberg et al.,
2001; Lepschy et al., 2007). One major
advantage of faecal samples for quantification
of stress sensitive molecules is that faeces are
voided voluntarily and there is no need to
handle animals, and even if there is some
response to the collection procedure, the delay
before corticosteroids appear in the faecal
pellets ensures that corticosteroid levels in the
samples collected are not affected by events
associated with the sampling procedures
(Bamberg et al., 2001; Möstl & Palme, 2002).
Prolonged
stress
may
lead
to
immunosuppression. The levels of secretory
immunoglobulin A (IgA) in saliva have been
used to assess welfare status associated with
different housing conditions (Guhad & Hau,
1996). Another option is to quantify faecal
IgA (Eriksson et al., 2004; Pihl & Hau, 2003;
Royo et al., 2004). There are few studies in
rats assessing the value of furniture items
using corticoid measurements and these are
often conflicting. Belz et al. (2003) showed
that singly housed SD rats of both sexes
housed with environmental enrichment had
lower baseline plasma corticosterone levels
than rats in standard cages, whereas Moncek
et al.(2004) detected significantly higher
corticosterone levels in Wistar male rats
housed in enriched cages. However, in the
latter study, multiple combinations of various
items were used and the effect of any single
item could not be differentiated. Nevertheless,
the combination did not seem to improve the
housing environment of the rats in terms of
lowering corticosterone levels.
There is ample evidence that ad libitum
feeding causes obesity, increases the
incidence of disease, and shortens lifespan in
rats (Hubert et al., 2000; Roe, 1994; Roe et
al., 1995). Current legislation mandates group
housing (Council of Europe, 2006; European
Union, 2007), and there is presently no
practical, effective way to restrict feeding
when rats are group housed. When food is
available ad libitum rats eat predominantly
102
housed in IVCs and used in the study with the
same method as in the present study
(Kemppinen et al., in press).
Animal housing and care. Rats were
housed in the same-strain groups of three in
solid bottom polysulfone cages (Tecniplast,
Buguggiate, Italy, ***Eurostandard IV S, 44.5
x 33.5 x 21.0 cm) with a stainless steel wire
mesh lid. The cage floor was covered with 3 l
aspen chip bedding (size of 4 x 4 x 1 mm,
4HP, Tapvei Oy, Kaavi, Finland). The rats
were moved to clean cages on day eight at
noon in every two-week-period. Tap water
was provided in polycarbonate bottles which
were changed once a week at the cage change
and refilled once in between.
The room temperature was 21.2 ± 0.3°C
(mean + SD) and the relative humidity (RH)
53 ± 6 %. Lighting with fluorescent tubes was
on from 06.00 to 18.00 with light intensity in
cages 1 m above floor of 16-18 lx. The sound
level, adjusted with R-weighting for the
hearing sensitivity of rats, in empty cages was
12-18 dB(R), with the corresponding adjusted
A-weighting - adjusted for human hearing
sensitivity - being 46-49 dB(A) (Björk et al.,
2000). For a more thorough description, see
Kemppinen et al (2008b).
Cage furniture and study design. The
experiment utilized a crossover design with
two week periods and a rotational order.
Within both strains there were two different
kinds of dividers made of intersecting two
aspen boards (34.0 x 14.7 x 3.2 cm; 21.1 x
14.7 x 3.2 cm), a rectangular aspen tube (20.0
x 12.0 x 12.0 cm, external dimensions), or
controls without any furniture. One divider
type included drilled holes for food pellets,
where the rats had to gnaw for food (diet
during the dark phase (Spiteri, 1982; Strubbe
& Alingh Prins, 1986; Strubbe et al., 1986).
Feeding rats only during the light phase may
have an impact on gastrointestinal motility
and physiology. The diet board, where food is
available ad libitum, but rats had to gnaw
through wood to obtain food, resulted in 1518 % less food being consumed and a reduced
body weight gain in the rats (Kemppinen et
al., 2008a).
We hypothesized that a combination of
cardiovascular telemetry, measurement of
locomotor activity, combined with assays of
faecal corticosteroid metabolites and faecal
IgA, would constitute efficient tools for the
assessment of refinement and reduction
potential of various enrichment items for rats.
This study was designed to evaluate the
impact of dividing aspen walls with or without
restricted feeding and an aspen tube on
laboratory rats using these parameters.
Additional aims were to determine whether
there would be a genetic component in
responses and whether the rats habituate to the
items chosen for scrutiny.
Materials and Methods
The study took place in the Laboratory
Animal Centre, University of Helsinki. The
protocol of the study was reviewed and
approved by the Animal Ethics Committee of
the University of Helsinki.
Animals. A total of 12 BN (BN/RijHsd)
and 12 Fischer344 (F344/NHsd) male rats
(Harlan, Horst, The Netherlands), were used
in this study. The rats were 33 weeks old and
weighed 310 - 430 g (BN) or 380 - 480 g
(F344), respectively, at the beginning of the
study. Before the experiments, all rats were
103
(Hypnorm®, Janssen Pharmaceutica, Beerse,
Belgium)
+
midazolam (Dormicum®,
Hoffmann-La Roche AG, Grenzach-Wyhlen,
Germany, 0.15 - 0.20 ml/100g SC). The
abdominal area was clipped and then scrubbed
with MediScrub®, 1 % triclosan solution
(Medichem International, Sevenoaks, UK)
solution and disinfected with chlorhexidine
solution (Klorohexol® 5 mg/ml, Leiras, Turku,
Finland), and an ocular lubricant (Viscotears®,
Novartis Healthcare, Copenhagen, Denmark)
was applied to both corneas. A sterile drape
was placed over the surgical area and a small
area cut away to enable a 3 cm incision to be
made through the skin along the abdominal
midline. The sterile transmitter was presoaked in sterile saline for at least 20 min
before the surgery and then placed into the
abdominal cavity, and the catheter into the
abdominal aorta. The transmitter was sutured
into the abdominal wall with 4-0 Ethicon®
Ethilon®II (Johnson & Johnson Intl, StStevens-Woluwe,
Belgium)
and
the
abdominal and skin incisions were closed with
5-0 Ethicon® Vicryl® (Johnson & Johnson
Intl, St-Stevens-Woluwe, Belgium). The
surgery procedure lasted about 20-30 minutes.
After the surgery, the animals were given,
twice a day, 0.01 – 0.05 mg/kg SC
buprenorphine (Temgesic®; Schering-Plough
Europe, Brussels, Belgium) and once a day a
dose of 5 mg/kg SC carprofen (Rimadyl®;
Vericore Ltd., Dundee, UK) and parenteral
fluids for three days. The pain medication for
each rat was titrated according to individual
response. All rats were given initially
buprenorphine at the highest dose; this was
continued for at least two days; and carprofen
medication for at least three days. On these
board); the other type was without drilled
holes and food pellets (plain board). The items
were made of aspen as was the bedding; both
have the same volatile compound emissions,
in this case especially the absence of α- and βpinenes (Nevalainen & Vartiainen, 1996), and
aspen furniture endures several bouts of
sanitation (Voipio et al. 2008). The day when
the rats were moved between cages differing
with respect to furniture item was designated
as day one in every study period. Diet boards
had to be renewed on day eight in order to
ensure that food was always available. The
order of the cage items was set at random, and
the first item in each group was randomly
allocated. The illustration of the items and
item order can be seen elsewhere (Kemppinen
et al., in press; Kemppinen et al., 2009).
Irradiated (25 kGy) pelleted feed (2016
Global Rodent Maintenance, Harlan Teklad,
Bicester, UK) was provided to three groups
(plain board, tube and control groups) ad
libitum, while the diet board group had the
food pellets embedded snugly in drilled holes
(diameter 12 mm) of the aspen board.
Surgical procedure. Eight rats (one rat in
each group) were implanted with a radio
telemetry transmitter (model TA11PA-C40;
Data Sciences International, St.Paul, MN,
USA). The cylinder shape transmitter body
(3.0 cm, diameter 1.5 cm) monitored pressure
and activity via a fluid filled catheter (8.0 cm
long) sending the signals to an electronics
module. The electronics module translated the
signals into a digitized form and transmitted
them to the receiver plate located under the
cage.
The rats were anesthetized with the
combination
of
fentanyl/fluanisone
104
freedom for error per strain, well within the
optimum range, i.e. 10-20. Means and
coefficient of variation (CV) for MAP and HR
were calculated at 30 min intervals for light
and dark phases on days two, six, ten and 14
of each period. The mean values in all groups
and differences in MAP and HR of the three
furniture groups as compared to the control
were calculated for these days; LA values
were used as such. Mixed-model repeated
measures ANOVA (SPSS Windows, version
14.0, SPSS Inc., Chicago, IL, USA) was used
combined with Bonferroni correction. For
LA, group was used as main effect and age as
a covariate. For means and CV of both MAP
and HR, LA was used as second covariates.
Significant CV differences between groups
were processed further to point estimates [=
(CV1/CV2)2].
Significance
for
mean
comparisons was set at p < 0.05, and for
increased certainty at p < 0.01 for CV
comparisons. In order to determine which of
the statistically significant cardiovascular
mean changes were biologically meaningful,
i.e. have welfare value; the mean night-day
difference of day 14 was calculated for the
control group of both rat strains.
The faecal corticosterone and faecal IgA
results of each rat from the same cage were
averaged at the cage level and calculated with
repeated measures mixed-model ANOVA
with Bonferroni correction, using age as a
covariate.
three days the implanted rats were housed
alone and then placed back into their home
cages.The animals were allowed to recover for
ten days before the experiment was started.
Sampling. Values of mean blood pressure
(MAP), heart rate (HR) and locomotor activity
(LA) were transmitted every 75 seconds to the
computer throughout the study. For LA
measurements the telemetric receiver had two
perpendicular antennas, and the receiver
detected signal strength change when the rat
moved in relation to these antennas.
At the end of each period, the rats were
housed singly for six hours (06.00-12.00) and
all faecal pellets voided from each individual
were collected and frozen (-18°C).
Faecal corticosterone quantification. The
extraction of both corticosterone and IgA was
performed as described by Pihl and Hau
(2003). The corticosterone ELISA was
performed using a commercial corticosterone
kit (DRG Diagnostics, Marburg, Germany)
using the manufacturer’s instruction manual.
The quantification of IgA was performed
using the assay described by Pihl and Hau
(2003) and reagents were obtained from AbD
Serotec, (Kidlington, Oxfordshire, UK;
(purified rat IgA standard, PRP01,
concentrations 0-1000 ng/ml); coating
antibody (mouse anti rat IgA heavy chain,
MCA191); and detection antibody (mouse anti
rat kappa/lambda light chain: HRP,
MCA1296P) diluted 1:1000).
Data processing and statistical analysis.
The number of animals needed in the study
was estimated with the resource equation
method (Festing, 2002). The cage was used as
an experimental unit, and with the crossover
design used, this resulted in 12 degrees of
Results
F344 rats were more active than the BN
rats during the dark phase, whereas LA during
the light phase was similar in these strains
(Figure 1). The F344 rats, on day ten and in
105
The HR of the F344 rats was significantly
(P < 0.05) higher in the diet board cage
compared to the tube cage on day two in the
light phase. On day 14, both lighting phases
exhibited the highest HR with the tube; with
lights on the HR was significantly higher in
the tube (P < 0.01) compared to the plain
board; in the dark compared to the diet board
(P < 0.001, Figure 2b). Similarly to MAP, no
HR comparison of F344 was close to
biological significance.
The F344 rats showed no differences in
MAP coefficient of variation (CV), and only
one significant HR CV value was observed,
i.e. between the tube and controls during the
light phase of day 14 (P < 0.01). These results
are shown graphically in Figure 3 and
corresponding point estimates in Table 1.
BN rats. In the BN rats, the day 14 nightday difference for the control group was 3
(±3) mmHg for MAP and 21 (±4) BPM for
HR. Between item comparisons showed
significant MAP differences only on day 14;
the tube was significantly (P < 0.05) less
effective in lowering MAP than the other two
items. These statistical significances are
unlikely to be of biological significance, since
they are about 3 mmHg, similar to the nightday difference value for day 14. Although all
furniture groups' MAP appeared to be below
the control values, only on day 14 did both
board
groups
consistently
achieved
significance (P < 0.05 - 0.01); and but this
lowering effect compared to tube didn’t
exceeded 3 mmHg (Figure 2c). On day two, in
the light phase, rats in the tube group had
significantly (P < 0.05) higher HR than the
diet board rats. Additionally, also in the light
phase, both on days six and 14, the HR was
the dark, were significantly (P < 0.001-0.05)
more active with the tube compared to the two
boards, and in the control group compared to
the plain board group.
F344 rats had significantly (P < 0.001)
higher MAP and HR than BN rats throughout
the study. MAP exhibited a significant (P <
0.001-0.05) group*strain interaction in the
dark on days six, ten and 14, and in the light
on days two, ten and 14. HR showed
significant (P < 0.001-0.05) group*strain
interaction in dark and light phases throughout
the study, except in the dark on day ten.
Because of the multiple interactions
encountered, the following results will be
presented separately for both strains and for
each lighting phase. The MAP and HR daily
30 min means of the control group were
subtracted from those of the rats with the
different cage items. Hence values above the
control baseline designate an elevation of the
parameter, and values below the control
baseline represent the opposite.
.F344 rats. The night-day difference on day
14 in the F344 control group was 10 (±3)
mmHg for MAP and 60 (±29) beats per
minute (BPM) for HR. On day two in the dark
phase, the F344 rats in the diet board cages
showed significantly (P < 0.001) lower MAP
than the rats in the tube and plain board
groups, and on day 14 the same was true in
comparison to the tube group. On day 14, in
the light phase, the rats in the tube cages had
significantly (P < 0.001-0.05) higher MAP
than rats housed with the two other items
(Figure 2a). However, none of these MAP
differences in F344 rats reached biological
significance i.e. exceeded day 14 night-day
difference.
106
A.
Activity (mean+SEM) - F344 rats
Activity (counts/min)
10
8
Diet board
* ***
*
6
Tube
Plain board
4
Control
2
0
Dark
Light
Day 2
B.
Dark
Light
Day 6
Dark
Light
Dark
Day 10
Light
Day 14
Activity (mean+SEM) - BN rats
Activity (counts/min)
10
8
Diet board
6
Tube
Plain board
4
Control
2
0
Dark
Light
Day 2
Dark
Light
Day 6
Dark
Light
Day 10
Dark
Light
Day 14
Figure 1. Locomotor activity (SEM) of F344 (A) and BN (B) rats with different cage items and
controls during the dark and light phases. Abbreviations: * = P < 0.05, § = P < 0.001. Number of
experimental units within a strain = 16.
107
A.
MAP difference (+SEM) - F344 rats
MAP difference (mmHg)
6
*
4
***
Diet board
2
Tube
0
Plain board
-2
-4
***
Dark
Light
Day 2
B.
Dark
Light
Day 6
Dark
Light
Day 10
Dark
Light
Day 14
HR difference (+SEM) - F344 rats
20
HR difference (BPM)
***
***
**
15
*
10
Diet board
5
Tube
Plain board
0
-5
-10
Dark
Light
Day 2
Dark
Light
Day 6
Dark
Light
Day 10
108
Dark
Light
Day 14
MAP difference (mmHg)
C.
BN rats
2
0
Diet board
-2
Tube
-4
Plain board
-6
* *
-8
Dark
Light
Day 2
Dark
Light
Dark
Day 6
Light
Day 10
Dark
Light
Day 14
Days and light phases
D.
BN rats
HR difference (BPM)
15
*
10
5
#
Diet board
0
Tube
-5
Plain board
-10
*
-15
Dark
Light
Day 2
Dark
Light
Day 6
Dark
Light
Day 10
Dark
Light
Day 14
Days and light phases
Figure 2. The MAP and HR differences (SEM) of F344 (A and B) and BN (C and D) rats to
controls with three cage items during the dark and light phases. Abbreviations: MAP=Mean Arterial
Pressure, HR=Heart Rate, BPM=Beats Per Min, * = P < 0.05, # = P < 0.01, § = P < 0.001. Number
of experimental units = 12.
109
MAP CV - F344 rats
A.
MAP CV
0,15
0,1
Diet board
Tube
Plain board
0,05
Control
0
Dark
Light
Day 2
Light
Day 6
B.
Dark
Light
Day 10
Dark
Light
Day 14
HR CV - F344 rats
0,15
Diet board
0,1
HR CV
Dark
**
0,05
Tube
Plain board
Control
0
Dark
Light
Day 2
Dark
Light
Day 6
Dark
Light
Day 10
110
Dark
Light
Day 14
C.
MAP CV - BN rats
0,15
MAP CV
**
0,1
** *** ** **
Diet board
Tube
Plain board
0,05
Control
0
Dark
Light
Day 2
Dark
Light
Day 6
D.
Dark
Light
Day 10
Dark
Light
Day 14
HR CV - BN rats
0,15
0,1
HR CV
Diet board
Tube
Plain board
0,05
Control
0
Dark
Light
Day 2
Dark
Light
Day 6
Dark
Light
Day 10
Dark
Light
Day 14
Figure 3. Coefficient of variation (CV) of the MAP and HR for F344 (A and B) and BN (C and D)
rats with three cage items and control groups during the dark and light phases. Abbreviations:
MAP=Mean Arterial Pressure, HR=Heart Rate, # = P < 0.01, § = P < 0.001. Number of
experimental units = 16.
111
Table 1. P-values for significant mean arterial pressure (MAP) and heart rate (HR) coefficient of
variation (CV) comparisons between the groups and corresponding point estimates (PE) for both
F344 and BN rats and both light phases on observation days. Comparisons with no significances are
excluded from the table. NS = not significant.
F344 rats p < / PE
Diet board/
Control
Day 2
Plain board/
Control
Diet board/
Tube
Day 10
Diet board/
Control
Day 14
Tube/
Control
BN rats p < / PE
MAP dark
MAP light
MAP dark
MAP light
HR dark
NS
HR light
NS
HR dark
0.01 / 1.87
HR light
0.01 / 1.89
NS
NS
NS
NS
NS
0.001 / 2.25
NS
0.01 / 2.25
NS
NS
NS
NS
NS
0.01 / 1.91
NS
NS
NS
NS
NS
NS
NS
0.01 /1.91
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
0.01 / 1.56
NS
NS
those which are statistically significant with
corresponding point estimates in Table 1.
Corticosterone and IgA assays. The
number of faecal pellets collected varied from
none up to more than ten pellets per animal.
Neither of the studied rat strains exhibited
significant differences in amounts of
corticosterone nor IgA excreted via faeces
between the furniture item groups. However,
the F344 rats had significantly (P = 0.05)
higher faecal corticosterone outputs than BN
rats whereas the excreted amounts of faecal
IgA were significantly (P < 0.05) higher in the
BN rats (Figure 4). In neither strain were any
significant faecal assay CV differences found.
significantly (P < 0.01 - 0.05) lower in the diet
board group compared to rats in the plain
board groups (Figure 2d). However, none of
these significant HR comparisons reached the
threshold of biological significance.
The MAP CV of the control BN rats was
significantly (P < 0.001 - 0.01) lower than in
both board groups, both in the dark and light
phases of day two. During the second week of
the study period, the MAP CV was
significantly (P < 0.01) higher in the diet
board group on day ten in the dark compared
to the controls and tube groups (Figure 3c). In
the BN rats, the HR CV exhibited no
significant results. All possible comparisons
are illustrated graphically in Figure 3 and
112
A.
Fecal corticosterone (mean+SEM)
0,5
nmol/h x kg
0,4
Diet board
0,3
Tube
Plain board
0,2
Control
0,1
0
F344
B.
BN
Fecal IgA (mean+SEM)
20
ug/h x kg
15
Diet board
Tube
10
Plain board
Control
5
0
F344
BN
Figure 4. Corticosterone (A) and Immunoglobulin A (IgA, B) excreted via feces in F344 and BN
rats with three cage items and control rats. Values expressed as cage means ± SEM nmol
corticosterone or μg IgA excreted per hour per kg body weight. Number of experimental units = 16.
The F344 rats displayed significantly (p = 0.05) higher fecal corticosterone levels than BN rats,
while the opposite was true for fecal IgA levels (p < 0.05).
113
Table 2. Comparison of cage types: mean MAP and HR in F344 and BN rats. Arrows show the cage
type with higher value. * < 0.05, ** < 0.01, *** < 0.001
F344
MAP
Day 2
Dark
ns
Light
OPEN ↑ ***
Day 6
Dark
ns
Light
ns
Day 10
Dark
ns
Light
ns
Day 14
Dark
ns
Light
OPEN ↑ **
ns = not significant
BN
HR
MAP
HR
OPEN ↑ ***
OPEN ↑ ***
ns
ns
OPEN ↑ ***
OPEN ↑ ***
OPEN ↑ ***
OPEN ↑ **
ns
ns
OPEN ↑ ***
OPEN ↑ **
OPEN ↑ ***
OPEN ↑ ***
ns
ns
OPEN ↑ ***
OPEN ↑ ***
OPEN ↑ **
OPEN ↑ ***
ns
ns
OPEN ↑ *
ns
lower than in the groups with furniture items
without restricted feeding (Figure 1). When
rats were housed in individually ventilated
cages (IVC), the highest LA on day ten was
also found in the tube group (Kemppinen et
al., in press), but in both cases, between
groups LA differences had disappeared by day
14.
In the study of Kemppinen et al. (in press)
the same methodology and the same animals,
albeit younger, as in the present study were
used to evaluate the effect of furniture items
in IVCs. In the IVCs, the F344 rats with the
tube exhibited higher values of MAP
compared to both board groups throughout the
two-week period. In the conventional cages,
the same was observed only on day 14 (Figure
2a). A major difference between conventional
and IVCs was observed in the BN rats; rats in
Discussion
The telemetry study of Sharp et al. (2005)
assessed the outcome from an enrichment
program on heart rate (HR), systolic blood
pressure and activity. It concluded that lower
HR in SHR rats was attributable to their
enrichment program, but with SD rats no
effect was detected. Their housing refinement
items were a combination of several smaller
items added to the cage at a few days
intervals.
Irrespective of the housing system and
enrichment program, Wistar rats seem to be
more active in the dark phase than in the light
phase, and they rest and sleep more in an
enriched environment (Batchelor, 1994). In
the present study, the diurnal variation of LA
in F344 rats was similar. On day ten during
the dark phase, the control group LA was
114
study; both rat strains had significantly higher
HR in the open cages (Table 2) compared to
the IVCs, where the temperature was 1-4 oC
higher (Kemppinen et al. 2008b).
However, in the present study the rats were
eight weeks older than in the IVC study and
perhaps the differences cannot be attributed to
cage type alone. Zhang & Sannajust (2000)
reported a decrease in the nocturnal HR in old
Wistar rats, an apparently opposite finding to
our study, with higher HR in older rats with
both strains. This may be due to the fact that
the old Wistar rats were two years old, in
contrast to the 8-10-months old rats examined
here.
Rats are nocturnal animals and blood
pressure and HR are elevated during the night
(Sharp et al., 2005; Zhang & Sannajust, 2000;
Lemaire et al., 1995; van den Brant et al.,
1999). Many studies have described
considerable differences in basal blood
pressure and HR between different rat stocks
or strains. This study used two rat strains,
F344 and BN, for enhanced precision and
applicability (Festing et al., 2002). These
strains differ in various aspects of physiology,
e.g. systolic and diastolic blood pressure, HR
(van den Brant, 1999), plasma corticosterone
(Armario et al., 1995; Sarrieau & Mormède,
1998),
and
brain
and
pituitary
mineralocorticoid receptor levels (Gómez et
al., 1998; Marissal-Arvy et al., 1999). The
F344 and BN rats exhibit differences in level
and diurnal rhythm of locomotor activity
(Kemppinen et al., 2008a; van den Brant et
al., 1999; Ramos et al., 1997), and in
behaviour (Spangler et al., 1994; Rex et al.,
1996; van den Staay & Blokland, 1996).
the IVC displayed small, 1-2 mmHg, MAP
differences between the groups, while in the
open cages they were as much as 6 mmHg
(Figure 2c), and additionally MAP levels of
all rats in groups with furniture items were
below the values of the controls.
When the two types of cages are compared,
the group MAP differences of the F344 rats
were smaller in the conventional open top rat
cages (Figure 2a) than in IVCs, but in BN rats
the situation was opposite (Figure 2c)
(Kemppinen et al., in press). Nonetheless,
overall LA and HR differences between the
strains were of the same magnitude in the
open top rat cages and IVCs. The CV for the
MAP and HR in BN seemed larger in open
cages than in IVCs, whereas in F344 rats CVs
appeared to be about the same amplitude
(Kemppinen et al., in press).
The IVC has become a common housing
system for laboratory rodents. However, it is
not always appreciated that the physical
environment inside the cages may be very
different from that of conventional cages in
the same room. Indeed, differences have been
found in illumination, sound level,
temperature and RH (Kemppinen et al.,
2008b). The higher sound level due to
ventilation in the IVCs may affect the
behaviour of the rats, while animals in the
open cages are more likely to hear noises
originating from care routines (Voipio et al.,
2006), and research procedures.
Even small changes in ambient temperature
can have an impact on cardiovascular
parameters in rats; when temperature
increases, MAP and HR of SD female rats
decreases (Swoap et al., 2004). This effect
was seen in HR of this study throughout the
115
(Kemppinen et al., in press). The trend seen in
the present study on day 14 was similar, but
due to increased night-day MAP difference, it
lacked biological significance. No habituation
to the furniture items was detectable in this
study.
Even if Iin the BN rats there wasn’t any the
only biologically significant result was
observed on day 14 in the light phase in MAP,
when the rats had the lowest value in the
groups with the boards. Moreover, on all
recorded days and both light phases, the
boards and also tube MAP values were in
most cases significantly lower than
corresponding control values (Figure 2c). We
conclude that in BN rats all furniture items
tested were beneficial, and the boards were
superior to the tube.
Krohn et al. (2003a) reported that systolic
blood pressure and HR in rats increase by 6-7
% when rats are housed on grid or solid
bottom floors compared to solid floor with
bedding, but they concluded that these
differences may not possess biological value
although they may be statistically significant
(Krohn et al., 2003b). We suggest that a
constant percentage as a cut off level for what
is biologically relevant or not may not be
applicable to all strains. Strain-specific
measures, such as night-day difference are
more valid for the purpose of determining
what is biologically meaningful and what is
not.
In CV of F344 rats there was only one
significant finding; day 14 light phase HR CV
of the tube group was higher than that in the
controls (Figure 3b). The corresponding point
estimates showed that with the tube, the
number of animals needed would be 1.56
There was a significant group*strain
interaction in MAP and HR on nearly every
day examined during the two-week study
period demonstrating strain differences. The
F344 rats exhibited higher blood pressure and
HR than BN rats with all items and the same
was true for LA of the F344 rats during the
dark phase (Figure 1). Van den Brant et al.
(1999) detected a similar trend in
telemetrically measured systolic and diastolic
blood pressure, HR and night activity. They
concluded that the BN rats have lost the
typical night activity pattern typical for
rodents. The present study shows that the
F344 rats had considerably larger night-day
difference both in the MAP and HR values
than the BN rats. Presumably pigmented BN
rats do not avoid light to the same extent as
the albino F344 rats do. Albino rats prefer a
cage with a shelter to one without (PattersonKane, 2003; Townsend, 1997) and they tend
to remain in the tube during the light phase
(Eskola et al., 1999).
This study compared all MAP and HR
between the furniture groups to the
corresponding
strain-specific
night-day
differences of the controls. We suggest that
when between-the-group differences are
smaller than the night-day differences, a result
displaying statistical significance is not
biologically important (Kemppinen et al., in
press; Kemppinen et al., 2009). On this basis,
none of the F344 rats’ cardiovascular
parameter differences between the furniture
items were biologically meaningful, i.e. they
should be considered as below the threshold
of reliable differences (Figures 2a-b). In the
earlier report with similar furniture in IVCs,
both boards were superior to the tube
116
times that of controls when HR is the result
parameter (Table 1). The BN rats had higher
MAP CV in the boards compared to the
controls on day two in both light phases
(Figure 3c). The same was seen in F344 rats
in the IVCs (Kemppinen et al., in press), and
this may be due to novelty effect of the item
introduced into the cage on the previous day.
The point estimates in the BN rats were 1.87 2.25 and throughout the study they were
higher in the open top than in the IVCs
(Kemppinen et al., in press) and they were
higher compared with the F344 rats as well
(Table 1). It seems that the BN rats have
larger variation in the open cages than in
IVCs. These results and those from our
previous study show that cage furniture has
strain specific consequences on within-group
variation and hence on number of animals
needed in blood pressure studies.
Ferrari et al. (1987) reported that a
cholinergic blockade in rats results in 30 %
reduction in HR CV and an increase in MAP
CV, but that a reduction in HR CV induced by
sympathetic blockage is not accompanied by a
change in MAP CV. Similarly to the IVC
results (Kemppinen et al., in press), in the
open cages there were only minor HR CV
changes in both strains and none of them
conformed to the scheme proposed by Ferrari
et al. (1987) It may be that rats require more
challenging stimuli than those resulting from
the furniture items used. Moreover, it has to
be borne in mind that results of Ferrari et al.
(1987) were seen in a situation where a part of
autonomic nervous system was blocked.
In this study no differences in faecal
corticosterone or faecal IgA could be
attributed to cage furniture. Eriksson et al.
(2004) have shown that the proportion of the
corticosterone and IgA excreted into faeces
and urine is at its highest during the dark
phase. Other studies have also shown the
same to be true with faecal corticosterone,
(Bamberg et al., 2001; Lepschy et al., 2007;
Pihl & Hau, 2003, Royo et al., 2004) and
faecal IgA (Royo et al., 2004). Royo et al.
(2004) stated that stress-induced changes in
excreted IgA concentrations are slower than
changes in corticosteroids and consequently
faecal IgA may be more useful for assessing
long-term
well-being
while
faecal
corticosterone is better at monitoring acute
stress events. The finding that F344 rats have
higher corticosterone and lower IgA excretion
into faeces than BN rats, supports the overall
concept that F344 strain is the more stress
prone of the two strains.
Moncek et al. (2004) reported higher
circulating corticosterone levels in Wistar
male rats housed in enrichment cages
compared to the non-enriched cages.
However, comparisons to this study are
complicated, because they used a combination
of objects as enrichment; toys, tunnels, swings
and running wheels and the enriched cage was
more than twice the size of the control cage.
Furthermore, the enriched cage had ten rats
and the control cage 3-4 rats; but nonetheless
cage density was lower in the enriched cages.
Krohn & Hansen (2002) suggested that
corticosterone may have only limited value
for assessing the effects of small
environmental changes on laboratory rodents.
The present study confirmed that F344 rats
have higher circulating corticosterone levels
than BN rats (Sarrieau & Mormède, 1998),
(Figure 4a). Siswanto et al. (2008) have
117
Based on the MAP results for F344 rats, the
tube appeared to be a poor choice for cage
furniture, while in BN rats all furniture items
seemed beneficial, but both the board types
were superior to the tube. Cage furniture may
result in increasing variation in the
physiological parameters studied and may
thus increase the numbers of animals needed
in blood pressure studies, albeit a lack of
consistency in the results were obvious in the
present study. In conclusion, it may be futile
to aim for general guidelines for optimal cage
furniture in terms of environmental
enrichment for laboratory rats due to their
genetic variation in responses, and the wide
variety of housing systems in different
laboratory animal facilities.
shown that there needs to be quite substantial
changes in serum corticosterone for these to
be detectable in faeces, and the HPA-axis may
not be stimulated enough by changes in cage
environment to be seen in faecal
corticosteroid excretion.
In the present study, two rat strains, F344
and BN, were used to achieve the better
presentation for the rat as a species (Festing,
2002), and as the results showed that the
strains did not respond to the cage items
equally and they responded differently to tube
and dividing boards. These findings show that
the environment effects to the study results in
rats. The study of Richter et al. (2009) argues
that genetic and environmental variations
cause the poor reproducibility of experimental
outcomes and thus, the environmental
standardization can contribute to spurious and
conflicting findings and unnecessary animal
use. However, environment and animals used
in the study are controlled to a large extent by
the researcher, otherwise than the random
variability, like inter-individual differences
(Festing, 2002).
In summary, cardiovascular parameters are
more sensitive than faecal corticosterone and
faecal IgA for assessing the physiological
impact of various types of cage furniture.
References
Abelson KSP, SS Fard, J Nyman, R Goldkuhl & J Hau.
Distribution of [H-3]-coricosterone in urine, feces and
blood of male Sprague-Dawley rats after tail vain and
jugular vein injections. In Vivo, 2009, 29, 381-386.
Armario A, A Gavaldá & J Martí. Comparison of the
behavioural and endocrine response to forced
swimming stress in five inbred strains of rats.
Psychoneuroendocrinology, 1995, 20, 879-890.
Acknowledgements
This study was supported by the Academy
of Finland, the Finnish Ministry of Education,
ECLAM and ESLAV Foundation, the NorthSavo Cultural Foundation, the Finnish
Research School for Animal Welfare, the
Oskar Öflund Foundation and the University
of Kuopio. We are thankful to Mr Heikki
Pekonen for surgical procedures and to Ms
Virpi Nousiainen for daily caretaking of the
rats.
Bamberg E, R Palme & JG Meingassner. Excretion of
corticosteroid metabolites in urine and faeces of rats.
Lab Anim, 2001, 35, 307-314.
Batchelor GR. The rest/activity rhythm of the laboratory
rat housed under different systems. Anim Technol,
1994, 45, 181-187.
Belz, EE, JS Kennell, RK Czambel, RT Rubin & Rhodes
ME. Environmental enrichment lowers stressresponsive hormones in singly housed male and female
rats. Pharmacol Biochem Behav, 2003, 76, 481-486.
118
Björk E, T Nevalainen, M Hakumäki & H-M Voipio. Rweighting provides better estimation for rat hearing
sensitivity. Lab Anim, 2000, 34, 136-144.
van den Brant J, P Kovács & I Klöting. Blood pressure,
heart rate and motor activity in 6 inbred rat strains and
wild rats (Rattus norvegicus): a comparative study. Exp
Anim, 1999, 48, 235-240.
Council of Europe. Appendix A of the European
Convention for the Protection of Vertebrate Animals
used for Experimental and Other Scientific Purposes
(ETS No. 123) Guidelines for accommodation and care
of animals. Strasbourg 2006. Access through:
http://conventions.coe.int/Treaty/EN/Treaties/Html/123
-A.htm
Eriksson E, F Royo, K Lyberg, H-E Carlsson & J Hau.
Effect of metabolic cage housing on immunoglobulin A
and corticosterone excretion in faeces and urine of
young male rats. Exp Physiol, 2004, 89, 427-433.
Eskola S M, Lauhikari, H-M Voipio & T Nevalainen. The
use of aspen blocks and tubes to enrich the cage
environment of laboratory rats. Scand J Lab Anim Sci,
1999, 26, 1-10.
European Union. Commission Recommendation on 18
June 2007 on guidelines for the accommodation and
care of animals used for experimental and other
scientific purposes (2007/526/EC). Brussels 2007.
Access
through:
http://eurlex.europa.eu/JOHtml.do?uri=OJ:L:2007:197:SOM:EN
:HTML
Ferrari AU, A Daffonchio & F Albergati. Inverse
relationship between heart rate and blood pressure
variabilities in rats. Hypertension, 1987, 10, 533-537.
Festing M, P Overend, R Gaines Das, M Cortina Borja &
M Berdoy. The design of animal experiments.
Laboratory Animal Handbooks No. 14. RSM Press,
London, 2002.
Gómez F, ER De Kloet & A Armario. Glucocorticoid
negative feedback on the HPA axis in five inbred rat
strains. Am J Physiol Regul Integr Comp Physiol,
1998, 274, 420-427.
Guhad FA & J Hau. Salivary IgA as a marker of social
stress in rats. Neurosci Lett, 1996, 216, 137-140.
Hubert M-F, P Laroque, J-P Gillet & KP Keenan. The
effects of diet, ad libitum feeding, and moderate and
severe dietary restriction on body weight, survival,
clinical pathology parameters, and cause of death in
control Sprague-Dawley rats. Toxicol Sci, 2000,
58,195-207.
Kaliste EK, SM Mering & HK Huuskonen. Environmental
modification and agonistic behavior in NIH/S male
mice: nesting material enhances fighting but shelters
prevent it. Comp Med, 2006, 56, 202-208.
Kemppinen N, A Meller, K Mauranen, T,Kohila & T
Nevalainen. Work for food – a solution for restricted
feeding in group housed rats? Scand J Lab Anim Sci,
2008a,
35,
81-90.
Access
through:
http://biomedicum.ut.ee/sjlas/35_2_81-90.pdf
Kemppinen N, A Meller, E Björk, T Kohila & T
Nevalainen. Exposure in the shoebox: comparison of
physical environment of IVC- and open rat cages.
Scand J Lab Anim Sci, 2008b, 35, 97-103. Access
through: http://biomedicum.ut.ee/sjlas/35_2_97-103.pdf
Kemppinen N, A Meller, K Mauranen, T Kohila & T
Nevalainen. The effect of dividing walls, a tunnel and
restricted feeding on cardiovascular responses to cage
change and gavage in rats (Rattus norvegicus). J Am
Assoc Lab Anim Sci, 2009, 48(2), 157-165.
Kemppinen N, J Hau, A Meller, K Mauranen, T Kohila & T
Nevalainen. Impact of aspen furniture and restricted
feeding on activity, blood pressure, heart rate, and
faecal corticosterone and IgA excretion in rats (Rattus
norvegicus) housed in individually ventilated cages.
Lab Anim, in press.
Kramer K, L Kinter, B Brockway, H-P Voss, R Remie &
BLM van Zutphen. The use of telemetry in small
laboratory animals: recent advances. Contemp Top Lab
Anim Sci, 2001, 40, 8-16.
Krohn TC & AK Hansen. The application of traditional
behavioural and physiological methods for monitoring
of the welfare impact of different flooring conditions in
rodents. Scand J Lab Anim Sci, 2002, 29, 79-89.
Krohn TC, AK Hansen & N Dragsted. Telemetry as a
method for measuring the impact of housing condition
on rats’ welfare. Anim Welf, 2003a, 12, 53-62.
Krohn TC, AK Hansen & N Dragsted. The impact of cage
ventilation on rats housed in IVC system. Lab Anim,
2003b, 37, 85-93.
Lemaire V & P Mormède. Telemetered recording of blood
pressure and heart rate in different strains of rats during
chronic social stress. Physiol Behav 1995, 58, 11811188.
Lepschy M, C Touma, R Hruby & R Palme. Non-invasive
measurement of adrenocortical activity in male and
female rats. Lab Anim 2007, 41, 372-387.
Manser CE, DM Broom, P Overend & TH Morris. Operant
studies to determine the strength of preference in
laboratory rats for nest-boxes and nesting materials.
Lab Anim, 1998, 32, 36-41.
Marissal-Arvy N, P Mormède & A Sarrieau. Strain
differences in corticosteroid receptor efficiencies and
regulation in Brown Norway and Fischer 344 rats. J
Neuroendocrinol, 1999, 11, 267-273.
Moncek F R Duncko, BB Johansson & D Jezova. Effect of
environmental enrichment on stress related systems in
rats. J Neuroendocrinol, 2004, 16, 423-431.
Möstl E & Palme R. Hormones as indicators of stress.
Domest Anim Endocrinol, 2002, 3, 67-74.
119
Nevalainen T & T Vartiainen. Volatile organic compounds
in commonly used beddings before and after
autoclaving. Scan J Lab Anim Sci, 1996, 23, 101-104.
Patterson-Kane EG. Shelter enrichment for rats. Contemp
Top Lab Anim Sci, 2003, 42, 46-48.
Pihl L & J Hau. Faecal corticosterone and immunoglobulin
A in young adult rats. Lab Anim, 2003, 37, 166-171.
Ramos A, O Berton, P Mormède & F Chalouff. A multipletest study of anxiety-related behaviours in six inbred rat
strains. Behav Brain Res, 1997, 85, 57-69.
Rex A, U Sondern, JP Voight, S Franck & H Fink. Strain
differences in fear-motivated behaviour of rats.
Pharmacol Biochem Behav, 1996, 54, 107-111.
Richter SH, JP Garner & H Würbel. Environmental
standardization: cure or cause of poor reproducibility in
animal experiments? Nat Methods, 2009, 6, 257-261.
Roe FJC. Historical histopathological control data for
laboratory rodents: valuable treasure or worthless trash?
Lab Anim, 1994, 28, 148-154.
Roe FJC, PN Lee, G Conybeare, D Kelly, B Matter, D
Prentice & G Tobin. The biosure study: Influence of
composition of diet and food consumption on
longevity, degenerative diseases and neoplasia in
Wistar rats studied for up to 30 months post weaning.
Food Chem Toxic, 1995, 33, 1S-100S.
Royo F, N Björk, H-E Carlsson, S Mayo, J Hau. Impact of
chronic catheterization and automated blood sampling
(Accusampler) on serum corticosterone and fecal
immunoreactive corticosterone metabolites and
immunoglobulin A in male rats. J Endocrinol, 2004,
180, 145-153.
Sarrieau A & P Mormède. Hypothalamic-pituitary-adrenal
axis activity in the inbred Brown Norway and Fischer
344 rat strains. Life Sci, 1998, 62, 1417-1425.
Sharp J, T Azar & D Lawson. Effects of a cage enrichment
program on heart rate, blood pressure, and activity of
male Sprague-Dawley and spontaneously hypertensive
rats monitored by radiotelemetry. Contemp Top Lab
Anim Sci, 2005, 44, 32-40.
Siswanto H, J Hau, HE Carlsson, R Goldkuhl & KS
Abelson. Corticosterone concentrations in blood and
excretion in faeces after ATCH administration in male
Sprague-Dawley rats. In Vivo, 2008, 22, 435-440.
Spangler EL, KS Waggie, J Hengemihle, D Roberts, B
Hess & DK Ingram. Behavioral assessment of aging in
male Fischer 344 and brown Norway rat strains and
their F1 hybrid. Neurobiol Aging, 1994, 15, 319-328.
Spiteri NJ. Circadian patterning of feeding, drinking and
activity during diurnal food access in rats. Physiol
Behav, 1982, 28, 139-147.
van den Staay F & A Blokland. Behavioural differences
between outbred Wistar, inbred Fischer 344, Brown
Norway and hybrid Fischer 344 x Brown Norway rats.
Physiol. Behav, 1996, 60, 97-109.
Stauffacher M, A Peters, M Jennings, R Hubrecht, B
Holgate, R Francis, H Elliott, V Baumans & AK
Hansen (Convener). Future principles for housing and
care of laboratory rodents and rabbits. Report for the
revision of the Council of Europe Convention ETS 123
Appendix A for rodents and rabbits. Part B. Council of
Europe 2002.
Access
through:
www.coe.int/t/e/legal_affairs/legal_cooperation/biological_safety%2C_use_of_animals/Labor
atory_animals/
Strubbe JH & AJ Alingh Prins. Reduced insulin secretion
after short-term food deprivation in rats plays a key role
in the adaptive interaction of glucose and fatty free acid
utilization. Physiol Behav, 1986, 37, 441-445.
Strubbe JH, NJ Spiteri & AJ Alingh Prins. Effect of
skeleton photoperiod and food availability on the
circadian pattern of feeding and drinking in rats.
Physiol Behav, 1986, 36, 647-651.
Swoap SJ, M Overton & G Garber. Effect of ambient
temperature on cardiovascular parameters in rats and
mice: a comparative approach. Am J Physiol Regul
Integr Comp Physiol, 2004, 287, R391-R396.
Tsai PP, U Pachowsky, HD Stelzer & H Hackbarth.
Impact of environmental enrichment in mice. 1: effect
of housing conditions on body weight, organ weights
and haematology in different strains. Lab Anim, 2002,
36, 411-419.
Tsai PP, HD Stelzer, HJ Hedrich & H Hackbarth. Are the
effects of different enrichment designs on the
physiology and behaviour of DBA/2 mice consistent?
Lab Anim, 2003, 37, 314-327.
Townsend P. Use of in-cage shelter by laboratory rats.
Anim Welf, 1997, 6, 95-103.
Voipio HM, T Nevalainen, P Halonen, M Hakumäki & E
Björk. Role of cage material, working style and hearing
sensitivity in perception of animal care noise. Lab
Anim, 2006, 40, 400-409.
Voipio HM, T Korhonen, T Koistinen, H Kuronen, S
Mering & T Nevalainen. Durability and hygiene of
aspen tubes used for providing environmental
complexity for laboratory rats. Scan J Lab Anim Sci,
2008,
35,
107-115.
Access
through:
http://biomedicum.ut.ee/sjlas/35_2_107-115.pdf
Zhang B & F Sannajust. Diurnal rhythms of blood
pressure, heart rate, and locomotor activity in adult and
old male Wistar rats. Physiol Behav, 2000, 70, 375380.
120
CHAPTER VII
GENERAL DISCUSSION
CHAPTER VII
GENERAL DISCUSSION
7.1 IVC vs. open cage
IVC systems provide protection against
animal infections and confer occupational
benefits for personnel; this latter effect
through a reduction in the levels of ammonia
and airborne allergens (Keller et al. 1983,
Lipman et al. 1992, Renström et al. 2001,
Teixeira et al. 2006). It is generally believed
that the IVCs and open cages share the same
physical environment whenever they are in
the same room. This study found that this is
not the case, but that they represent rather
different
physical
environments;
the
magnitude of the difference being large
enough to have impact on the animals inside
the cages. Table 7.1 presents a summary of
physical cage environment between the caging
systems.
Due to the additional cover, the light
intensity in IVCs was lower than in open top
cages. Not surprisingly the brightest cages
were on the top row of both racks and the
dimmest on the bottom. The placements of
fluorescent tubes on the ceiling were decisive
in determining the light intensity in the cages;
cages closer to light source were brighter
within both cage types. Similar illumination
results have been reported Clough et al.
(1995) in transparent, individually ventilated
cages; while in the translucent, conventional
cages, the illumination level was higher than
in the cages used in this study. The dimmer
lightning in the present study may be due to
the black plastic sheet that was placed on top
of the open top cage racks to equalize lighting
in both cage types. In some conventional
cages examined in this study, the lighting may
have been too bright (see Chapter II), even to
the extent that over a longer period it could
have caused retinal damage in albino rats
(Gorn & Kuwabara 1967, Stotzer et al. 1970,
Weisse et al. 1974).
Since sound levels went down when
approaching 16 000 Hz; it is fair to assume
that no ultrasounds (> 20 000 Hz) are emitted
into either cage type. Accordingly, the
measured R-weighted sound pressure levels
monitor accurately the sound level as heard by
the rats. In open top cages, the R-weighted
sound levels were about 7 dB(R) lower than in
IVCs; i.e. loudness was about half and energywise the difference was fivefold. This
different sound environment between the
cages is presumably due to the extra lid and
air fans of the ventilation machine in the IVCracks. The difference in sound levels audible
to the rat (R-weighted) appears to be large, yet
it remains unclear whether the levels
measured (< 25 dB(R)) have any impact on
the animals.
The climatic conditions in the cage depend
on those of the surrounding room as well as
on the air supply to the cage-rack (Scheer et
al. not dated). In the present study, when there
were animals in the IVCs, this increased the
temperature by 3-4 °C and RH by about 6 %
in the cages, while temperature and RH in
IVCs were the same as in the animal room
when measured without animals. However,
temperature and RH in the IVCs (Table 7.1)
did not exceed the limits of the European
Kuopio Univ. Publ. C Nat. and Environ. Sci. 258:121-140 (2009)
123
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
Table 7.1 Physical environment characterization of IVCs and open top rat cages in the present study.
Illumination in empty cage
Sound level in empty cage
Temperature (empty cage)
RH (empty cage)
Temperature (3 rats in cage)
RH (3 rats in cage)
IVC
Open cage
Top row: 16.4-29.2 lx
Bottom row: 3.8-4.9 lx
20-25 dB(R)
45-47 dB(A)
21.8-22.1 °C
30-55 %
22.3-26.0 °C
39-65 %
Top row: 53.8-91.0 lx
Bottom row: 14.3-25.3 lx
12-18 dB (R)
46-49 dB(A)
21.3-22.1 °C
32-57 %
20.8-22.9 °C
35-67 %
lesions as they grow older; BN rats in adrenal
guidelines (Council of Europe 2006). Swoap
glands, kidneys, lungs and pancreas and F344
et al. (2004) showed that even small changes
rats in eyes, heart, lungs and kidneys
in
ambient
temperature
can
alter
(Lipmann et al. 1999), none of these problems
cardiovascular parameters in rats; when
were seen in our rats during the 16 week
temperature increased, MAP and HR of SD
study. This is not surprising since these
female rats decreased. This effect was seen in
lesions typically occur in rats older than those
HR of the present study throughout the study;
examined here.
both rat strains had significantly higher HR in
The similarity of the temperature and RH
the open cages than in the IVCs, where the
in the open top cages and in the animal room
temperature was 1-4 oC higher. However, in
is in line with the results of Clough et al.
the open cages, the rats were eight weeks
(1995), who essentially used the same type of
older than those in the IVCs, hence the
IVCs as ours in terms of physical environment
differences cannot be attributed to the cage
and compared them to rats housed in
type alone. Zhang & Sannajust (2000)
conventional cages. Also Reeb et al. (1998)
reported a decrease in the nocturnal HR in old
have reported lower temperature and RH in
Wistar rats, an apparently opposite finding to
empty control mouse IVC when compared to
this study, with higher HR in older rats with
room air. Moreover, microenvironment
both strains. However, their old Wistar rats
temperature and RH in mouse IVCs are
were two years old, whereas our rats were
known to be significantly higher with lower
only 8-10-months old in the open top cages.
ventilation rates, such as 30-40 air changes/h
The rats used of this study were older than
(Reeb et al. 1998). This shows that IVCthe types of young adult animals most
ventilation is not capable of removing
commonly used in basic biomedical research.
excessive heat produced by the animals and
However, now than genetically altered rat
heat generated from urine-faeces-bedding
strains are becoming more popular, this age
fermentation. The fermentation reaction is an
class will become increasingly common in
unlikely candidate for heat generation because
many facilities in addition to those used in
ventilation tends to keep bedding quite dry.
long term safety studies. It is known that both
However, the heat emission from one animal
BN and F344 rats develop spontaneous
Kuopio Univ. Publ. C Nat. and Environ. Sci. 258:121-140 (2009)
124
General discussion
be a better restricted feeding method than the
“foraging device” used in the study of
Johnson et al. (2004); the weight gain of the
rats was higher with the foraging device than
that encounters in ad libitum fed controls, and
when the rats had limited access to food, their
body weights remained unchanged, both
indications of method failure.
Other ways to decrease the weight gain of
rats have been tried; e.g. sugar beet pulp fibre
made from water soluble polysaccharides
(Eller et al. 2004). In that study, the rats grew
less when fed with the fibre diet, but an
autopsy after the study revealed an enlarged
digestive tract in the rats that had received the
fibre enriched diet, especially the caecum was
enlarged. This may have been caused by
hygroscopic effect of the fibre.
7.2 Diet board as a restricted feeding
In early studies it has been demonstrated
method in rats
that rats are willing to work for food; if the
In the present study, the rats with the diet
rats had free access to food they would rather
board grew less than those on ad libitum
earn their food as long as the work demands
feeding in both cage types. The diet board
were low (Carder & Berkowitz 1970,
worked especially well with the F344 rats in
Neuringer 1969). Johnson et al. (2004) also
controlling body weight. The F344 rats with
reported that the rats preferred to eat mostly
the diet board even lost weight in the open
from the foraging device which required
cages, when they were older, but the
digging gravel in order to gain access. This
magnitude of loss was marginal; only a few
may reflect the rats’ need to perform foraging
grams during two weeks, most likely fat
behaviour as they would in their natural
tissue. The rats ate 12 - 18 % less with the diet
environment.
board as compared to the respective controls.
In the present study, the rats had to gnaw
The same was seen in a study with the outbred
the wood if they wished to eat the food pellets
Wistar rats; with the diet board they ate
from the diet board. Thus, the rats gnawed the
significantly less and had lower body weights
wood most with the diet board as compared to
than ad libitum feeding controls (Kasanen et
plain board and tube groups in both cage
al. 2009a).
types. The F344-rats gnawed wood
The rats in the open cages ate more with
significantly more than the BN rats - these rats
the plain board than with the two other
hardly gnawed any wood from plain board
controls - apparently because the plain board
and tube - this may related to a difference in
round followed the diet board round, and the
the natural behaviour of these two strains (Rex
animals regained their weight loss in that
round. Nevertheless, the diet board seems to
et al. 1996, Ramos et al. 1997). Eskola et al.
Kuopio Univ. Publ. C Nat. and Environ. Sci. 258:121-140 (2009)
125
has been estimated to be about 4 W (Heine
1998), and this is a likely source of excessive
heat in the cage.
It appears that the transition from traditional
open top cages to IVCs may cause changes in
the physical environment, especially if the
incoming air into the IVC-system is drawn
from the room air. This makes any
comparisons between these caging systems
problematic without characterization of the
physical parameters e.g. lighting intensity,
sound environment, temperature and RH. In
the present study, several differences were
found in the responses of rats housed in IVCs
and open-top cages; these will be discussed
later in this chapter.
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
activity, similarly to the other day-night
(1999) showed that outbred Wistar rats would
rhythms is controlled by the circadian
spontaneously gnaw aspen blocks and tubes
oscillator located in the suprachiasmatic
but this opportunity for gnawing combined
nuclei. It has been suggested that there are
with ad libitum feeding had no effect on their
other oscillators involved in the circadian
growth; a similar situation to F344 rats with
system and this accounts for the flexibility
plain board and tube. Sørensen et al. (2004)
needed for adaptation to different external and
have suggested that excessive gnawing in rats
internal stimuli (Anglés-Pujolrás et al. 2006).
is related to escape or frustration – in the
If the rats are given meals for a few hours
present study in the F344 rats, the gnawing
daily,
the
food
is
eaten
almost
could be more related to escaping.
instantaneously; this impairs their natural
In both cage types, the F344 rats were
feeding activity and consequently the
significantly more active in the dark compared
associated gastrointestinal physiology. The
to the BN rats with all cage items. Since there
diet board used for food restriction in the
were no differences in the activity between the
present study allowed rats to retain a natural
plain board and diet board groups, we
feeding pattern and the feeding activity was
conclude that work associated with the diet
similar to that encountered with the plain
board was not overly laborious to the animals.
board. Kasanen et al. (2009a, 2009b) have
Furthermore, the activity of the rats that had to
also shown that when rats have access to the
work for food was no different from their
diet board, they maintain their normal diurnal
activity during ad libitum feeding. In previous
eating rhythms. Furthermore, the diet board
studies, it has been shown that rats with
appears to result in higher serum
limited access to food spend more time
corticosterone and decreased IgA levels in rats
feeding, but with the ”foraging device” the
compared to the ad libitum feeding controls,
time spent feeding was markedly decreased
yet no obvious stress pathology was
(Hawkins et al. 1999; Johnson et al. 2004).
associated with its use (Kasanen et al. 2009b).
There were no significant changes in the total
activity levels between the groups. In the rat
groups, there were no changes in the social
7.3 Cage change and IG-gavage
The results of the present study show that
hierarchy nor any increased fighting or
cardiovascular responses to cage change and
stereotypic behaviour when rats had limited
gavage can be modified by provision of the
access to food (Hawkins et al. 1999).
cage furniture items in both strains studied.
Rats consume most of their food during the
Overall, there are very few previous studies
night; Spiteri (1982) reported that 94 % of
on the impact of cage items to procedural
food intake takes place during the dark. A
responses. In most of the published studies on
rat’s typical feeding rhythm shows two peaks
cage change or IG-gavage, the cage items
in the dark phase, the first peak at the
used are either not mentioned or all the
beginning of the dark and the other at the end
studied groups have had the same objects
of that period (Spiteri 1982; Strubbe et al.
(Saibaba et al. 1996, Schnecko et al. 1998,
1986). Light shifts the clock in a circadian
time-dependent way ensuring entrainment
Brown et al. 2000, Duke et al. 2001, Sharp et
according to the illumination cycle. Eating
Kuopio Univ. Publ. C Nat. and Environ. Sci. 258:121-140 (2009)
126
General discussion
these activities increase the cardiovascular
parameters and LA after the cage change. The
cage furniture can serve as an odour cue
making the new cage environment more
familiar to rats.
The cage change procedure increased
MAP, HR and activity instantly in all study
groups, but the values returned close to
baseline within one hour. The same
phenomenon was seen in the results of Duke
et al. (2001) and Sharp et al. (2002, 2003),
who also detected a notable elevation in blood
pressure and heart rate after the cage change,
but in these studies both parameters returned
to baseline within 60-180 min. HR of the rats
has been shown to increase only by moving
the cage from the cage rack (Gärtner et al.
1980). Schnecko et al. (1998) reported that if
the change took place in the morning, during
the resting period, the systolic and diastolic
blood pressure and HR responses were larger
compared to situation if the change occurred
during the activity period in the evening. A
recent study of Abou-Ismail et al. 2008
reported that if rats experienced a cage change
during the light period, they slept less and had
more chromodacryorrhea, reduced thymus
weight, increased aggression, and less objectdirected behaviour. The late timing for
husbandry procedures is unlikely to work in
practice; hence in this study the procedure was
carried out during afternoon within working
hours.
In the present study, assessment on whether
statistically significant differences detected
possess biologic or welfare relevance, we
utilized strain-specific reference values, the
night–day differences in MAP and HR
calculated for the control group. Based on this
comparison, the statistically significant MAP
or HR responses to both cage change and IGKuopio Univ. Publ. C Nat. and Environ. Sci. 258:121-140 (2009)
127
al. 2003, Bonnichsen et al. 2005, Õkva et al.
2006).
The MAP and HR responses to gavage
during the first hour after the procedure
appear to be smaller in magnitude to those
associated with the first cage change. In the
study of Õkva et al. (2006), the immediate
responses in blood pressure and HR to cage
change and IG-gavage in outbred Wistar rats
were essentially the same as these seen in
F344 and BN rats in this study. Moreover,
Seggie & Brown (1975) found a higher
increase in the corticosterone level when rats
were moved to a novel environment compared
with short-term handling. The IG-gavage is a
short-term procedure, commonly considered
to be more stressful than handling. One
common factor with both procedures is that
the rats are returned back to the familiar home
cage. In the cage change procedure, the
animals are placed into a new environment
with the new odours; hence it is no surprise
that there are greater responses to cage
change.
Immediately after the rats had been placed
into clean cages, their activity increased. This
finding agrees with the several other studies
(Burn et al. 2006, Schnecko et al. 1998,
Saibaba et al. 1996). The behaviour repertoire
of the rats is also affected in clean cages;
walking and skirmishing frequency increase
immediately after the cage change, whereas
grooming, eating, drinking, resting, rearing
and bedding manipulation all decrease (Burn
et al. 2006). The skirmishing or fighting of
rats after the cage change is associated with
dominance hierarchies within the group, and
is related to their territory (Barnett 1958), e.g.
a new cage. Being exploratory animals, rats
investigate their new surroundings by
ambulating and rearing (Hughes 1968) and
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
change. The MAP response was less during
the period from one hour after cage change
until dark in BN rats with a diet board when
compared to the controls. The new diet board
may have allowed easier access to food than
the old, gnawed board, thereby leading to the
changes in MAP. In open-top cages, the only
observed effect in the BN rats was during the
first hour after the IG-gavage and with the
tube.
A summary of the MAP and HR
differences to the baseline during one hour
after cage change and in response to gavage in
F344 and BN rats is presented in Table 7.2. It
shows that significantly higher differences
were mostly seen in the IVCs after the cage
change. The reasons for this phenomenon
might be the rats’ younger age in the IVCs or
the difference in the physical environment in
the two cage types as discussed in Chapter II.
gavage for F344 rats in IVCs were not greater
than the night–day MAP or HR difference in
these parameters and thus they cannot be
considered
biologically
meaningful.
Therefore, a biologically valid effect
attributable to cage objects is not apparent in
the current study. In open-top cages, many
statistically significant differences were
detected in the responses to both procedures in
the F344 rats, but again, none of them were
large enough to achieve biologic significance.
In the light, when the BN rats had the plain
board in IVCs, the MAP response to IGgavage was lower compared to the other
items. This statistically significant difference
exceeded the BN-specific MAP night–day
difference,
thus
being
biologically
meaningful, but this was not the case for
corresponding HR differences. The BN rats
showed no significant between the cage
furniture item differences in HR after the cage
Table 7.2 Comparison of cage types: MAP and HR difference to the baseline during one hour after
cage change and IG-gavage in F344 and BN rats. Arrows show the cage type with the higher
difference value. * < 0.05, ** < 0.01, *** < 0.001
Cage change
F344
Diet board
Tube
Plain board
Control
BN
MAP
IVC ↑ ***
NS
NS
IVC ↑ ***
MAP
Diet board
NS
Tube
IVC ↑ *
Plain board
NS
Control
NS
NS = not significant
128
IG-gavage
HR
IVC ↑ ***
NS
IVC ↑ **
NS
MAP
NS
NS
NS
NS
HR
IVC ↑ *
NS
NS
NS
HR
MAP
HR
NS
IVC ↑ ***
NS
IVC ↑ *
NS
IVC ↑ *
OPEN ↑ **
IVC ↑ *
NS
IVC ↑ ***
NS
IVC ↑ **
Kuopio Univ. Publ. C Nat. and Environ. Sci. 258:121-140 (2009)
General discussion
significant, were judged to be false positive
results. All biologically meaningful results are
listed in the Table 7.3. The results of this
study suggest that a fixed percentage as in the
study of Krohn et al. (2003a) is not applicable
for all strains, and that a strain-specific
measure, such as a night-day difference may
be more valid for this purpose.
Some scientists have stressed the effect of
the cage items on the variation instead of
mean values. Some studies suggest that the
cage items or “enrichments” may increase
variation, resulting in a higher number of
animals needed in experiments (Eskola et al.
1999, Mering et al. 2001, Tsai et al. 2002,
Tsai et al. 2003a). The studies of Tsai et al.
(2002, 2003a, 2003b) showed that enrichment
items had an effect on the coefficient variation
(CV) of body weight, haematological data,
organ weights and breeding index in mice. In
our studies with rats and cage items, the CV
has not been used before as an indication of
the variation within the experimental groups.
In this study, the F344 rats in the IVCs had
the highest CV of MAP in the plain board on
the second day both in the dark and light
phases, and this may reflect the novelty of the
cage items. The point estimates (see Table
7.5) showed that with the plain board, the
number of animals needed would be about 1.5
times greater than with the other items or even
controls
when
the
corresponding
cardiovascular parameters were result
parameters. Surprisingly, the number of
animals needed to achieve the same results
with the plain board would have been higher
than with the tube although the MAP with the
tube was at the highest levels in F344 rats.
Hence, the high blood pressure in the F344
rats with the tube was not associated with
wide variation. In the open cages, the F344
Kuopio Univ. Publ. C Nat. and Environ. Sci. 258:121-140 (2009)
129
7.4 Cage furniture items and stress
indicators in rats
7.4.1 Cardiovascular parameters (MAP
and HR)
This study shows that the cardiovascular
responses of F344 rats and BN rats to the
added furniture items were not the same. The
F344 rats in the IVCs had the highest MAP in
the tube cages all through the two week period
irrespective of whether lights were on or off,
whereas in the open cages the same was seen
only in the last day of the period. The BN rats
displayed a considerable difference between
open cages and IVCs; in the IVC they were
very small, 1-2 mmHg, MAP differences
between the groups. In the open cages, they
were elevated up to 6 mmHg, and additionally
MAP levels of all rats in groups with cage
items were below the values of the controls.
Krohn et al. (2003a) showed that systolic
blood pressure and heart rate in SD rats rises
6-7 % in stressful housing conditions, and
concluded that changes below this percentage
in systolic blood pressure and heart rate may
not be biologically significant despite being
statistically significant (Krohn et al. 2003b).
The results of the present study suggest that a
fixed percentage may not be applicable to all
strains, and that a strain-specific measure,
such as the night-day difference may be more
valid for that purpose.
This study considered the cardiovascular
effect of housing items to be biologically
meaningful; if it were at least as large as the
difference between the corresponding night
and day values. The mean night-day
difference values were calculated from the
control group results from the day 14 data; i.e.
for both rat strains and for both cage types
separately. The results that were not
biologically meaningful, but statistically
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
rats displayed only one significant finding on
day 14 when the HR CV in the tube group
was higher than that in the controls.
The BN rats in the IVCs exhibited higher
CV values of both MAP and HR in the control
group compared to the diet board and the
point estimates were 1.45 and 1.68,
respectively. At the end of the study period,
the BN rats seemed to have adjusted to the use
of the diet board and the variation was
significantly lower compared to the controls.
In the open cages, the BN rats had higher
MAP CV in the boards compared to the
controls on day two in both light phases that
may be due, as in the F344 rats in the IVCs, to
the novelty of the item which had been
introduced into the cage on the previous day.
The point estimates of the BN rats in open
cages were 1.87 - 2.25 and throughout the
study; they were higher in the open top than in
the IVCs, and they were higher than in the
F344 rats as well. It seems that the BN rats
display a larger variation in the open cages
than in IVCs.
Table 7.3 Biologically meaningful telemetric changes attributable to cage furniture items compared to
controls in the present study. Statistical results are shown in Chapters IV, V and VI.
IVC
F344
Cage change -
IG-gavage
-
Day 2
All statistically
significant results
of MAP (V)
All statistically
significant results
of MAP (V)
All statistically
significant results
of MAP (V)
All statistically
significant results
of MAP (V)
Day 6
Day 10
Day 14
130
Open cage
BN
All statistically
significant results
of MAP (IV)
All statistically
significant results
of MAP (IV)
F344
-
BN
-
-
All statistically
significant results
of MAP (IV)
All statistically
significant results
of MAP; HR during
the first hour (IV)
-
-
-
-
-
-
-
All statistically
significant results
of MAP (V)
-
-
-
Kuopio Univ. Publ. C Nat. and Environ. Sci. 258:121-140 (2009)
General discussion
were housed in enriched cages than in nonenriched cages. However, comparisons of
these findings to the present study are
complicated, because they used a combination
of various objects as enrichment i.e. toys,
tunnels, swings and running wheels and the
enriched cage was more than twice the size of
the control cage. Furthermore, the enriched
cage housed ten rats and the control cage had
3-4 rats; but nonetheless cage density was
lower in the enriched cages. Rushen & de
Passillé (1992) have criticized the use of
corticosterone as an indicator of animal
welfare in different housing methods since
they are not always closely related to the
7.4.2 Faecal corticosterone and faecal IgA
mental or emotional states of animals and the
This study found no differences in faecal
measures of a single hormone ignore the
concentrations of corticosterone or IgA
complex physiological reactions of animals to
attributable to cage furniture in either of the
their environments. Furthermore, the study of
cage types. There are studies to show that the
Krohn & Hansen (2002) suggests that
highest amounts of corticosterone and IgA are
corticosterone may have only limited value
excreted into faeces and urine during the dark
for assessing the effects of minor
phase (Bamberg et al. 2001, Eriksson et al.
environmental changes on laboratory rodents.
2004, Royo et al. 2004, Pihl & Hau 2003,
Furthermore, Siswanto et al. (2008) showed
Lepschy et al. 2007). It has been stated that
that there needs to be quite substantial
stress-induced changes in excreted IgA
changes in serum corticosterone for these to
concentrations are slower than changes in
be detectable in faeces, and the HPA-axis may
corticosteroids and thus faecal IgA may be
not be stimulated enough by changes in cage
more useful for assessing long-term wellenvironment for this to be reflected in changes
being whereas the faecal corticosterone is
in the faecal corticosteroid excretion.
better for monitoring acute stress events
In summary, cardiovascular parameters
(Royo et al. 2004). We found that F344 rats
appear to be more sensitive than faecal
in the open cages have higher corticosterone
corticosterone and faecal IgA for assessing the
levels and lower IgA excretion into faeces
physiological impact of various types of cage
than BN rats; this supports the overall concept
furniture. Based on the MAP results for the
that F344 strain as being more sensitive for
F344 rats, the tube appeared to be a poor
experiencing stress and have higher
choice as a piece of cage furniture, while in
circulating corticosterone levels than BN rats
BN rats all furniture items seemed to be
(Armario et al.1995, Ramos et al. 1997,
beneficial, but both board types were better
Sarrieau & Mormède 1998).
than the tube. Cage furniture may result in
Moncek et al. (2004) found higher serum
increasing variation in the physiological
corticosterone levels in male Wistar rats that
Kuopio Univ. Publ. C Nat. and Environ. Sci. 258:121-140 (2009)
131
These results show that cage furniture has
strain specific consequences on within-group
variation. The same was described in the
studies of Tsai et al. (2002, 2003a, 2003b)
with the different mouse strains and
measurements of body weight, haematological
data, organ weights and breeding index.
Overall, cage items may result in increased
variation in the cardiovascular parameters
studied and may thus change the numbers of
animals needed in those studies, albeit the
lack of consistency in the results was obvious
in the present study.
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
parameters studied and may thus change the
numbers of animals needed in blood pressure
studies, albeit the lack of consistency in the
results was obvious in the present study.
i.e. rats, mice, gerbils, hamsters, and guinea
pigs (Council of Europe 2007). However, all
rodent species and, as shown in this study,
strains within a species, may have different
needs. These differences raise the possibility
that general guidelines, which may be valid
for one species, may have a different effect on
welfare in other species and strains.
The present study shows that for the F344
rats, the tube appeared to be a poor choice as a
piece of cage furniture, while for BN rats all
furniture items seemed to be beneficial, but
both the board types were better than the tube.
As with these two strains in two different cage
types, it can be seen that there is a genetic
component involved in rat responses to cage
items as well. Sharp et al. (2005) also showed
a difference between SHR and SD rats in their
responses to procedures with a specified
enrichment program, and the authors
speculated on the difficulties of making
generalized recommendations to the animal
care community regarding rat enrichment
programs. Thus, it may be necessary, on top
of general guidelines, to add stock- and strainspecific modifications about optimal cage
furniture for laboratory rats because of the
obvious genetic component involved and also
to be able accommondate the wide variety of
housing systems in use.
7.5 Differences in rat strains
In the present study, two inbred strains of
rat, F344 and BN, were used to achieve
greater precision and wider applicability of
the results (Festing et al. 2002). These strains
have been shown to differ in various aspects
of physiology, e.g. systolic and diastolic blood
pressure, HR (Van den Brant et al. 1999),
plasma corticosterone concentration (Armario
et al. 1995, Sarrieau & Mormède 1998) and
brain and pituitary mineralocorticoid receptor
levels (Gómez et al. 1998, Marissal–Arvy et
al. 1999). Moreover, the F344 and BN rats
exhibit different levels and diurnal rhythms in
locomotor activity (Ramos et al. 1997, Van
den Brant et al. 1999) and behaviour
(Spangler et al. 1994, Van den Staay &
Blokland 1996).
The results of this study confirm the results
of previous studies with respect to blood
pressure, HR and LA, but also that these
strains have different food consumptions,
weight gain and wood gnawing behaviour. In
summary, BN and F344 rats responded
differently to the cage furniture items and to
the procedures. These and previous findings
(Table 1.6 in Chapter I) in these two strains
7.6 The Two R value of the results
might be attributable to the activities of the
To achieve a better applicability towards the
rats with the items, and in the future, it would
rat as a species, two defined rat strains (F344
be interesting to study the behaviour of the
and BN) were used in this study (Festing
rats with the same cage items. The summary
2002), and the results reveal that the strains
of the results in F344 and BN rats is shown in
behaved quite differently. F344 and BN rats
Table 7.4.
exhibit differences in LA, MAP and HR levels
The European regulations for laboratory
and in responses to the cage items and to the
rodents, although they are rather specific, are
procedures. These findings suggest that that
generally the same for all laboratory rodents,
Kuopio Univ. Publ. C Nat. and Environ. Sci. 258:121-140 (2009)
132
General discussion
Table 7.4 The summary of the results of F344 and BN rat strains used in the study.
IVC
F344
OPEN
BN
F344
BN
PHYSIOLOGY (mean values of control group)
MAP
Light
110.8 mmHg
92.7 mmHg
103.4 mmHg
88.6 mmHg
Dark
115.7 mmHg
95.6 mmHg
113.6 mmHg
91.8 mmHg
HR
Light
335 bpm
284 bpm
330 bpm
282 bpm
Dark
388 bpm
309 bpm
390 bpm
303 bpm
Activity
Light
1.4 counts/min
1.0 counts/min
1.2 counts/min
1.2 counts/min
Dark
4.9 counts/min
2.2 counts/min
5.0 counts/min
2.0 counts/min
FOOD CONSUMPTION, WEIGHT GAIN AND WOOD GNAWED (mean values)
Food consumed / 2 weeks
Diet board
Ad libium
712 g
874 g
607 g
612 g
718 g
913 g
567 g
664 g
-1.5 g
10.4 g
7.6 g
9.1 g
-8.8
9.7 g
3.4 g
4.0 g
134 g
16 g
27 g
100 g
3g
0g
147 g
11 g
10 g
94 g
1g
1g
Weight gain / 2 weeks
Diet board
Ad libitum
Wood gnawed
Diet board
Plain board
Tube
RESPONSES TO THE CAGE ITEMS
MAP difference (mean) to the controls at day 14 (Light/Dark)
Diet board
-5.6/-2.2 mmHg
-3.4/-2.2 mmHg
2.1/-1.5 mmHg
-5.9/-6.5 mmHg
Tube
3.1/3.4 mmHg
-1.9/-0.4 mmHg
4.5/1.8 mmHg
-3.2/-3.7 mmHg
Plain board
-10.4/-5.9 mmHg -0.2/-0.5 mmHg
-0.1/-0.5 mmHg -5.9/-5.0 mmHg
HR difference (mean) to the controls at day 14 (Light/Dark)
Diet board
-13.1/0.5 bpm
-13.9/-6.6 bpm
2.9/-3.0 bpm
-8.4/4.3 bpm
Tube
-1.2/-3.7 bpm
-9.4/1.7 bpm
12.5/13.4 bpm
-6.0/-3.4 bpm
Plain board
-112.9/-6.4 bpm
4.9/3.9 bpm
-3.4/6.0 bpm
-1.5/0.4 bpm
Corticosterone and IgA (mean values)
Corticosterone
NS
F344 had higher values (P = 0.05)
IgA
NS
BN had higher values (P < 0.05)
Kuopio Univ. Publ. C Nat. and Environ. Sci. 258:121-140 (2009)
133
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
all cage furniture items are not equally good
for laboratory rats, irrespective of the strain,
and that welfare indicators should be used to
rank the feasibility of cage furniture.
The
present
study
showed
that
conventional open cages and IVCs differ in
temperature, RH, sound spectra level and light
intensity. These findings did not appear to
have any direct effect on the results of the
studies made in those cages, but age
difference of eight weeks between open cages
and IVC do not allow simple comparisons.
The European recommendations emphasize
that enrichment has to be applied irrespective
of cage system (Council of Europe 2006).
Moreover, the applicability of any furniture
brought into the cage requires that an item has
a similar effect in all housing systems (Richter
et al. 2009).
The current European guidelines on
laboratory animal housing and care are similar
for all rodent species i.e. rats, mice, guinea
pigs, hamsters and gerbils (EU 2007). Within
a species, they have to be implemented
irrespective of animal strain, and caging
system used. The results of this study
indicated that it may be nessessary to devise
strain specific guidelines for optimal cage
furniture in terms of environmental
enrichment for laboratory rats because of the
genetic component involved in the responses
of the animals to these items. Once strain
specific guidelines for rats have been
established, then it could be possible to see
whether one can then devise species-specific
guidelines. This is a labour intensive
approach, but is there a scientifically valid
shortcut within sight?
Environmental enrichment as a term refers
to a positive impact on animals e.g. as based
134
on biological functioning, behaviour and 2Rs
(Chamove 1989, Newberry 1995, Purves
1997). The present study showed that
unfortunately all items added to the cage do
not result in a positive impact for rats; and
indeed
by
definition
environmental
enrichment does not constitute enrichment
before it is proven to have this affect. It is
clear that all studies should carry detailed
description of the cage furniture items used,
and better designed studies are needed to rank
various furniture items and other cage
additions.
In animal welfare studies, it is essential to
use a variety of welfare indicators to assess
effects of animal housing and management
systems. In the case if one parameter displays
a negative impact to welfare, then the result is
explicit, but if the result has a positive impact,
more studies and more positive results are
needed (Broom 1991, Clark et al. 1997). In
the present study, four parameters (MAP, HR,
faecal corticosterone and faecal IgA) were
used to assess the animal welfare. To achieve
an even more valid welfare impact of studied
cage items, additional parameters should be
added to the palette (e.g. behavioural and
preference studies).
A recent study indicated that genetic and
environmental variations are responsible for
the poor reproducibility of experimental
outcomes (Richter et al. 2009). However, the
traditional opinion states that the wider range
of genetic backgrounds and environments
used in an experiment, the applicability of the
results. This is exactly the reason why we
used two different rat strains and two different
caging systems to assess the welfare value of
the cage items used.
Richter et al. (2009) stated that
environmental standardization is indeed a
Kuopio Univ. Publ. C Nat. and Environ. Sci. 258:121-140 (2009)
General discussion
animals needed would be 1.87 - 2.25 times
higher than there needed in the diet board or
plain board, a through 24 hour period of day
2. Since both of these findings were detected
early in the two week period, this may be
attributable to a novelty effect soon after the
item had been introduced into the cage.
Some of effects on the point estimates
carried over till the end of the fourteen day
period. The use of the plain board meant also
less animals would be needed than with the
tube when MAP was the result parameter, but
with HR the situation was reversed. The
results showed that the number of animals
needed could be reduced in blood-pressure
studies. Although the results were strain
specific and there were differences between
the cage types, the cage furniture has a strong
Reduction potential in biomedical studies.
In summary, the present study showed that
the cage items and restricted feeding have
Refinement and Reduction potential for
laboratory rats. The diet board is a better
method of restricted feeding compared to the
previous methods; where rats have access to
food only a few hours every day, or a certain
amount of food is offered once a day. With
the diet board, rats ate less and gained less
weight in comparison to ad libitum-feeding,
but it had no effect on daily feeding activity or
other diurnal rhythms in rats. With respect to
the use of the cage items, the cardiovascular
responses to cage change and IG-gavage can
be lessened and furthermore there is also an
effect which lasts for weeks. In cardiovascular
studies, number of animals needed can be
reduced with suitable cage items. Rat welfare
can be assessed with MAP and HR in relation
to cage furniture, whereas changes induced by
the items in faecal corticosterone and faecal
IgA excretion seem too small to be
Kuopio Univ. Publ. C Nat. and Environ. Sci. 258:121-140 (2009)
135
cause for weak reproducibility of study
outcomes. Standardization of experiments has
been claimed to reduce within- and betweenexperiment variation in order to facilitate
detection of treatment effect and maximize
reproducibility of results (Würbel 2000). The
term standardization fallacy is used to
describe the increase of reproducibility at the
expense of external validity; i.e. how
applicable the results are in some other
environmental context, population and species
(Würbel 2000). The study of Wolfer et al.
(2004) showed that environmental enrichment
does not disrupt the standardization; even
though there are studies to suggest that
complex cage environments may increase
aggression among male mice (Nevison et al.
1999), which may increase variability in the
aggressive strains of laboratory rodents
(Festing 2002). In conclusion, it is
questionable whether one can blame
standardization for the poor results of
reproducibility; on top of good standardization
one has to emphasize applicability – in terms
of variety of defined genetic backgrounds and
environments used - as shown by Festing et al.
(2002) and as used in this study.
This study used CVs and point estimates to
assess between-group variation in F344 and
BN rats. The summary of the point estimates
reflecting the results from the corresponding
significant CVs (p < 0.01) are presented in
Table 7.5. The point estimates of day 2
showed that in the IVCs, the number of the
F344 rats needed would be 1.36 - 1.81 times
greater with the plain board than with the diet
board, tube or control when the MAP was the
result parameter, both in dark and light
phases. On the other hand, in the open cages
the point estimates of the MAP in the BN rats
show that in the controls group, the number of
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
quantifiable. Based on the MAP results, the
tube is not to be recommended as a cage
furniture item for F344 rats, whereas the plain
board and the diet board appear to be suitable
for both strains.
Table 7.5 Point estimates for significant (p < 0.01) MAP CV and HR CV comparisons between the
groups for both F344 and BN rats and for both light phases on the observation days. Arrow between
the two cage items indicates the multiplier to be used for the number of animals needed for that
transition direction.
F344
Day 2
Plain board Æ Diet Board
Plain board Æ Tube
Plain board Æ Control
IVC
OPEN
MAP
dark
MAP
light
1.36
1.56
1.41
1.42
1.81
1.52
HR
dark
Day 10
Control Æ Diet board
Day 14
Plain board Æ Tube
Tube Æ Control
BN
HR
light
MAP
dark
MAP
light
1.45
0.70
1.56
MAP
light
Day 2
Diet board Æ Control
Plain board Æ Control
1.87
2.25
1.89
2.25
Day 10
Diet board Æ Tube
Diet board Æ Control
1.91
1.91
136
HR
light
1.75
OPEN
MAP
dark
Day 14
Control Æ Diet board
HR
dark
IVC
MAP
dark
MAP
light
1.45
HR
dark
HR
light
HR
dark
HR
light
1.68
Kuopio Univ. Publ. C Nat. and Environ. Sci. 258:121-140 (2009)
General discussion
7.7 Conclusions
The following specific conclusions were
drawn from the results of this study:
1. Conventional open cages and IVCs
differ in several physical parameters in the
cage environment when IVC-system draws air
from the room.
2. Rats eat less and gain less weight
when fed with the diet board compared to ad
libitum-feeding, and especially for the F344
rats, the diet board was an effective way to
control weight.
3. Cardiovascular responses to both cage
change and IG-gavage can be modified with
the cage items in both studied strains.
4. The immediate MAP and HR
responses to IG-gavage appear to smaller in
magnitude than those associated with cage
change.
5. Cardiovascular parameters can be used
to assess the welfare value of cage furniture,
whereas changes seen in faecal corticosterone
and faecal IgA excretion would appear to be
too small to be quantifiable.
6. F344 and BN rats have different LA,
MAP and HR levels and different responses to
the cage items and to the procedures.
7. The tube is not recommended as a
cage furniture item for F344 rats, whereas the
plain board and diet board are suitable for
both strains.
8. It may be nessessary to aim at strain
specific guidelines for optimal cage furniture
in terms of environmental enrichment for
laboratory rats because of the genetic
component involved in the responses of the
animals to these items.
Kuopio Univ. Publ. C Nat. and Environ. Sci. 258:121-140 (2009)
137
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
7.8 References
Abou-Ismail UA, Burman OHP, Nicol CJ, Mendl M. 2008.
Let sleeping rat lie: Does the timing of husbandry
procedures affect laboratory rat behaviour, physiology
and welfare? Applied Animal Behaviour Science
111:329-341.
Anglés-Pujolrás M, Chiesa JJ, Díez-Noguera A, Cambras
T. 2006. Motor rhythms of forced desynchronized rats
subjected to restricted feeding. Physiology & Behavior
88:30-38.
Armario A, Gavaldá A, Martí J. 1995. Comparison of the
behavioural and endocrine response to forced
swimming stress in five inbred strains of rats.
Psychoneuroendocrinology 20:879-890.
Bamberg E, Palme R, Meingassner JG. 2001. Excretion of
corticosteroid metabolites in urine and faeces of rats.
Laboratory Animals 35:307-314.
Barnett SA. 1958. An analysis of social behaviour in wild
rats. Proceedings of the Zoological Society of London
130:107-152.
Bonnichsen M, Dragsted N, Hansen AK. 2005. The
welfare impact of gavaging laboratory rats. Animal
Welfare 14:223-227.
Broom DM. 1991. Animal welfare: Concepts and
measurement. Journal of Animal Science 69:41674175.
Brown AP, Dinger N, Levine BS. 2000. Stress produced by
gavage administration in the rat. Contemporary Topics
in Laboratory Animal Science 39:17-21.
Burn CC, Peters A, Mason GJ. 2006. Acute effects of cage
cleaning at different frequencies on laboratory rat
behaviour and welfare. Animal Welfare 15:161-171.
Carder B, Berkowitz K. 1970. Rats' preference for earned
in comparison with free food. Science 167:1273-1274.
Chamove AS. 1989. Environmental enrichment: A review.
Animal Technology 40:155-178.
Clark JH, Rager DR, Calpin JP. 1997. Animal well being
II. An overview of assessment. Laboratory Animal
Science 47:580-585.
Clough G, Wallace J, Gamble MR, Merryweather ER,
Bailey E. 1995. A positive, individually ventilated
caging system: A local barrier system to protect both
animals and personnel. Laboratory Animals 29:139-51.
Council of Europe. 2006. [Internet] Appendix A of the
European Convention for the Protection of Vertebrate
Animals used for Experimental and Other Scientific
Purposes
(ETS
No.
123).
Guidelines
for
accommodation and care of animals. Strasbourg.
http://conventions.coe.int/Treaty/EN/Treaties/Html/123
-A.htm
Duke JL, Zammit TG, Lawson DM. 2001. The effects of
routine cage-changing on cardiovascular and
behavioural parameters in male Sprague-Dawley rats.
Contemporary Topics in Laboratory Animal Science
40:17-20.
138
Eller E, Lawson J, Ritskes-Hoitinga M. 2004. The potential
of dietary fiber to achieve dietary restriction. Abstract.
Internationalisation and Harmonisation of Laboratory
Animal Care and Use Issues. 9th FELASA symposium,
Nantes, France.
Eriksson E, Royo F, Lyberg K, Carlsson H-E, Hau J. 2004.
Effect of metabolic cage housing on immunoglobulin A
and corticosterone excretion in faeces and urine of
young male rats. Experimental Physiology 89:427-433.
Eskola S, Lauhikari M, Voipio HM, Nevalainen T. 1999.
The use of aspen blocks and tubes to enrich the cage
environment of laboratory rats. Scandinavian Journal of
Laboratory Animal Science 26:1-10.
European Union. 2007b. [Internet] Commission
Recommendation on guidelines for the accommodation
and care of animals used for experimental and other
scientific purposes (2007/526/EC). Brussels. http://eurlex.europa.eu/JOHtml.do?uri=OJ:L:2007:197:SOM:EN
:HTML
Festing M, Overend P, Gaines Das R, Borja MC, Berdoy
M. 2002. The design of animal experiments. Laboratory
Animal Handbooks No. 14. The Royal Society of
Medicine Press Limited, London.
Gómez F, De Kloet ER, Armario A. 1998. Glucocorticoid
negative feedback on the HPA axis in five inbred rat
strains. American Journal of Physiology – Regulatory,
Integrative and Comparative Physiology 274:420-427.
Gorn RA, Kuwabara T. 1967. Retinal damage by visible
light: A physiologic study. Archives of Ophthalmology
77:115-118.
Gärtner K, Büttner D, Döhler K, Friedel R, Lindena J,
Trautschold I. 1980. Stress response of rats to handling
and experimental procedures. Laboratory Animals
14:267-274.
Hawkings P, Heath M, Dickson C, Wrightson D, Brain P,
Grant G, Hubrecht R. 1999. Report of the 1998
RSPCA/UFAW rodent welfare group meeting. Animal
Technology 50:41-49.
Heine WOP. 1998. Environmental management in
laboratory animal units. Basic Technology and Hygiene
Methods and Practice. Pabst Science Publishers, Berlin.
Hughes RN. 1968. Behaviour of male and female rats with
free choice of two environments differing in novelty.
Animal Behaviour 16:92-96.
Johnson SR, Patterson-Kane EG, Niel L. 2004. Foraging
enrichment for laboratory rats. Animal Welfare 13:305312.
Kasanen IHE, Inhilä KJ, Nevalainen JI, Väisänen SB,
Mertanen AMO, Mering SM, Nevalainen TO. 2009a. A
novel dietary restriction method for group-housed rats:
Weight gain and clinical chemistry characterization.
Laboratory Animals 43:138-148.
Kasanen IHE, Inhilä KJ, Vainio OM, Kiviniemi VV, Hau
J, Scheinin M, Mering SM, Nevalainen TO. 2009b. The
diet board: Welfare impacts of a novel method of
Kuopio Univ. Publ. C Nat. and Environ. Sci. 258:121-140 (2009)
General discussion
dietary restriction in laboratory rats. Laboratory
Animals 43:215-223.
Keller GL, Mattingly SF, Knapke Jr FB. 1983. A forced-air
individually ventilated caging system for rodents.
Laboratory Animal Science 33:580-582.
Krohn TC, Hansen AK. 2002. The application of
traditional behavioural and physiological methods for
monitoring of the welfare impact of different flooring
conditions in rodents. Scandinavian Journal of
Laboratory Animal Science 29:79-89.
Krohn TC, Hansen AK, Dragsted N. 2003a. Telemetry as a
method for measuring the impact of housing conditions
on rats’ welfare. Animal Welfare 12:53-62.
Krohn TC, Hansen AK, Dragsted N. 2003b. The impact of
cage ventilation on rats housed in IVC system.
Laboratory Animals 37:85-93.
Lepschy M, Touma C, Hruby R, Palme R. 2007. Noninvasive measurement of adrenocortical activity in male
and female rats. Laboratory Animals 41:372-387.
Lipman NS, Corning BF, Coiro MA. 1992. The effects of
intracage ventilation on microenvironmental conditions
in filter-top cages. Laboratory Animals 26:206-210.
Lipman RD, Dallal GE, Bronson RT. 1999. Effects of
genotype and diet on age-related lesions in ad libitum
fed and calorie-restricted F344, BN and BNF3F1 rats.
Journal of Gerontology: Biological Sciences
54A:B478-B491.
Marissal–Arvy N, Mormède P, Sarrieau A. 1999. Strain
differences in corticosteroid receptor efficiencies and
regulation in Brown Norway and Fischer 344 rats.
Journal of Neuroendocrinology 11:267-273.
Mering S, Kaliste-Korhonen E, Nevalainen T. 2001.
Estimates appropriate number of rats: Interaction with
housing environment. Laboratory Animals 35:80-90.
Moncek F, Duncko R, Johansson BB, Jezova D. 2004.
Effect of environmental enrichment on stress related
systems in rats. Journal of Neuroendocrinology 16:423431.
Neuringer AJ. 1969. Animals respond for food in the
presence of free food. Science 166:399-401.
Nevison CM, Hurst JL, Barnard CJ. 1999. Strain-specific
effects of cage enrichment in male laboratory mice
(Mus musculus). Animal Welfare 8:361-379.
Newberry RC. 1995. Environmental enrichment:
Increasing the biological relevance of captive
environments. Applied Animal Behaviour Science
44:229-243.
Pihl L, Hau J. 2003. Faecal corticosterone and
immunoglobulin A in young adult rats. Laboratory
Animals 37:166-171.
Purves KE. 1997. Rat and mice enrichment. In: Animal
Alternatives, Welfare and Ethics, ed. LFM van Zutphen
and M Balls. Elsevier Science B.V. pp. 199-207.
Ramos A, Berton O, Mormede P, Chalouff F. 1997. A
multiple-test study of anxiety-related behaviours in six
inbred rat strains. Behavioural Brain Research 85:5769.
Reeb CK, Jones RB, Bearg DW, Bedigian H, Myers DD,
Paigen B. 1998. Microenvironment in ventilated animal
cages with different ventilation rates, mice populations,
and frequency of bedding changes. Contemporary
Topics in Laboratory Animal Science 37:43-49.
Renström A, Björing G, Höglund U. 2001. Evaluation of
individually ventilated cage systems for laboratory
rodents: Occupational health aspects. Laboratory
Animals 35:42-50.
Rex A, Sondern U, Voigt JP, Franck S, Fink H. 1996.
Strain differences in fear-motivated behavior of rats.
Pharmacology Biochemistry, and Behavior 54:107-111.
Richter SH, Garner J, Würbel H. 2009. Environmental
standardization: Cure or cause of poor reproducibility
in animal experiments? Nature Methods 6:257-261.
Royo F, Björk N, Carlsson H-E, Mayo, Hau J. 2004.
Impact of chronic catheterization and automated blood
sampling (Accusampler) on serum corticosterone and
fecal immunoreactive corticosterone metabolites and
immunoglobulin A in male rats. Journal of
Endocrinology 180:145-153.
Rushen J, de Passillé AMB. 1992. The scientific
assessment of the impact of housing on animal welfare:
A critical review. Canadian Journal of Animal Science
72:721-743.
Saibaba P, Sales GD, Stodulski G, Hau J. 1996. Behaviour
of rats in their home cages: Daytime variations and
effects on routine husbandry procedures analysed by
time sampling techniques. Laboratory Animals 30:1321.
Sarrieau A, Mormède P. 1998. Hypothalamic-pituitaryadrenal axis activity in the inbred Brown Norway and
Fischer 344 rat strains. Life Sciences 62:1417-1425.
Scheer M, Heinekamp C, Thull B, Wagner R, Scheuber
HP. [Internet, not dated]. Performance evaluation of
IVC systems - Part 1: Test instructions.
www.lal.org.uk/IVC/index.html
Schnecko A, Witte K, Lemmer B. 1998. Effects of routine
procedures on cardiovascular parameters of SpragueDawley rats in periods of activity and rest. Journal of
Experimental Animal Science 38:181-190.
Seggie JA, Brown GM. 1975. Stress response pattern of
plasma corticosterone, prolactin and growth hormone in
rat, following handling or exposure to novel
environment. Canadian Journal of Physiology &
Pharmacology 53:629-637.
Sharp J, Zammit T, Azar T, Lawson D. 2002. Stress-like
responses to common procedures in male rats housed
alone or with other rats. Contemporary Topics in
Laboratory Animal Science 41:8-14.
Sharp J, Zammit T, Azar T, Lawson D. 2003. Stress-like
responses to common procedures in individually and
group-housed female rats. Contemporary Topics in
Laboratory Animal Science 42:9-18.
Kuopio Univ. Publ. C Nat. and Environ. Sci. 258:121-140 (2009)
139
Niina Kemppinen: 2Rs outcomes of cage furniture and restricted feeding in laboratory rats
Sharp J, Azar T, Lawson D. 2005. Effects of a cage
enrichment program on heart rate, blood pressure, and
activity of male Sprague-Dawley and spontaneously
hypertensive rats monitored by radiotelemetry.
Contemporary Topics in Laboratory Animal Science
44:32-40.
Siswanto H, Hau J, Carlsson HE, Goldkuhl R, Abelson
KSP. 2008. Corticosterone concentrations in blood and
excretion in faeces after ATCH administration in male
Sprague-Dawley rats. In Vivo 22:435-440.
Sørensen DB, Ottesen JL, Hansen AK. 2004.
Consequences of enhancing environmental complexity
for laboratory rodents – a review with emphasis on the
rat. Animal Welfare 13:193-204.
Spangler EL, Waggie KS, Hengemihle J, Roberts D, Hess
B, Ingram DK. 1994. Behavioral assessment of aging in
male Fischer 344 and brown Norway rat strains and
their F1 hybrid. Neurobiology of Aging 15:319-328.
Spiteri NJ. 1982. Circadian patterning of feeding, drinking
and activity during diurnal food access in rats.
Physiology & Behavior 28:139-147.
Stotzer H, Weisse I, Knappen F, Seitz R. 1970. Die RetinaDegeneration der Ratte. Arzneimittelforschung 20:81117.
Strubbe JH, Keyser J, Dijkstra T, Alingh Prins AJ. 1986.
Interaction between circadian and caloric control of
feeding behaviour in the rat. Physiology & Behavior
36:489-493.
Swoap SJ, Overton M, Garber G. 2004. Effect of ambient
temperature on cardiovascular parameters in rats and
mice: A comparative approach. American Journal of
Physiology. Regulatory, Integrative and Comparative
Physiology 287:R391-R396.
Teixeira MA, Chaguri LCAG, Carissimi AS, Souza NL,
Mori CMC, Saldiva PHN, Lemos M, Maccihione M,
Guimarães ET, King M, Merusse JLB. 2006. Effects of
an individually ventilated cage system on the airway
integrity of rats (Rattus norvegicus) in a laboratory in
Brazil. Laboratory Animals 40:419-431.
Tsai PP, Pachowsky U, Stelzer HD, Hackbarth H. 2002.
Impact of environmental enrichment in mice. 1: Effect
of housing conditions on body weight, organ weights
and haematology in different strains. Laboratory
Animals 36:411-419.
Tsai PP, Stelzer HD, Hedrich HJ, Hackbarth H. 2003a. Are
the effects of different enrichment designs on the
physiology and behaviour of DBA/2 mice consistent?
Laboratory Animals 37:314-327.
Tsai P-P, Oppermann D, Stelzer HD, Mähler M, Hackbarth
H. 2003b. The effects of different rack systems on the
breeding performance of DBA/2 mice. Laboratory
Animals 37:44-53.
Van den Brant J, Kovács P, Klöting I. 1999. Blood
pressure, heart rate and motor activity in 6 inbred rat
strains and wild rats (Rattus norvegicus): A
comparative study. Experimental Animals 48:235-240.
140
Van den Staay FJ, Blokland A. 1996. Behavioural
differences between outbred Wistar, inbred Fischer
344, Brown Norway and hybrid Fischer 344 x Brown
Norway rats. Physiology & Behavior 60: 97-109.
Weisse I, Stotzer H, Seitz R. 1974. Age and lightdependent changes in the rat eye. Virchows Archiv für
pathologische Anatomie und Physiologie und für
Klinische Medizin 362:145-56.
Wolfer DP, Litvin O, Morf S, Nitsch RM, Lipp HP,
Würbel H. 2004. Cage enrichment and mouse
behaviour. Nature 432:821-822.
Würbel H. 2000. Behaviour and the standardization
fallacy. Nature Genetics 26:263.
Zhang B, Sannajust F. 2000. Diurnal rhythms of blood
pressure, heart rate, and locomotor activity in adult and
old male Wistar rats. Physiology & Behavior 70:375380.
Õkva K, Tamoševičiūtė E, Čižiūtė A, Pokk P, Rukšėnas O,
Nevalainen T. 2006. Refinements for intragastric
gavage in rats. Scandinavian Journal of Laboratory
Animal Science 33:243-252.
Kuopio Univ. Publ. C Nat. and Environ. Sci. 258:121-140 (2009)
Kuopio University Publications C. Natural and Environmental Sciences
C 238. Pinto, Delia M. Ozonolysis of constitutively-emitted and herbivory-induced volatile organic
compounds (VOCs) from plants: consequences in multitrophic interactions.
2008. 110 p. Acad. Diss.
C 239. Berberat née Kurkijärvi, Jatta. Quantitative magnetic resonance imaging of native and
repaired articular cartilage: an experimental and clinical approach.
2008. 90 p. Acad. Diss.
C 240. Soininen, Pasi. Quantitative ¹H NMR spectroscopy: chemical and biological applicatons.
2008. 128 p. Acad. Diss.
C 241. Klemola, Kaisa. Cytotoxicity and spermatozoa motility inhibition resulting from reactive
dyes and dyed fabrics.
2008. 67 p. Acad. Diss.
C 242. Pyykönen, Teija. Environmental factors and reproduction in farmed blue fox (Vulpes
lagopus) vixens.
2008. 78 p. Acad. Diss.
C 243. Savolainen, Tuomo. Modulaarinen, adaptiivinen impedanssitomografialaitteisto.
2008. 188 p. Acad. Diss.
C 244. Riekkinen, Ossi. Development and application of ultrasound backscatter methods for the
diagnostics of trabecular bone.
2008. 79 p. Acad. Diss.
C 245. Autio, Elena. Loose housing of horses in a cold climate: effects on behaviour, nutrition,
growth and cold resistance.
2008. 76 p. Acad. Diss.
C 246. Saramäki, Anna. Regulation of the p21 (CDKNIA) gene at the chromatin level by 1 alfa,25dihydroxyvitamin D3.
2008. 100 p. Acad. Diss.
C 247. Tiiva, Päivi. Isoprene emission from northern ecosystems under climate change.
2008. 98 p. Acad. Diss.
C 248. Himanen, Sari J. Climate change and genetically modified insecticidal plants: plant-herbivore
interactions and secondary chemistry of Bt Cryl Ac-Toxin producing oilseed rape (Brassica napus L.)
under elevated CO2 or O3.
2008. 42 p. Acad. Diss.
C 249. Silvennoinen, Hanna. Nitrogen and greenhouse gas dynamics in rivers and estuaries of
theBothnian Bay (Northern Baltic Sea).
2008. 98 p. Acad. Diss.
C 250. Degenhardt, Tatjana. An integrated view of PPAR-dependent transcription.
2009. 120 p. Acad. Diss.
C 251. Häikiö, Elina. Clonal differences of aspen (Populus spp.) in responses to elevated ozone and
soil nitrogen.
2009. 49 p. Acad. Diss.
C 252. Hassinen, Viivi H. Search for metal-responsive genes in plants: putative roles in metal
tolerance of accumulation.
2009. 84 p. Acad. Diss.