Publishing History
First edition titled: Principles of Animal Technology 1
edited by P J Kelly, K G Millican and Pam Organ
Published by the IAT in 1988 reprinted 1992
Second edition titled: Introduction to Animal Technology
Revised by Stephen W Barnett
Published by Blackwell Science Ltd in 2001
Third edition titled: Introduction to Laboratory Animal Science and
Technology
Revised by Stephen W Barnett
Published by the IAT in 2016
© 2016 by Institute of Animal Technology
5 South Parade, Summertown, Oxford, OX2 7JL
www.iat.org.uk
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Preface
Since 2001, when the second edition of this book appeared, there
have been great changes in laboratory animal science and
technology. The changes have included developments in cage
design and the materials used to make them, methods used in
environmental control, an expansion in the use of genetically
altered animals and of course, amendments to the Animals
(Scientific Procedures) Act 1986. These, and other developments,
made a revision of the book over due. The reissue of the book also
allowed us to change the title in keeping with the terminology used
for the laboratory animal science and technology qualifications.
While it is hoped this book will be of interest to all people starting
work in laboratory animal facilities it has been written to support
the IAT level 2 Diploma syllabus. The contents should cover the
topics mentioned in the syllabus but it will not provide all of the
information that will be needed to complete assignments, students
will still have to listen to their tutors and carry out their own
literature research.
Although there have been changes in both technology, law and
educational requirements the principles of animal technology remain
the same and this book is still firmly based on the first edition,
Principles of Animal Technology 1 edited by John Kelly, Keith
Millican and Pam Organ.
For the first time the Institute of Animal Technology is making the
book available as an ebook. A printed version will be available later
in 2017. I would be interested in hearing the comments all those
who use the book.
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Since the last edition was published two distinguished pioneers of
Animal Technology education have passed away. John Kelly was one
of the editors of the first edition he made a major contribution in
establishing and developing animal technician education as a
teacher, examiner, member of the IAT Council and long time
member of the IAT Examination Board.
Kevin Dolan was another inspiring teacher and writer. He was
generally acknowledged as an expert in laboratory animal law and
ethics and he wrote two, well received, books on those subjects as
well as many articles. Many people have reason to be grateful to
these two men for their knowledge, generosity and guidance but
most of all for their friendship.
SWB 2016
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Sources and Further Information
As this book is an introduction it is not filled with lots of references.
The information it contains has come from a variety of sources as
well consultations with individuals and personal experience.
Students wishing to expand their knowledge of topics discussed
here can look at the IAT Manual of Animal Technology (edited by
Stephen W Barnett, published by Blackwells). The print edition is no
longer available but it can still be obtained as an e-book. A new
edition is in preparation.
A more academic treatment of laboratory animal science subjects
can be found in the UFAW Handbook on the Care and Management
of Laboratory Animals published by Blackwells. It is not
recommended you purchase a copy of this as it is very expensive
but it can be consulted in libraries.
If you would like to follow up ethical and philosophical ideas
connected with the use of animal in science, Ethics, Animals and
Science by Kevin Dolan, published by Blackwells Science may be of
interest.
The nc3r’s website is a good, free source of information on a variety
of useful subjects (www.nc3r’s.or.uk). The Home Office website
(www.gov.uk/guidance/research-and-testing-using-animals)
provides useful information on the law and guidance on care and
accommodation of animals.
The IAT Journal, Animal Technology and Welfare provides useful
reviews and research reports on the latest topics of interest on a
variety of subjects.
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Trawling the internet can provide useful information but, as
anybody can put information on the web, care must be taken to
evaluate the information. A good practice is to look at where the
author comes from, if it is a respected University or other research
laboratory the information can generally be relied on. If the author
has no affiliation the article should be treated with suspicion. A lot
of good information comes out of the USA but they do not have an
equivalent of the Animals (Scientific Procedures) Act 1986 as
amended 2012 and what is acceptable there may not be in the UK.
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Acknowledgements
The third edition of this book still follows pattern set out in first
edition so I am pleased to acknowledge the work of the original
editors, John Kelly, Keith Millican and Pam Organ.
I am grateful for the help I have received from my long term
collaborator, Jas Barley and from Patrick Hayes, whose reviewing
and proof reading skills have added greatly to the value of the book.
I asked several people to revise specific chapters and I am grateful
for this help, they were, Tina O’Malley, Gary Childs, Ian Garrod and
Carol Fox. Stephen Woodley provided the SOP that appears in
chapter 5. Allan Thornhill is responsible for turning my inexpert
typing into the e-book, for which I express my thanks.
I have consulted a number of people in the production of the book
and many have allowed me into their departments to take
photographs or allowed me to use their own photographs. They
include: Ken Applebee, Allan Thornhill, Wendy Steel, Mandy Thorpe,
Sarah Lane, David Spillane, Ross Millard, Wayne Russell, Gemma
Marshall, James Wilson, Michael Evans, Steven Cubitt, James
Bussell, Nicola Goodwin, Jim Scott.
All of the above and many of others have been a great help in the
production of this book and I wish to express my thanks to all of
them. We have made every effort to make sure all mistakes have
been removed, if any persists they are my responsibility.
Page | vii
I am grateful to the following for providing photographs
James Bussell Figs. 1.1, 1.2, 1.7
A Buckwell Fig. 1.5
P A Flecknell Figs. 1.8, 11.2, 11.3
Nicola Goodwin Fig. 1.10
Wendy Steel Fig. 2.3
Allentown Inc Fig. 2.6
Tecniplast UK Figs. 2.7, 6.2, 6.3, 6.5, 6.6, 11.4
Gary Childs Fig. 2.9
Sarah Lane Fig. 2.11
Imperial College Figs. 3.2, 3.4
The Cube Figs. 4.4, 6.4
LBS Ltd Figs. 5.1, 5.2, 5.4, 5.5, 5.6, 5.8
Rentokil Initial Figs. 7.9, 7.10, 7.11, 7.12, 7.15, 7.16
Edstrom Inc. Figs.7.18 a & b, 7.19
Roger Francis Fig. 8.2
Tina O’Malley 10.6
Chapter 1
ANIMAL HEALTH
The production and maintenance of healthy animals is a primary
function of animal technicians and technologists. Many of the topics
discussed later in this book (e.g. cleaning routines, hygiene
measures, environmental controls, barriers and the provision of
adequate diets) contribute to ensuring animals stay free from
disease.
Disease can be defined as any condition that interferes with the well
being of an animal. Diseases are divided into two main groups,
infectious or non-infectious. Infectious diseases are caused by
specific agents (e.g. bacteria, viruses, fungi and invertebrate
parasites) and can be passed from one animal to another by direct
means (contact) or indirect means (in the air, on food, by vectors
etc.). Some infectious diseases can be passed from animals to man,
these are called zoonoses. They are rare in laboratory animal
facilities and, where they may be a risk, establishment licence
holders will put measures in place to protect the staff (see chapter
15).
A brief mention should be made here of unusual infectious agents
called prions. They appear to be abnormal proteins that can cause
other, normal, proteins to alter their structure and therefore alter
the structure and function of the tissues they are part of; normally
the brain. The disturbance of structure leads to holes appearing in
the brain giving it a sponge like appearance and leading to the
name of the group of diseases caused by these agents,
transmissible spongiform encephalopathies (TSE’s). The individual
diseases are probably better known, bovine spongiform
encephalopathy (BSE) in cattle, scrapie in sheep and Creutzfeldt-
Jacob disease (CJD) in humans. There is little chance that people
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working in scientific procedure establishments will come across
these agents unless they are being studied there.
All other causes of disease are non-infectious and include physical
injury or trauma (e.g. wounds from fighting [Fig. 1.1]), inherited
abnormalities (e.g. malocclusion), nutritional imbalance (e.g.
vitamin C deficiency in guinea pigs and primates), inappropriate
environment (e.g. ringtail in rats) and absorption of toxic
substances.
Fig. 1.1 traumatic damage to mouse tail
Diseases of all kinds are accompanied by stress. Stress can also
occur in other situations examples of which are listed in table 1.1.
Environmental factors
Noise
Vibration
Transport
Boredom
High stocking densities
Experimental and husbandry factors
Cage cleaning
Sudden change of diet
Regulated procedures
Poor handling
Table 1.1. Some causes of stress in laboratory animals
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Stress occurs when an animal is exposed to worrying or potentially
dangerous situations. Hormones are released in the body which
enables the animal to cope with the situation. It is a normal
response and occurs in all animals, including humans. Once the
perceived danger has passed the hormonal levels return to normal
and the stress disappears. Problems arise when the stressful
conditions persist and the ‘stress’ hormones remain elevated. The
animals behaviour and physiology is affected in a number of ways
including:
Reduced resistance to disease
Overeating or refusing to eat
Lack of grooming or over grooming
Exhibiting stereotypies (repetitive behaviour patterns with no
obvious purpose) e.g.
o pacing
o bar chewing
o over grooming
Pain
Pain often accompanies disease. It is important to recognise signs
that indicate when an animal is in pain:
Appearance
o Animals in pain may isolate themselves from cage or
pen mates and sit hunched in the corner of their cage.
They may not groom so their coats become scruffy (Fig.
1.2) and there may be soiling around the anus. Mostly
they are immobile but when they do move they may
have an abnormal gait.
Food and water intake
o There is a marked reduction in food and water intake
which, in single housed animals, should be noticeable by
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the amount of food left in the hopper and water in the
bottle. There will be a consequential loss of body mass
and lower amount of faeces and urine produced.
Temperament
o Animals that are usually easy to handle may become
aggressive, particularly when the region that is painful
is touched.
Vocalisation
o The amount of sound produced by an animal in pain
differs with species and degree of pain being
experienced. Few species make a noise all the time
although dogs may whine for long periods. Animals in
pain may cry out when being handled; in some cases,
these cries may be in ultra sound which cannot be
perceived by humans.
Interpreting some of the signs of pain can be difficult,
wherever there is doubt more experienced colleagues should
be consulted.
Fig. 1.2. Mouse with hunched appearance and with hair standing on
end (staring coat)
Effects of Disease in an Animal Facility
Disease in an animal facility results in the suffering of individual
animals and, if the disease is an infectious one, the whole colony
could be affected. Experimental results will be jeopardised because
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physiological changes in the body of an animal caused by the
disease alter the response to experimental procedures. Similarly
breeding is affected by disease because sick animals do not breed
well, the life of the female is put at risk and if young are produced
they will be weak and sickly. It is necessary, therefore, to take
measures to keep disease out of the animal facility and to ensure
that if any animal is in poor health it is identified as quickly as
possible.
The Animals (Scientific Procedures) Act 1986 (as amended) (see
chapter 13) requires that a competent person checks all the animals
in a facility at least once a day. The Act also expects the
establishment licence holder to establish a strategy to ensure the
health status of all the animals.
Microbiological Screening
In order to be fully aware of the infectious organisms that may have
gained entry to their facilities, managers put in place a system of
microbiological screening. Periodically representative numbers of
animals from each room are sent to specialist microbiological
laboratories where they undergo a series of tests that show whether
specific infectious organisms are or have been present in the
colony. The tests can identify organisms before any clinical signs
show in the animal and are also able to identify the presence of
organisms that may affect experimental results but do not cause
any observable clinical signs.
Microbiological screening is expensive and may, therefore, be
restricted to rodent and rabbit colonies kept in full barrier units.
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Assessment of Health Status
Animals indicate they are unwell by changes in their normal
behaviour. Behavioural changes associated with disease are called
clinical signs. Many of these changes can only be detected by
people who have a thorough knowledge of normal behaviour of the
species, and ideally, the individual animal concerned. This
knowledge is gained by spending time working with animals and
observing how they react when they are in their cages, moving,
eating, drinking, interacting with other animals etc. Divergence
from the norm could indicate a problem and the animal should be
inspected more closely.
All staff, no matter how inexperienced, should be alert to signs that
indicate an animal is unwell and should be encouraged to report
their observations to a senior colleague. A detailed health inspection
should be carried out by a person who is trained to do so. Particular
care needs to be taken in handling animals that may be injured or
otherwise sick.
Monitoring health status is a continuous process. Every time a cage
is passed, every time an animal is handled or fed and watered they
are observed (Figs. 1.3 & 1.4). Inspection of the cage can be as
revealing as inspecting the animal. Some of the factors that should
be noted are shown on table 1.2.
Fig 1.3 Healthy, alert rabbit
in its cage
Fig.1.4 Healthy, alert rat
in its cage
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Factor
Possible abnormality
Animal
movement
Abnormal gait, lack of
movement
posture
Hunched, head or limb held at
an odd angle
isolation
Separation from other animals
in the cage, pen or field
Coat condition
Hair rough and erect (staring
coat, fig 1.2), lack of grooming
respiration
Rate abnormal, laboured or
breathing noisy
temperament
Signs of apprehension and
aggression in an otherwise
docile animal
Cage or pen
food
Ignored, mouthed (food taken
into the mouth and then
dropped so it is wet), excessive
intake
water
Ignored or excessive intake
Faeces and urine
Excessive amounts, too little,
diahorrea, presence of blood.
discharges
Haemorrhage, mucus, vomit
(note that rodents, rabbits and
horses cannot vomit)
hair
Excessive hair in the cage.
Table 1.2. Signs that could indicate ill health
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Detailed Health Inspection
In addition to the constant observations described above, it is
necessary to carry out more thorough inspections of individual
animals on a regular basis. All laboratory animal facilities will
incorporate health assessments into their routines. The regularity
and nature of the inspection will depend on a number of factors e.g.
the species, if they are undergoing or are about to undergo
regulated procedures, if they are being selected for breeding or it is
a routine inspection.
Although there may be some variation in the way inspections are
carried out, the basic principles are common to all mammals. It is
necessary to follow a routine so that no point is overlooked. Any
animal giving cause for concern, however trivial, should be reported
to a senior member of staff without delay.
The inspection starts when the animal is in its cage or pen where it
can be observed undisturbed. Movement, posture and respiration
can be noted without disturbing the animal. Respiration rates of
small rodents may be too fast to record accurately without special
equipment but in larger species it is much easier. Table 1.3 has
typical respiration rates of common laboratory species.
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Species
Respiratory rate
(breaths per minute)
Mouse
180
Rat
80
Rabbit
55
Dog
25
Cat
26
Rhesus monkeys
35
Table 1.3: Respiratory rates of some laboratory animal species
(taken from Flecknell P (1996) Laboratory Animal Anaesthesia (2nd Edition),
Academic Press.
The cage or pen should also be inspected at this point to ensure
none of the signs mentioned in table 1.2 are present.
For close inspection individual animals should be removed from the
cage by a method appropriate for the species (see handling and
sexing chapter) and placed on a non-slip surface where it will feel
safe and can be restrained if necessary. The inspection should
follow a logical sequence beginning with the head and working the
way down the upper surface of the body to the underside.
The mouth
Lips, gums and tongue should have a clean appearance, all teeth
should be present and aligned properly. Examples of abnormalities
that could be seen are lesions (sores, cuts), inflammation (redness,
swelling and heat), misaligned (malocclusion), overgrown (Figs 1.5,
1.6 & 1.7), broken or missing teeth. Soreness or wetness on the
chin could indicate problems in the mouth.
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The nose
The nostrils and surrounding area should be clean and clear. If the
animal has a respiratory infection there may be signs of discharge
causing blocked nostrils and matted fur. Rabbits and rodents wipe
their noses with their paws and matted fur may also be found inside
their fore legs (Fig. 1.8).
Fig.1.8: Rabbit with respiratory disease. Note the discharge around the
nose and the matted fur on the fore paws.
The eyes and eyelids
The eyelids should be clean. Any signs of swelling, redness and
discharge of any kind should give cause for concern. The eye should
be clear and bright with no sign of redness or discolouration in the
sclera (the white of the eye) or cloudiness in the cornea.
Fig.1.5 Normal
teeth of a rabbit
Fig.1.6 Bottom incisor
overgrown
Fig.1.7 Malocclusion
in mouse
Page | 10
If a problem is found in one eye it could be due to a foreign body or
traumatic damage. If a problem is found in both eyes it is more
likely to be an infection.
The ears
The shape, thickness, size and the amount of fur they carry differs
widely between species and so the normal appearance will differ
too. In general ears should be clean and alert (responding to the
source of sound). There should be no sign of traumatic damage that
could be caused by fighting or scratching. Ear mites colonise all
species if they get the chance, these cause intense irritation and
could be the cause of scratching. Mites can cause hair loss,
thickening of the skin and discharges that become encrustations.
Signs may be on the edges or deep down inside the ear depending
on the type of mite.
An example of an ear mite that may infest a laboratory species is
the rabbit ear canker mite (scientific name Psoroptes cuniculi). A
yellow/brown crust deep inside the ear may indicate the presence of
this mite (Fig.1.9).
Fig.1.9: Brown/yellow exudate in the inner ear indicating ear canker.
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The body, coat, back and sides
Hands or fingers (depending on the species) are passed over the
back and body of the animal to check for lumps and bumps which
could indicate abscesses, cysts, wounds that are covered by the fur.
This process may also indicate weight loss obscured by fur in some
species. The coat should be well groomed. Brushing the coat the
wrong way (from tail to head) in thickly furred species allows the
downy fur to be inspected. If it is less dense than it should be it
could indicate the presence of body mites who tend to affect the
softer downy hair before the tough guard hair.
The loose skin on the back should be gently pinched up and allowed
to fall (a process called tenting). In a healthy animal the skin should
return to normal quickly, if it takes a long time the animal may be
dehydrated.
The tail and anus
At this point smaller species may need to be turned over to inspect
the underside of the body.
The anus and the region surrounding it should be clean. There
should be no faecal staining or evidence of discharge, blood or
prolapse (where the wall of the rectum protrudes through the
anus).
The genitalia
Females The vulva should be clean with no discharges or traumatic
damage. However the appearance varies during the oestrous cycle
so what is observed must be correlated with the stage of the cycle
the female is in. Oestrous cycles are discussed in the chapter on
breeding (chapter 8).
Males Sexually mature males should have two well-developed
testes. Occasionally one or both do not descend into the scrotum.
In some species injury to testes may result from keeping males
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together. The penis should show no signs of inflammation and the
prepuce should be clean.
The hind legs
The hind legs should be checked for swollen, damaged or stiff
joints. The feet should be checked for broken, torn or overgrown
claws. In some species pressure sores are occasionally seen (e.g.
sore hocks in rabbits).
The abdomen, thorax and axillae
The fur is thinner on the underside of the body in many species and
therefore some conditions may be easier to see e.g. scratches, hair
loss and inflammation. It may also be easier to see evidence of
parasites. In breeding animals mammary glands should be checked
to ensure there are no signs of lumps or inflammation that could be
due to mastitis or tumours.
The fore legs
These should be checked as described for the hind legs.
Example of Non-Mammalian Species
Zebra fish are used as an example of a non-mammalian species in
this chapter because fish are the second most commonly used
laboratory species and zebra fish make up 65% of all fish used. The
same principles of health assessment mentioned for mammals
applies to fish, identification of ill health is based on a deviation
from normal appearance and behaviour. Healthy fish will be active
in the tank, eating and interacting with other fish (in the wild they
travel in shoals). Like mammals they are susceptible to both
infectious and non-infectious diseases. Efficient water treatment
equipment is essential to eliminate infectious organisms and to
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ensure water quality remains high. Table 1.4 lists some signs of ill
health in fish with some possible causes.
Sign
Possible cause
Pale, loss of colour, lack of
movement, sitting on bottom of
the tank
General signs
Damage to and/or reddening to
fins
Could be due to fighting (fin
nipping) or bacterial infection.
Lesions on the body
Could have infectious cause or
problems with water quality (e.g.
pH levels).
Protruding scales
Could be early sign of dropsy*
(Fig. 1.10)
Red, swollen abdomen
Later sign of dropsy*
Enlarged abdomen
Could indicate female is egg
bound (may happen when no
male is present).
Loss of balance
Usually infection in swim bladder
Table. 1.4 Some signs of ill health in Zebra Fish
* Dropsy is the name given to the condition where there is an
accumulation of fluid in tissues and body cavities. It is not a specific
disease but a clinical sign that can have many causes.
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Fig. 1.10 Normal Zebra fish on left. Fish with dropsy on right, showing
enlarged abdomen and raised scales.
Microbiological monitoring is carried out in Zebra fish facilities. For
instance, tests are carried out for Mycobacterium species (which
cause fish tuberculosis, a zoonosis) and Microsporidiosis a fungus
like protozoan which is associated with emaciation in fish (known as
skinny disease).
Action to be taken after completing the Health Assessment.
The results of a health assessment must be reported in accordance
with local rules. Some conditions may be able to be ameliorated
(improved or made better) by relatively minor interventions. Two
examples of those are given here but it should be emphasised that
these procedures should only be carried out by experienced and
authorised staff.
Overgrown teeth
Incisors of rodents and rabbits grow continually. They are kept
short and sharp by the upper and lower sets wearing against each
other. The teeth can become overgrown which affects the ability of
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the animal to eat. Overgrowth may start with one or more teeth
being broken so the opposite tooth has nothing to wear down on.
Alternatively, the condition may be due to malocclusion (failure of
the teeth to meet properly) which is an inherited trait so animals
with the condition should not be used for breeding (Fig 1.7).
A competent person can trim the overgrown teeth if it is deemed
appropriate. If the cause of the overgrowth is malocclusion the
condition will return so the animals need to be regularly inspected
and the treatment repeated.
Overgrown claws
All species are at risk of overgrown claws. These can be painful and
affect the way the animal walks. Claws need regular checking
because if they are allowed to grow too long they become
impossible to trim them back to their correct length. In most cases
clipping is a straightforward process but should only be attempted
by a trained and authorised person.
Trimming must be done with clippers specifically designed for the
species concerned. The animal must be restrained in an appropriate
manner (Fig.1.11); although the procedure is not painful if carried
out properly, many species object to it. Running down the centre of
each claw is the ‘quick’, made up of blood vessels and nerves.
Cutting the quick causes pain, bleeding and may lead to infection so
care needs to be taken to avoid damaging it. The longer the claw
grows the further down the quick will grow. In light coloured
animals the quick can be seen but in dark animals it cannot. Claws
are best clipped little and often.
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Body Temperature
Measuring body temperature can be a useful indicator of the health
of an animal. A variation of more than ± one degree Celsius in
homoeothermic animals could indicate ill health. Table 1.5 lists
temperature ranges for a number of laboratory species. Care should
be taken when using tables of physiological values because there
are strain and individual differences.
Species
Temperature °C
Mouse
37.4
Rat
38
Rabbit
38
Dog
38.3
Cat
38.6
Rhesus monkey
39
Table 1.5 Body temperatures of some laboratory animal species
(taken from Flecknell P (1996) Laboratory Animal Anaesthesia (2nd Edition),
Academic Press.
Body temperature is measured by inserting an electronic
thermometer probe into the rectum. Before the thermometer is
used it should be checked to ensure the battery has enough power
Fig.1.11 Restraining a rabbit to
clip claws
Fig.1.12 Clipping a rabbit’s claw
Page | 17
to record an accurate reading. The thermometer probe should be
clean and disinfected with a suitable agent such as 70% alcohol.
Fig. 1.13: Electronic thermometer
The animal must be removed from its cage or pen and be restrained
in a suitable manner e.g. a dog may happily stand still without
restraint, a rat will probably need scruffing and a rabbit can be
wrapped in a towel. Whichever method is used, the techniques
should be done with the minimum of fuss because if the animal is
excited it could cause the temperature to rise.
The bulb of the thermometer probe should be lubricated with
Vaseline or other suitable lubricant and gently inserted into the
rectum while holding the tail out of the way (Fig.1.14). Rotating the
instrument may ease the insertion. Force must not be used. The
thermometer should be inserted until the bulb is completely
inserted in the rectum but no further. The bulb must be in contact
with the wall of the rectum. It is held in place until the thermometer
indicates the temperature has been recorded. After the animal has
been replaced in its cage or pen the temperature can be read and
recorded and the thermometer cleaned and disinfected ready for
the next use.
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Fig.1.14: Taking the rectal temperature of a rabbit.
Summary
Disease can either be infectious (caused by specific organisms
and can be passed from one animal to another) or non-
infectious.
Both kinds of disease compromise animal welfare and
experimental results.
Stringent hygiene measures minimise the risk of infectious
disease entering the animal facility
Continual vigilance ensures any disease is identified as early
as possible thereby reducing the harmful effects on animals
and experiment results.
Detailed health inspections should be carried out in a
systematic way to ensure nothing gets missed.
Any cause for concern found in routine or detailed health
assessments should be reported according to local rules
immediately.
Some problems may be easily ameliorated but this should
only be done by properly trained and authorised people.
Page | 19
Page | 20
Chapter 2
CAGING AND HOUSING
Laboratory animals spend almost all of their lives in a cage or pen.
It is necessary, therefore, their accommodation provides for all of
their physical needs and as many of their behavioural needs as
possible.
Animal housing must allow enough area for exercise and to
accommodate materials for environmental enrichment. Space must
be available to allow grooming, social activity, privacy, urination,
defaecation and, where appropriate, to give birth and rear young. It
must also allow for easy access to food and water. The cage or pen
must allow adequate light and ventilation so that appropriate
temperature and humidity levels reach the occupants. To ensure
the continual safety of the animals their accommodation must be
checked regularly for damage, sharp edges etc. that may cause
them injury.
In addition to the requirements for the animals, the housing must
allow for the following essential factors:
The safe containment of the animals.
Be easy to use and service e.g.
o The cage should be easy to clean, to provide with food
and water and to get the animals into and out of safely.
Animals should be able to be seen so that their welfare
can be assessed.
Be economic
o Cages must be able to withstand the attention of their
occupants and the routine wear and tear (e.g. cage
wash, autoclave, disinfectants etc.) they will be
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exposed to. Note that economy and cheapness are not
the same thing. Economic considerations involve a
balance of the initial cost and the length of time they
can be used.
Meet experimental requirements.
Where there is difficulty in providing for all behavioural or
experimental needs in one cage or pen, other measures have to be
taken. For instance, it is both impossible and inadvisable to provide
enough space in a dog’s pen for its housing and exercise
requirements, so an exercise area must be provided elsewhere in
the facility and an exercise period built into the facility routines (Fig.
2.1) or, if experimental protocols demand 24-hour urine collection
from an animal, it would be necessary to house it in a metabolism
cage (see chapter 11) for the period.
Fig. 2.1 Dogs being exercised in a facility corridor
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Caging Material
A variety of materials are used to manufacture cages and pens for
laboratory animals. Consideration of the physical properties of these
materials helps to assess their usefulness for particular purposes.
Strength rigidity and ability to resist animal activity and normal
wear and tear in the animal unit. For instance, metals are very
strong e.g. stainless steel is used for primate accommodation.
Plastics are less strong and are suitable for housing smaller
species.
Density the density of a material is defined as its mass per
unit volume. It is a useful measure to compare the heaviness of
objects of different shapes. Metals are much more dense than
plastics (e.g. the density of stainless steel is approximately
8g/cm
3
and polysulphone 1.25 g/cm
3
).
Thermal conductivity thermal conductivity is the ability of a
material to conduct heat. Metals are good conductors and
therefore conduct heat rapidly. Plastics and wood are poor
conductors; poor conductors are good insulators. Where
temperatures vary greatly thermal conductivity is of great
importance.
Example: if a rabbit was housed in a metal hutch outdoors in
the winter, it would probably die of hypothermia because the
metal would conduct heat away from the rabbit and its body
temperature would drop. If the rabbit was housed at the same
temperature, but in a wooden hutch, it would survive because
the wood would act as an insulator enabling the rabbit to
conserve its body heat.
In modern temperature controlled animal units the thermal
conductivity of a material is a less important consideration.
Page | 23
Reactions with acids, alkalis and detergents at concentrations
encountered in the animal units a number of chemicals are
used in the animal unit (e.g. descalers, detergents and
disinfectants). The materials used to construct cages and pens
must be able to resist any damaging effects.
Ability to withstand repeated autoclaving.
Most cages and pens are constructed from metal or plastic or a
combination of both. Properties of commonly used caging materials
are summarised in table 2.1.
Material
Properties
Examples of
Uses
Wood and wood
products
(including
cardboard)
Construction of cages and boxes is
easy with wood, however it is
difficult to clean, impossible to
sterilise and may rot.
Wood is a good insulator but
animals may gnaw it.
Limited use in
the animal unit
but can be used
for travelling
boxes,
shelves and
perches (Fig.
2.2)
Metals in
general
Metals are strong, easily cleaned
and will withstand autoclaving. They
are good conductors of heat, which
should not cause a problem in
temperature controlled animal
rooms. Most are heavy. They
usually require specialist
manufacturing techniques to
construct. They generate noise
when dropped or rattled.
Galvanised mild steel and
aluminium were common in animal
Page | 24
facilities but have fallen in
popularity. Stainless steel is more
widely used.
Stainless steel
Very strong material that is
resistant to most substances found
in animal units. It is very long
lasting and the most expensive
material used for caging.
Several grades of stainless steel are
available for different purposes e.g.
grade 316L is used for bottle spouts
and housing for aquatics; the lower
grade 304L for racking.
Large cages for
primates
(Fig.2.3).
Racking, cage
doors and tops,
food hoppers
and water
bottle spouts. It
is also used for
IVC plenums.
Plastics in
general
Light weight materials that are
good insulators with variable
resistance to damage by chemicals.
Plastics make much less noise than
metal either in use or if dropped.
Plastic cage bases are made in one
piece and are easy to stack.
Traditional plastics become brittle
with repeated autoclaving and
exposure to chemicals but modern
high temperature/chemical tolerant
plastics are much more resistant.
Polycarbonate
A clear plastic that will withstand
autoclaving at 121
o
C. It becomes
discoloured and brittle with
repeated treatments. Detergents
and water denature the material
over time.
Used for
conventional
rodent boxes.
(Fig. 2.4)
High Heat
Polycarbonate
This material withstands
autoclaving to 132°C otherwise it is
similar to polycarbonate.
Page | 25
Polyethylene
(polyethylenetere
phthalate PET)
This material will melt at 93°C so
cannot be autoclaved. These are
available irradiated or non-
irradiated.
Disposable
rodent cages
and cage liners.
Polysulfone*
Transparent material. Withstands
autoclaving at temperatures up to
140
o
C. Heat and chemical resistant.
Caging
Polyphenylsulfone
*
Very high temperature and chemical
resistance.
Caging
Polystyrene
Cheap and disposable. It is brittle
and cannot be autoclaved.
Disposable
cages
Glass
Transparent and impervious to all
chemicals used in the animal house.
It is fragile and is easily broken
resulting in dangerous sharp edges.
Metabolism
cages
Table 2.1: Properties of common caging materials
* Polysulfone and polyphenylsulfone are more expensive than polycarbonate
cages but their heat and chemical resistance mean they have a longer life than
other plastic materials. Polysulphone has a life of over three years and
polyphenylsulphone of eight years.
Page | 26
Fig. 2.2. Wooden nest box and perches in a marmoset cage.
Fig. 2.3 Stainless steel primate cage
with environmental enrichment items.
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Fig. 2.4 Polycarbonate mouse cage on a stainless steel rack.
The design of cages evolves over time to the benefit of the animals
housed in them and the people who work with them. The most
significant design development in recent years has been the
introduction of Individually Ventilated Cages (IVCs) that house most
of the small rodents used in research. IVCs are sealed units fitted
into racks that carry the conditioned air supply directly to and
extract air directly from the cage (Figs. 2.5, 2.6). The air can be
supplied at positive or negative pressure. If the animals are
microbiologically clean and need to be protected from external
organisms the IVC is kept at positive pressure. If the animals are
being used to study harmful organisms the units can be maintained
at negative pressure. IVC units also help to minimise the dispersion
of laboratory animal allergens into the environment. Cage cleaning
is carried out in a cage cleaning station so that there is no cross
contamination between animals and the environment.
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Fig. 2.5 Individually ventilated cage
Fig. 2.6 Rack of IVCs showing air filtration unit on top.
Page | 29
Another development involves using disposable caging. As
mentioned in table 2.1 cages made with materials such as PET are
considered disposable. Instead of cleaning and sterilising cages the
soiled ones are disposed of and are replaced by new ones. Using
disposable caging reduces the need for large cagewash rooms and
equipment. Theoretically the soiled cages can be recycled but this
would mean they have to be cleaned first and so defeat one of the
main reasons for using them. They are usually incinerated.
Thin liners that sit within IVC cages are available. When cages need
to be changed the liners containing the soiled bedding are removed
and disposed of. Liners are particularly useful if an experiment
involves the use of toxic or infectious materials because they
minimise the risk of staff contact.
Design developments also include double-decker cages for rats (Fig.
2.7), which improve the conditions especially when long term
housing is required.
Fig. 2.7 Double-decker rat cage
Page | 30
Arrangement of Cages
A number of methods can be used to arrange cages in animal
rooms.
Racks can be mobile (wheeled), free standing or fixed to the
walls. Mobile racks are the most useful because they can easily be
moved to the cagewash area for processing. They also make the
room more flexible because it is easy to change one set of racks for
those suitable for another species (providing the Home Office
approves the change of use).
Cages can also be arranged on shelves (Fig. 2.8) or in batteries
(where banks of cages are connected to one another e.g. chicken
batteries).
Fig. 2.8 Cages arranged on shelves.
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Where high volumes of animals are housed library racking can be
used (Fig 2.9). In this system racks are mounted on runners and
are situated close to each other. When a particular rack needs to be
worked on the other racks can moved along the runners so a gap
appears, wide enough for a person to gain access.
Fig. 2.9 Library racking. Banks of racks can be moved by
Turning the grey handles shown at the end of the racks.
Pens are used to house larger species such as dogs or pigs or
smaller ones such as rabbits. For large species permanently built
structures are necessary (Fig. 2.10 ) but for smaller species
temporary structures can be built within animal rooms (Fig. 2.11).
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Fig. 2.10 Permanent dog pen.
Fig. 2.11 Rabbits in temporary floor pen.
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Summary
Cages and pens must provide for all of the physical needs and
as many of the behavioural needs of the animals they house
as possible.
In addition they must be easy to use and service, meet
experimental needs and be economic.
The materials used to construct cages, pens and racks must
be strong, able to withstand exposure to chemicals used in
the facility, repeated autoclaving and animal activity.
Cage and pen design continues to develop in order to improve
animal welfare, ease of use and safety (e.g. IVCs and double-
decker rat cage).
Cages are usually arranged on wheeled racks.
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Chapter 3
THE ANIMAL FACILITY
An animal facility is any structure that accommodates animals and
the service equipment and facilities necessary to maintain them. In
the United Kingdom facilities intended to house animals being used,
bred or supplied for scientific procedures must hold the appropriate
Establishment Licence (see Chapter 13) issued by the Home Office.
This Licence will only be given if the facilities reach and maintain the
standard described in the current edition of the Home Office Code of
Practice for the Housing and Care of Animals Bred, Supplied or Used
for Scientific Purposes.
Licenced establishments range in size from one or two rodent rooms
to facilities accommodating thousands of animals of many different
species. Whatever the size or type of the unit the aim must be to
provide a stable environment so that the animals physiological and
behavioural needs are met and they are able to be bred and studied
for experimental purposes.
Ideally animal facilities should be purpose built and should be
physically separate from other buildings. This minimises the risk of
animals being disturbed by extraneous noise and it increases
security. Where separate facilities are not feasible they should be as
isolated as possible from other parts of the building. The design of
the unit should provide maximum flexibility so that a room can be
changed from one species to another without major alterations
(although such changes must have Home Office authorisation).
The rooms should contain the minimum amount of fixed equipment;
all surfaces should be covered with an impervious material that will
withstand animal activity and the husbandry routines necessary in
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an animal facility. All junctions between walls, floor and ceiling
should be coved so that places where dust can collect are minimised
and the room can be easily cleaned. All material used in animal
rooms must be harmless to the occupants.
The layout of the rooms in a facility should ensure animals are
disturbed as little as possible and are kept free from the risk of
disease-causing agents including parasites. Predator and prey
animals should be kept so that they are unable to see, hear or smell
one another. Naturally noisy animals (e.g. dogs) should be isolated
from naturally quiet animals (e.g. mice). The use of sound-proofing
and sound absorbing materials can help to reduce the sound from
noisy animals. Breeding animals are often very sensitive to
disturbance so they are usually isolated from experimental animals
and other parts of the unit. Animals that are brought into the unit
may have to undergo a period of quarantine to ensure they are free
from unwanted organisms before they are allowed contact with
established animals. Quarantine rooms must be isolated from other
rooms.
Areas set aside for cage cleaning, air conditioning plant and other
potentially noisy activities should be situated as far away from the
animals as possible to minimise disturbance. The cage wash area
should be arranged so there is a separated clean and dirty side to
prevent clean materials being contaminated with dirty ones.
Some units are arranged with a double corridor system, one
designated the clean corridor and one the dirty corridor. This
minimises the risk of cross contamination. All clean cages, food,
bedding etc. travel down the clean corridor. Dirty cages, cage soil
and other waste travel down the dirty corridor. This helps prevent
cross contamination and helps to maintain hygienic conditions.
Double corridors take up a lot of space and are not always
Page | 36
appropriate, so single corridors have to be used (Fig.3.2). The risk
of cross contamination with single corridor systems can be
minimised by separating clean and dirty materials by time rather
than space. Clean materials are delivered to the animalsrooms in
the morning and dirty materials moved in the afternoon after which
the corridor is disinfected ready for the next morning.
Fig. 3.2 Animal unit corridor showing environmental monitoring panel
Adequate storerooms should be provided and they must be
designed to safeguard the quality of the materials they store.
Different goods (e.g. food, bedding, cleaning materials, drugs etc.)
must be stored separately. The maintenance of food stores is
covered in chapter 7.
It is important that animal facilities are designed to eliminate
unauthorised entry of people (with electronic key systems), animals
(e.g. rodent barriers) and other pests (e.g. insect barriers) and the
escape of animals that should remain inside.
Page | 37
Other aspects of the environment of animal facilities (e.g. light,
humidity, temperature, noise etc. are covered in chapter 4)
Barriers
Animals within a unit must be protected from disease-causing
agents including parasites. Unwanted organisms can enter an
animal unit in a number of ways among which are:
on/in imported animals;
on/in food and bedding;
on/in staff;
on air.
When animals are kept in large numbers, in a confined area,
infectious diseases spread very rapidly. In order to prevent them
entering and travelling around the unit barriers are erected. A
barrier is any physical arrangement, procedures or routines set up
with the intent of minimising the likelihood of contamination of the
animal with unwanted organisms.
Examples of barriers are:
physical separation from other buildings;
restricted entry of personnel;
use of protective clothing;
showering into the unit;
rodent and insect barriers (Figs. 3.3, 3.4);
routine use of disinfectants;
appropriate use of sterilisation procedures;
air conditioning with filtered air and pressure differentials.
Page | 38
Fig. 3.3 Doors fitted with rodent barriers. Magnehelic monitors above the
doors showing the air pressure within the room can also be seen.
Fig. 3.4 Electrocution apparatus for flying insects
All animal units institute barriers of some kind but the extent of
them depends on the nature of the work that goes on in the unit
and the length of time the animals are kept. Where animals are
housed for a short time the risk of them contracting and suffering
Page | 39
disease is low. In this situation it may be only necessary to restrict
entry to non essential personnel, require staff to wear protective
clothing, institute routine cage cleaning and disinfection
programmes and have filtered air conditioning.
If the work within the unit involves long-term studies it is essential,
from both an animal welfare and from a scientific point of view that
unwanted organisms are kept out. In this case more stringent
barriers must be instituted. In addition to the measures mentioned
above anyone entering the unit may be required to shower and
dress in sterilised clothing before entering. All goods would be
sterilised on entry. Air supply would be filtered and at positive
pressure to the outside to prevent airborne micro-organisms
entering. All entry points would be protected with insecticutors and
rodent traps. These types of animal houses are called full barrier
units.
The barriers mentioned above are designed to keep unwanted
organisms out of the unit. In some cases, it is necessary to care for
animals that have been infected with known harmful agents and it is
necessary to prevent the organisms escaping from the animal room.
In these units, called isolation units, the same barriers are used but
they work in reverse, for instance air is supplied at negative
pressure to the outside air so organisms cannot escape, waste
materials are autoclaved before leaving the unit, staff shower on
leaving the unit.
Page | 40
Individually ventilated cages (see chapter 2) and isolators (Fig. 3.5)
can be used to both protect animals from unwanted organisms and
isolate those infected with organisms.
Fig. 3.5 Isolators
Summary
animals must be provided with an environment that provides
for their physiological and behavioural needs;
the facility must be secure, preventing access to unauthorised
people and unwanted animals while preventing housed
animals escaping;
materials used in the facility should be able to withstand the
day to day wear and tear of an animal facility and should not
be harmful to the animals that come in contact with them;
suitable barriers should be erected to prevent the entry or
escape of unwanted organisms.
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Page | 42
Chapter 4
ENVIRONMENT
Introduction
The environment of an animal is made up of everything that
surrounds or exerts an effect on it. The conditions immediately
surrounding the animal, bounded by the cage or pen, are referred
to as the micro-environment. The term macro-environment is used
when referring to the conditions outside the micro-environment.
Unlike wild animals, those kept in laboratory animal units have a
more limited opportunity to control their own environment; they are
dependent on humans to control it for them. Many species or
strains of animals used in scientific procedures have been so altered
by selective breeding that they would be unlikely to survive outside
the controlled environment of the animal unit.
Animals differ in the way they respond to their environment and
there are major differences in the way animals and man respond to
the same environment. Conditions that man might find tolerable
may not be acceptable to animals. For example, in most laboratory
mammals smell is a much more important sense than it is in
humans and some are able to hear sounds at a much higher
frequency (pitch) than man (ultrasound). These and other
differences must be considered when establishing a suitable
environment.
Page | 43
Many factors make up the environment of laboratory animals’. They
include:
cage, bedding, diet, water, other animals;
temperature, relative humidity, day length, light intensity;
air (the oxygen, carbon dioxide, water vapour, ammonia
and dust content);
routine care procedures, noise;
humans;
breeding systems or experimental procedures;
micro-organisms, diseases and treatments.
Some of these factors are easy to control, others much less so. The
factors listed above that are not discussed in detail in this chapter
are covered elsewhere in the book.
Humans control all aspects of the living conditions of laboratory
animals and as such are totally responsible for their environment;
animals suffer if staff are uncaring and unobservant. More directly
when animals are handled for routine maintenance tasks, e.g. when
changing cages or for the assessment of health status, good
handling techniques minimise the amount of disturbance. Poor
technique may create resentful or aggressive animals and can lead
to injury.
Most species react adversely to being caged singly; they appear to
miss social contact, e.g. being able to groom one another. They
may become aggressive and develop abnormal behavioural
patterns. When animals are housed in groups, whether for breeding
or experimental purposes, the groupings are unnatural as a human,
not the animals themselves, choose their cage mates. Initially in
new groups there may be some fighting but a hierarchy is rapidly
established and the animals soon settle down. However new groups
Page | 44
must be watched carefully in case the fighting goes on too long or
bullying occurs. Once a group has been established new animals
should not be added as the established animals may attack the
newcomer.
Some strains of animals are very aggressive particularly the males,
and special care must be taken when these are group housed.
Group housing will increase the amounts of waste, the temperature
and the relative humidity within the cage. Higher levels of ammonia
will be produced creating an unpleasant environment and increasing
the risk of disease so stocking levels and routines must be adapted
to deal with it.
Overcrowding in cages and pens causes stress and leads to
increased incidence of ill health and fighting. Acceptable stocking
densities are given in the Codes of Practice.
Home Office Codes of Practice
The environmental standards required to house laboratory animals
are detailed in the Home Office Code of Practice for the Housing and
Care of Animals Bred, Supplied or Used for Scientific Purposes
(December 2014) (CoP). This code can be downloaded from the
Home Office website. All students of Animal Technology are
recommended to download the CoP and become familiar with it.
The code is arranged in three sections, the first describes minimum
legal standards that are in force now, the second covers minimum
legal standards that come into force on January 1
st
2017 and the
third section provides advice on a wider range of topics.
The legal standards in sections one and two of the Code of Practice
are mandatory, that means they must be complied with, but they
Page | 45
are minimum standards. The Home Office encourages users to
continually review and improve the ways in which animals are kept
in keeping with the spirit of the 3Rs. The advice in section three is
not mandatory but it is based on well-documented research and
experience and therefore should be considered very seriously. It is
guidance on how the mandatory standards in sections 1 & 2 can be
complied with. Section three does not cover all eventualities and,
where circumstances outside the guidance occur, users are required
to consult the literature, technical staff, the appropriate Named
Persons and/or the local Home Office Inspector to establish
appropriate conditions for the animals.
Ventilation Systems
In order to provide a stable environment, help control disease
outbreaks and increase security, the majority of laboratory animal
facilities do not have windows that open. Filtered fresh air at the
required temperature and humidity is supplied and contaminated
waste air is removed through a ventilation system.
There is a mandatory requirement that insulation, heating and
ventilation of an animal holding room ensures that the air
circulation, dust levels and gas concentrations are kept within limits
that are not harmful to the animals housed, and are appropriate for
the housing system in operation.
A typical air handling system takes the air from outside passes it
through a number of filters, to remove up to 99.97% of the
particles in it, adjusts the temperature and humidity and distributes
it around the facility. Before entering the room the air passes
through secondary heaters and humidity regulators so that the
temperature and humidity can be adjusted for individual species.
Sensors in the room measure the temperature and humidity and
Page | 46
feed it back to the control centre so that adjustments can be made
to keep the levels stable.
Air extracted from the room is passed to the outside and is not re-
circulated. However, in some systems, heat is recovered from the
extracted air to help save energy. Additionally, some facilities have
filters attached to room extract ducts so that, in the event of a
system failure, potentially contaminated air in the ductwork cannot
leak back into the animal room.
Air has to be supplied to the room so that all animals are supplied
with air of the desired quality but no animal is in a draught.
The rate of supply and extraction of air can be adjusted so that
rooms can be at positive or negative pressure. If air is supplied to
the room at a faster rate than it is extracted the room will be at
positive pressure, this means that pathogenic agents and other
airborne matter cannot enter the room through gaps around the
door or even when the door is opened. If, however, the research in
the room involves work with infectious organisms or other harmful
material, the main focus must be on preventing escape from the
room so air is supplied at a lower rate than it is extracted resulting
in negative pressure in the room. Air pressure gauges (called
manometers or magnehelic) measure the air pressure within a room
compared with the corridor outside Fig. 4.1.
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Fig. 4.1 Magnehelic pressure gauge showing air pressure
in the room is slightly above that in the corridor.
Where facilities use potentially harmful organisms the air that is
extracted from the room must be treated and/or filtered before it is
released into the atmosphere.
The amount of fresh air supplied to a room in order to provide the
animals with their needs is measured in air changes per hour
(ACH).
To calculate the ACH you divide the volume of air entering the room
into the volume of the room. The volume of the air entering the
room can be found by multiplying the area of the air inlet by the
speed (or velocity) that the air is passing through the inlet. An
instrument called an anemometer is used to measure air velocity.
The number of air changes per hour required depends on the
species being housed and the stocking density. The guidance given
in the Home Office Code of Practice is that 15 20ACH is normally
adequate for a fully stocked room of rodents or lagomorphs
Page | 48
dropping to 8 10ACH if the stocking density is low. In rooms
housing cats, dogs and primates 10 12ACH may be sufficient.
However, these figures given above are only guides.
A good indication of whether the conditions in the room are suitable
is apparent when the room is entered. If the temperature and
humidity are correct and there is no build up of ammonia or other
noxious odours the conditions will be suitable. If there is a high
level of ammonia it could mean that the air changes are insufficient
but it could also mean the room is overstocked or the cleaning
routine is inadequate.
Ventilation systems can be a source of noise and vibration that have
been shown to disturb the animals. To minimise this disturbance,
the main air handling plant should be situated as far away from
animals as practicable and the system must be balanced to
minimise the audible and ultrasound being emitted.
Farm animals used for scientific purposes benefit from being kept
outdoors or in housing ventilated with natural air such as the
weatherboarding shown in Fig. 4.2. There is, however, a mandatory
requirement to make sure these animals are protected from
draughts and extreme weather conditions.
Page | 49
Fig. 4.2 Farm animal housing showing a solid wall base topped with
weatherboarding. This arrangement allows an adequate airflow above the
animals but protects them from draughts.
Many laboratory rodents are kept in individually ventilated cages
(IVCs) where air is supplied and extracted directly to the cage. They
can be kept at positive or negative pressure. The ACH depends on
the design of the cage and stocking density but will be either 50 or
75ACH.
Sensors connected to alarms are fitted to IVCs to warn of system
failure. It is essential that problems are dealt with as quickly as
possible because in the confined space of a sealed cage the welfare
of animals will deteriorate very rapidly
Temperature
Animals are classified as being either ectothermic or endothermic.
Reptiles, amphibians, fish and invertebrates are ectotherms, which
means they cannot regulate their body temperature by physiological
means, although they are efficient at doing it by behavioural means
(e.g. basking in the sun to warm (Fig.4.3) or seeking shade to
cool).
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Fig. 4.3 Agama lizard basking in the morning sun.
Mammals and birds are endotherms. They can regulate their body
temperature within a narrow range by physiological and behavioural
means, so if the ambient temperature increases or decreases their
internal body temperature remains constant.
If the ambient temperature drops body heat can be conserved by
constricting surface blood capillaries (lessening the loss of heat from
the blood), piloerection (raising the body hair to trap a layer of
insulating air), sitting hunched up (lessening the amount of body
surface exposed to the cold outside air), nest building and sitting
close to cage mates. Shivering helps because heat is produced as a
by-product of the energy used to cause the muscles to move.
If ambient temperature rises body temperature can be kept stable
by dilating surface blood capillaries (bringing more blood to the
surface and increasing the loss of heat from the blood), panting and
or sweating (due to the latent heat of vaporisation) and lying
spread out (exposing the maximum surface area from which heat
can be lost).
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The thermoregulatory mechanisms of endotherms only work over a
fixed range of ambient temperatures, outside this range they will
suffer from heat and cold stress. If the extreme temperatures go on
for long they can prove fatal. Small animals like rodents have a fast
metabolic rate that makes them much more sensitive to
temperature changes. Larger species like dogs and cats can tolerate
a wider range of ambient temperatures.
The mechanisms needed to maintain a stable body temperature
(called thermoregulation), although a perfectly normal physiological
response, affect the metabolism and behaviour of animals that can
influence results obtained from some types of experiments. For this
reason, as well as for the comfort of the animals, it is important to
select a suitable temperature range to keep them in and to make
sure once a temperature has been selected it does not vary more
than ±2
o
C. Table 4.1 gives temperature ranges for various animals
that are taken from the guidance section of CoP. These figures are
suitable for adult, healthy animals and would need to be adjusted
for young, hairless or otherwise compromised animals. There is an
overriding mandatory requirement to ensure that temperature in a
holding room is adapted to the species and age groups of the
animals housed. There is also a requirement to measure and record
temperature on a daily basis.
Farm animals housed outside can tolerate very cold weather,
particularly if they are outside all the time and grow thick coats in
winter but no animal can survive long periods exposed to wind or
draughts. They must be provided with access to shelter from both
wet and wind. Some old world primate units give the animals access
to outside pens. The animals appear to gain benefit from this but
Page | 52
they must have access to shelter and to heated inside
accommodation when needed.
Species
Temperature range (
o
C)
Mice, rats, gerbils, hamsters
20 24
Guinea pigs
15 21
Rabbits
15 21
Cats
15 21
Dogs
15 24
Ferrets
15 24
Marmosets and tamarinds
23 28
Table 4.1 Suitable temperature ranges for adult uncompromised animals.
Humidity
Humidity refers to the amount of water vapour in the air. Relative
humidity (RH) is the amount of water vapour contained in a volume
of air expressed as a percentage of the water vapour that would be
in the same volume of air if it was saturated and at the same
temperature.
Put more simply it is the amount of water vapour in the air divided
by the amount that would be in the same volume of air at the same
temperature if it could hold no more water, multiplied by 100.
The amount of water vapour present in air is dependent on its
temperature. Warm air can contain more moisture than cool air so if
the temperature rises the relative humidity will fall even though the
amount of water in the air remains the same. The opposite is also
true, cold air can contain less water vapour than warm air, which
explains why moisture in the air in a warm room condenses on cold
windows.
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As with all environmental factors wide variations in humidity or long
periods at either low or high levels will stress animals and may
create the conditions in which disease may develop e.g. low
humidity may cause a condition known as ring tail in young rats and
encourages some types of respiratory infections, high humidity can
lead to facial dermatitis in gerbils. Dampness caused by high
humidity can provide conditions in which moulds will grow. High
humidity also speeds up the production of ammonia.
Rodents, especially rats and gerbils are more sensitive to variations
in humidity than species such as dogs and cats. The CoP states
prolonged periods below 40% or above 70% should be avoided for
all species and give more specific advice for individual species (see
Table 4.2).
Species
RH%
Rodents (except gerbils)
45 65
Gerbils
35 55
Rabbits
Not less than 45%
Cats
Can tolerate a wide fluctuation in RH
Dogs
Can tolerate a wide fluctuation in RH
Ferrets
Avoid high humidity levels
Non-Human Primates
40 70
Table 4.2 : Suggested RH% for laboratory animals (source CoP)
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Light
Most laboratory animals are kept under artificial light. Artificial light
can be controlled to suit animal husbandry and experimental needs.
However the CoP advises that the provision of non-opening windows
should be considered in holding rooms because natural light
provides environmental enrichment for some species e.g. non-
human primates, dogs, cats, farm animals and other large
mammals (Fig. 4.4). The light environment is made up of three
elements - intensity, photoperiod and wavelength - all of which
have the potential to affect animal welfare, breeding performance
and experimental results if not adjusted correctly.
Fig. 4.4 Dog unit fitted with glass bricks to let in
daylight while maintaining security.
Intensity
Light intensity (or brightness) is measured in units of lux. To give
an idea of extreme measures of lux natural light in full sun can
produce a reading of about 100 000 lux falling to less than 20 lux in
full moonlight. The CoP requires that light levels in an animal room
should be sufficient to ensure the animals can be inspected properly
and that husbandry can be carried out. Levels of 350400 lux
measured at one metre above the ground have been found to be
suitable for this purpose (Fig.4.5). Light intensity within the room
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will vary due to the position of the light source and the distance
from the source. Near the light source the reading may be as high
as 700 lux and in a cage at the bottom of a rodent rack could be as
low as 10 lux. Low light levels in cages and pens are beneficial for
rodents.
Fig. 4.5 Light meter reading 419 lux.
Many laboratory species are nocturnal or crepuscular so their eyes
are adapted to function in dim light. Exposure to bright light can
cause retinal damage. The situation is made worse if the animals
are albino as they have no protective pigment in their eyes.
Damage of this kind has been seen to
occur in rodents housed at
the top of a
rack near the light source, for this reason the top of
rodent racks should have a cover to shade the light.
Animals benefit from situations where a two level light system
operates, the higher intensity being used to provide staff with
adequate light to carry out their duties and a lower level when the
work has been done and the animals are on their own. A method of
varying light levels to imitate dawn, dusk and moonlight has also
been found beneficial for some species e.g. non-human primates.
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Photoperiod
Photoperiod is the length of time the animals are exposed to light
during the day. Photoperiod affects a number of biological
processes that vary over a 24hour period. These variations are
called circadian rhythms and include sleep, feeding patterns, body
temperature and hormone levels. An uncontrolled photoperiod
could affect breeding and experimental results. Included in the
hormones affected are those that control the reproductive cycle. If
they are kept in natural light some species will be affected by the
seasonal variation in the light/dark cycle. These variations often
disappear with a constant photoperiod of constant length e.g. 12
hours light: 12 hours dark. The Syrian hamster, for example, will
go out of breeding condition in winter when the daylight hours get
short if kept under natural light but they will continue to breed all
year if kept in a constant 12L:12D photoperiod.
It is possible to adjust artificial lighting systems to reverse day
and night or half reverse day and night if it is required for
experimental purposes.
Continuous darkness has the effect of lengthening the oestrous
cycle in many animals and continuous light can produce
continuous oestrus. Both situations will result in a drop in
breeding productivity and eventual cessation of breeding.
Even a short flash of light in the dark phase of the day can affect
the circadian rhythms of some animals and that, in turn, may
affect experimental and breeding results. If these animals need to
be inspected in the dark phase it is better to use a red light rather
than to turn on the normal lights as many species are not
sensitive to red light.
Wavelength
The wavelength of light determines colour perception. It has long
been known cats, some birds and non-human primates have
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colour vision. It is now known that many other species also have
the ability distinguish different wavelengths of light, however they
probably do not perceive it in the same way humans do.
As has been mentioned above daylight is known to be beneficial
to a number of species. There is also some experimental evidence
to show that a source of ultra violet light has a beneficial effect on
animals housed indoors. Imitation daylight lights that contain an
ultra violet element are recommended when animals are kept
under artificial lighting.
Noise
Noise is unwanted sound. Levels of noise should be kept low so
that animals are not startled and so they can communicate by
sound without interference. Sound has two properties, intensity
and frequency.
Intensity and frequency
Sound intensity (or volume) is measured on a logarithmic scale in
decibels (dB) (Fig. 4.6). The pitch of a sound depends on its
frequency, the higher the frequency the higher the pitch.
Frequency is measured in hertz (Hz). Not all species are sensitive
to the same sound frequencies. Humans with good hearing can
hear a range of frequencies from 0.2-20 kHz, but are most
sensitive to sounds in the range of 0.5-5 kHz. These sensitive
frequencies appear to be louder than other frequencies to the
human ear, even though the intensity is the same. For this
reason, straight dB measurement is not very useful when
assessing how annoying a sound is to humans as it treats all
sound the same whatever its frequency. To overcome this
problem sound meters have been developed that filter sound so
that extra consideration is given to the frequencies that humans
are most sensitive to.
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Measurements on this scale are called dB (A weighted scale) or
dBA.
Fig. 4.6 Sound meter reading 58.2 dBA
Humans cannot perceive sounds lower than 0.2 kHz (infrasound)
and higher than 20 kHz (ultrasound) at all. Many species of
animals can hear outside the human range of frequencies, for
instance rodents and rabbits hear in ultrasound as do cats,
marmosets and tamarinds. As an example rats have a hearing
range of 0.2576 kHz and are most sensitive between 35 and
40kHz and cats can perceive sound in a range from 0.0791 kHz
with a peak sensitivity of 140kHz.
Loud or long lasting noise or noise of a frequency that is annoying
as well as sudden irregular noise will cause stress, compromise
welfare and will be evident in changes in behaviour, a drop in
breeding performance and unexpected experimental results. In
susceptible strains of mice some noise may cause audiogenic
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seizure (fits).
Sounds produced by animals form an important element in their
communication repertoire for instance, it is important during
courtship, mothering, excitement, aggression and defence. High
ambient noise levels will interfere with this communication.
It follows that it is necessary to control sound levels to make sure
they remain within acceptable levels. The starting point in
reducing noise is to identify the sources in animal facilities. For
instance:
Animals themselves most species make some noise but
usually it is fairly low level and does not cause problems.
However, some, e.g. dogs, primates and pigs, produce
sounds that often exceed 90dBA, a level that will cause
permanent hearing loss in humans. Rooms holding these
animals should be fitted with sound insulation and sound
absorbing materials. Quiet animals should be housed away
from the noisy ones so that they will not be disturbed by
them. Humans must wear ear protectors when entering
rooms of noisy species, particularly at feeding time.
Appropriate apparatus traditional metal cages, bins etc.
have given way to modern plastic versions which has
drastically reduced day to day noise production in the
animal facility. Human activity can still generate noise so
staff have to be aware and work as quietly as possible.
‘Silentone’ fire alarms produce sound at a frequency that
humans can hear but that the more sensitive animals
cannot, the CoP requires these to be fitted as long as they
do not compromise human safety.
Routines it is impossible not to generate noise with
some animal facility activities, for instance cage cleaning
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and loading and unloading goods. These activities should
be carried out as far away from animals as possible.
Controlling noise that humans can hear is usually straight
forward, it is more difficult with ultrasound. Ultrasound is
generated by a number of common activities e.g. filling
water bottles, using hoses or vacuum cleaners, VDU
screens, trolley wheels. All of these activities should be
done away from animals. Even a dripping tap will produce
ultrasound so it is important to ensure taps are turned off
properly.
Vibration
Most animals are sensitive to vibration, like noise it causes stress.
In addition to the welfare consideration it has an adverse effect on
reproduction and experimental results. Animals must be housed
away from sources of vibration such as cage wash areas and lifts.
Monitoring the Environment
The animal facility environment must be monitored to ensure the
system is working properly. Temperature and humidity must be
done daily, other aspects of the environment such as noise requires
special equipment and is done less frequently.
Most laboratory animal facilities are fitted with Building
Management Systems (BMS). These are computer-controlled
systems that deliver and monitor the required environmental levels.
The monitoring system consists of electronic sensors, arranged in
the room, which collect information on temperature, humidity etc.
and passes it to a computer. The computer not only keeps a
continuous record of these values but also gives warnings if any
value falls outside a pre-set limit (Figs. 4.7a & b).
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Even though computerised monitoring systems are almost
universally used in animal facilities, instruments that have to be
read and the readings manually recorded still have an important
role to play in ensuring a stable environment. They are essential if
the computerised system fails and can be used to check the
accuracy of the computerised system. They also give the people
servicing the room immediate information on the levels within it.
Fig.4.7a BMS Screen showing environmental levels for a
section of animal rooms.
Fig. 4.7b Small section of a BMS screen showing warning
of environmental readings outside the desired levels
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Monitoring Instruments
Monitoring systems used within the room should be sited in a
position that is most likely to record the general conditions of the
room which means away from the influence of sources of heat, cold
or moisture e.g. air ducts, doors, sinks etc.
Measuring temperature and humidity
Temperature is measured in degrees Celcius (
o
C) and relative
humidity in percentages (%). An instrument that measures both is
called a thermohygrograph (Fig. 4.8). Electronic versions of these
instruments record the maximum and minimum temperature in the
room since the instrument was last read and reset as well as the
current room temperature. They also give a reading for the relative
humidity at the time of reading. These readings must be manually
recorded.
Fig. 4.8 Electronic Thermohygrograph
A traditional thermohygrograph (Fig. 4.9) keeps a permanent
record of the variations of the temperature and humidity over the
period of a week. In these instruments the temperature sensor is a
bimetalic strip arranged in a coil so that the coil opens when the
temperature rises and closes when the temperature falls. The
humidity sensor is hair, which expands or contracts depending on
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how much moisture is in the air. These sensors are fixed at one end
and attached to levers at the other. At the end of the lever is a pen
in contact with a sheet of ruled paper wrapped around a drum. The
drum is turned by clockwork and takes seven days to do a complete
revolution. As the drum turns the pens record the changes in
temperature and humidity. The paper is ruled in hours and days on
the horizontal axis and the temperature and humidity on the
vertical axis.
Fig. 4.9 Thermohygrograph
The charts show either temperature or % RH on the vertical axis
and time on the horizontal axis.
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Summary
The environment of a laboratory animal consists of
everything that surrounds and exerts an effect on it.
It is necessary to control the environment to provide for the
animals physiological and behavioural needs, to exclude
unwanted disease causing organisms, provide security and
control environmental variables that can invalidate
experimental results.
The minimum environmental standards suitable for
protected animals are detailed in the Home Office Code of
Practice for the Housing and Care of Animals Bred, Supplied
or Used for Scientific Purposes.
Air quality, temperature, humidity, light and noise all have
the potential to harm animals or influence experiments if
not maintained within acceptable limits.
Environmental monitoring ensures the right levels are being
provided that is achieved usually by computer controlled
building management systems.
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Chapter 5
ROUTINE ANIMAL HOUSE PROCEDURES
Routine animal house procedures refer to the aspect of work that
are concerned with the care of the animal in its cage or pen.
A routine is an established method of completing a task or series of
tasks. Routines are prepared for all the work necessary to maintain
laboratory animals because:
they ensure no element is forgotten;
they ensure all staff carry out the work in the same way;
they enable work to be planned efficiently;
animals become accustomed to the work and are less
disturbed by it.
Basic routines are the same for all animals and all types of unit,
they include:
checking and recording the environment (temperature,
humidity, air pressure);
checking the condition of the animals;
feeding and watering;
cleaning out pens, cages or trays;
changing cages;
cleaning equipment;
checking equipment for damage, especially in-contact
equipment;
cleaning the room and its fittings.
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Although the basic elements of an animal facility routine are similar
they must be adapted to fit the needs of each species, method of
housing and type of work. Differences that affect the routines
include:
the amount and consistency of faeces (dogs wet and
smelly need cleaning at least once a day, mice relatively
dry need cleaning less often);
type of food (dry pellets can stay in hopper for several
days, wet food or fresh food must be changed every day);
cage stocking density (multi-occupied cages need cleaning
more often than singly housed animals);
type of work (breeding units need pairing, weaning etc
included into the routine);
experimental demands.
Animal facilities tend to have the way routines should be carried out
written down and available for staff members to read, often in the
form of Standard Operating Procedures (SOPs). SOPs can be used
by all types of facilities but are a legal requirement for units
involved in studies covered by Good Laboratory Practice Regulations
(GLP).
Although this chapter concentrates on the care of the animal in its
cage or pen, routines and SOPs are established for all aspects of
work in an animal facility for instance work in the cage wash, food
store management, handling animals. Some of these topics are
covered in other chapters of this book. Below is a typical SOP
covering a daily room checks for an animal room.
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Standard Operating Procedure
Daily Routine 3 Animal Room AM & PM Checks
Introduction
This SOP covers the basic responsibilities when first entering an animal room
(AM) and when leaving at the end of the working day (PM) to ensure that
environmental, husbandry and welfare standards are being met.
1. Health & Safety
1.1. Risk of laboratory
animal allergens (LAA) is minimised by wearing personal
protective equipment (PPE) and Local Exhaust Ventilation Systems.
1.2. Additional PPE:
1.2.1. Respiratory mask.
1.2.2. Overshoes.
1.2.3. Gown.
1.2.4. Gloves.
1.3. Training in animal handling will reduce the risk of being bitten.
2. Equipment/Supplies
2.1. Individually Ventilated Cages (IVC).
2.2. IVC air handling unit.
2.3. IVC change station.
2.4. Daily Record Book.
2.5. Diet.
2.6. Bottled sterilised water.
2.7. Hard surface disinfectant/detergent as specified.
2.8. Hand disinfect/detergent as specified.
2.9. Sterile environmental enrichment i.e. Bed’r’Nest and fun tunnels.
3. Preparation
3.1. Enter room with daily record book.
3.2. Apply appropriate PPE.
3.3. Turn on change station.
4. Method
Animal technicians are responsible for the health and welfare of the animals in
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the room that they are checking and confirm that the appropriate standards are
met when signing the daily record book.
All AM checks should be completed by 11.00 hours to ensure that any subsequent
actions can be taken within a timely manner i.e. Environment
al conditions out of
specification.
5. Environment
5.1.
Record the temperature and humidity of all air handling units within the
room in the daily record book.
5.1.1. Note
: If any conditions are out of specification suggested by the
Code of Practice (CoP) alert the
area manager and record in the
comments section.
5.2. Ensure that all IVC pipework is connected correctly.
5.3.
Ensure all IVC racks are positioned appropriately and that all cages are
connected to the circulating air supply, coloured tabs may be present on the
racks to flag cages which aren’t properly connected.
5.4.
Prior to commencing husbandry and welfare checks, turn on the change
station and confirm it is clean and functional before use.
6. Welfare
6.1. Begin at the top of the first rack, checking cages from left to right
and work
downwards until all rows have been checked.
6.1.1. Note:
working in an organised methodical way ensures easy
communication of completed work.
6.2.
Prior to inspecting the cage interpret the cage card in order to most
appropriately assess the cage. Consider t
he following points and act
accordingly:
6.2.1. How many mice are in the cage?
6.2.2. Is it a breeding cage?
6.2.3. Is the female pregnant?
6.2.4. Does a litter need to be weaned?
6.2.5. Do the females need to be plug checked?
6.2.6. Have the animals been used in an experimental procedure/s?
6.2.7. Are the animals newly housed in the cage?
6.3.
Lift up the cage label and check that each mouse is accounted for and is in
the expected state of health. Assessment of each cage can be made by
looking directly at the mice, but also by looking at the state of t
he
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environment e.g. blood in cage, reduced nesting behaviour.
6.3.1. Note:
Cages do not necessarily need to be removed if all mice can
be assessed in position. This will help reduce the stress and noise
levels.
6.4. Upon discovering new litters, record the date of b
irth on the cage and place
wean labels on with recommended wean dates.
6.4.1. Note:
If the cage already contains a litter which is old enough,
wean them to avoid trampling and the prolonged disturbance of the
new litter.
6.5. Remove to the change station any cage whic
h has a green or red health
card label and open the cage to carry out a thorough inspection of the
animal/s in question. If there is a significant change in condition or you are
concerned contact the area manager/NACWO ahead of their normal daily
checks.
6.6. Sign the day book to indicate the completion of the above tasks.
7. Husbandry
7.1.
After completing the welfare assessment for each cage, ensure that the
following conditions are met.
7.1.1.
Sufficient volume of diet available for the number of animals in the
cage to last until the next weekly welfare assessment.
7.1.2.
Sufficient volume of water has been consumed and is available for
the number of animals in the cage until the next empty/refill or
whole bottle change.
7.1.3. A suitable amount of nesting material is present.
7.1.4. A fun tunnel is provided and is in good condition.
7.2. Sign the day book to indicate the completion of the above tasks.
8. PM Checks
To ensure that all basic requirements are met after daily use of an animal room
and that the environment is left in a clean condition for the following morning.
9. Technician checks
9.1. Check all cages have adequate food and water bottle.
9.2.
Check all cages are entire and connected properly to the airflow of the AHU.
If present, use red tab indicators as a visual aid.
9.3.
Commence second daily check on all welfare concerns as required. If
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necessary contact the area manager or NACWO.
9.4. Fill any gaps in the IVC racks with new cages.
9.5. Leave the change station in a clean state and attach the hatch
start UV
cycle if the function is available.
9.6. Hoover floor and then mop with appropriate detergent/disinfectant.
9.7. Sign the day book to indicate which tasks have been performed.
10. Area Manager/NACWO checks
10.1.
Check the welfare record book to ensure all appropriate actions have been
taken. i.e. Have PILs informed etc…
10.2.
Ensure all AM and Technician PM Checks have been signed for and
completed as necessary.
10.3. Sign the day book to indicate the completion of the above tasks.
SOP Provided by Stephen Woodley and Stuart Newman
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Bedding Material
One of the most important routine tasks is to ensure the animals
are kept clean and comfortable. This involves regular cleaning and
changing of bedding material. Bedding material may be placed on
the floor of a solid bottomed cage or pen and so be in direct contact
with the animal or, it may be used to line a tray underneath a cage
and so be separated from the animal by a grid or perforated floor
(non-contact bedding).
Characteristics of an ideal bedding material
An ideal bedding material should be:
harmless to animals i.e. non-toxic (containing no poisonous
material), non-injurious (containing no sharp pieces to wound),
dust free, free from living organisms (especially those that cause
disease - this implies it must be sterilisable);
absorbent - to soak up urine and spilt water so animals keep dry
and to cover faeces to prevent animals becoming soiled*;
a thermal insulator - to reduce conduction of body heat through
the floor of the cage (insulating properties decrease with
wetness);
comfortable;
readily available - so that sufficient quantities of the required
quality can be obtained throughout the year;
packed conveniently for handling and storage;
easily disposable - preferably by incineration as large volumes
are involved and some may be chemically or biologically
contaminated;
reasonably priced - true cost is a function of the purchase price,
storage costs, amount used, frequency it has to be replaced and
cost of disposal.
In addition to the above, bedding materials should be inedible,
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should not stain and should be easy to remove and handle when
cleaning out.
*Ammonia is produced by bacteria acting on the nitrogen components of urine
and faeces. These nitrifying bacteria work best in damp conditions and are
inhibited in dry conditions. Very absorbent bedding materials slow the production
of ammonia. Lower ammonia levels means the bedding needs changing less
often.
Nesting materials share many of the same properties as bedding
materials with the obvious addition of them being able to be built
into nests by the animals.
Examples of bedding and nesting materials
A wide variety of bedding materials are available from specialist
laboratory animal suppliers. When purchased from these suppliers
the products will fulfil the characteristics of the ideal materials
mentioned above. Care must be taken when using non-specialist
suppliers as their standards may not meet those required in bio-
medical research.
All bedding materials are of plant origin and are therefore
biodegradable. They come in the form of wood products, paper
products and cellulose.
Wood and wood products
Wood chips and shavings of various grades and soft wood wool.
Wood for bedding should only come from soft, white wood
(conifers), because some hardwoods contain toxic chemicals such
as phenols that could be absorbed by the animals. Specialist
suppliers ensure the wood has not been chemically treated with
preservatives and have analyses carried out to determine the level
of other harmful contaminants. Specialist manufacturers will also
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grade products to the desired particle size, exclude dust and large,
potentially harmful, pieces.
Wood materials are dried to reduce the moisture content to less
than 10%, so they will absorb a considerable amount of moisture
before they become soggy. The degree of absorbency is related to
the particle size, shavings being less absorbent than smaller sized
particles (Figs. 5.1-2).
Wood itself is a poor conductor of heat and, in particle form, the
extra air trapped between the particles makes it a very good
insulator.
A continuous supply of wood products of the quality and in the
packaging (e.g. compressed, shrink-wrapped or steam permeable
bags) the user requires is available. Soiled bedding is easy to
dispose of by incineration.
Wood products can be used as contact or non-contact bedding. The
cost of the material depends on the quality of the product and the
extent of the chemical analysis that is required.
Most species can use soft wood wool to construct nests and
shavings can be used by species, like rats, who construct open
nests.
Fig.5.1 Wood chips
Fig.5.2 Wood shavings
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Paper and cellulose products
The manufacturing process ensures that paper and related cellulose
fibre materials have a very low microbial load, they are easily
stored, can be sterilised if necessary, and can be disposed of by
incineration. A major advantage of using these products is the very
low levels of dust and the consequent reduction of allergens.
Because the material is specifically manufactured its quality is
constant and can be assured.
Although clean waste paper (e.g. shredded office paper) may have
a limited use with some larger animals it is not recommended in
laboratory animal facilities. The paper may be too harsh leading to
sharp edges that can cause injuries to animals and the inks may be
toxic. Some paper products available from specialist suppliers are
recycled but these are treated to ensure they are harmless.
Paper sheet can be used as a non-contact bedding and is useful
when it is necessary to estimate food wastage, find vaginal plugs or
investigate urine and faeces (Fig. 5.3).
Fig. 5.3 Paper sheet used as a non-contact bedding
Other specifically manufactured paper and cellulose bedding
materials in chip, strand or compressed form are available. These
materials are cut and folded to provide a soft, comfortable substrate
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with good insulating properties. They can be used as contact
bedding and as nesting materials. They are very absorbent
(Fig.5.4).
Fig. 5.4 Cellulose based bedding material
Paper wool and shavings make good nesting materials (Fig. 5.5).
Fig.5.5 Paper shavings
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Corn cob
Corn cob is produced by crushing a maize cob after the maize has
been extracted and the resulting particles are graded into required
sizes (Fig. 5.6). The bedding is a good insulator and appears to be
comfortable for the animals. Urine drains through the bedding to
the bottom of the cage and is absorbed by the bottom layer of corn
cob keeping the animals on the top clean and dry. The absorbency
of corn cob is very good and animals need cleaning out less often
than those on wood products. It has low dust levels.
Fig. 5.6 Corn Cob
Other nesting materials
Straw
Straw is traditionally used for farm animals. It is not absorbent but
allows urine to drain through so the top stays dry. Even the softest
straw is too harsh for small laboratory species.
Hay
Hay is a natural nesting material and food for many species. In the
animal unit care must be taken because it may contain coarse
plants such as thistles and could be contaminated with mould,
bacteria, chemicals or other wild life. Good quality meadow hay
should be free from weeds and will provide a soft nesting material
for animals, however all hay contains microbiological contaminants
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and should be sterilised by autoclaving or irradiation before being
given to laboratory animals. Rabbits will build nests with hay and it
gives privacy to guinea pigs. Both species will eat it so it will need
frequent replenishment (Fig. 5.7).
Fig. 5.7 Young rabbit in nest made with hay and
lined with the mother’s fur.
Cotton Fibre
Nesting material supplied as sheets of cotton fibre divided into 5x5
cm pieces can be added to the cage so the animal can shred and
construct nests from them. The material is soft, non-irritating, inert
and non-digestible (Fig.5.8).
Fig. 5.8 Cotton fibre nesting materials which rodents
are able to shred to make nests.
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Summary
A routine is an established, standard method of completing a
task or series of tasks;
Routines ensure nothing is forgotten, everybody carries out
the task in the same way, work can be planned efficiently and
allows animals to know what to expect;
The basic elements of a care routine are similar for all species
although details may need to be adapted to account for
different species and conditions;
Routines are usually written down, often in the form of SOPs,
and are available to be consulted by staff;
Selecting appropriate bedding and nesting materials is an
important part of routines;
Bedding and nesting materials must meet the standards
required for the bio-medical research;
A variety of bedding and nesting materials are available from
specialist suppliers.
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Chapter 6
HYGIENE
Hygiene is concerned with the maintenance of health. Hygienic
conditions are achieved by the use of routine cleaning, sterilisation
and disinfection. The aim of these routines is to reduce the levels of
substances likely to cause allergic reactions (see chapter 15) and
the numbers of agents that could cause disease, thereby providing
a healthy environment for animals and people within the animal
facility.
The agents that cause disease are viruses, bacteria, fungi (moulds
and yeasts) and invertebrate parasites, their eggs and larvae
(protozoa, worms, insects and arachnids). Of these, bacterial spores
are the most difficult to destroy. Spores are formed by some groups
of bacteria (e.g. Bacillus and Clostridium) when their environment
becomes hostile; they are very resilient and can exist in spore form
for very long periods. When the environment improves they begin
to grow and reproduce again. Bacterial spores are resistant to many
disinfectants but will be killed by autoclaving.
Servicing animals from facilities studying prions and other
potentially dangerous organisms requires special handling and
cleaning techniques which are outside the scope of this book.
Cleaning
To achieve hygienic conditions suitable cleaning regimes must be
applied in a rigorous way. Inadequate attention to detail or the use
of unsuitable cleaning agents or disinfectants will result in spreading
the agents intended to be removed or destroyed.
All cleaning processes involve moving dirt from one place to
another. Efficient methods are those which remove the dirt from the
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environment of the animals to the outside e.g. via a drain or in a
polythene bag to an incinerator.
A combination of methods are employed to clean the unit.
Factors involved in the choice of methods are:
a) the desired state of cleanliness, e.g. sterile, disinfected or
merely free from obvious dirt;
b) the properties of the surface being cleaned, e.g. absorbency,
resistance to scratching;
c) the nature of the item and the safety of the animals and the
operators, e.g. cleaning electrical equipment such as
electronic balances and electric clippers;
d) the nature of the dirt to be removed, e.g. grease, dust and
limescale;
e) the availability of services, e.g. water, drains, power, vacuum
cleaners;
f) the properties of chemicals used, e.g. toxicity, staining,
corrosiveness and rinsing ability;
g) experimental or special requirements, e g. the use of a
chemical recommended by the DEFRA in quarantine facilities.
Each animal unit devises procedures for cleaning which take
account of all these factors in order to achieve the desired state of
cleanliness.
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Cleaning Agents
A cleaning agent is any physical or chemical means by which dirt is
removed from an article or surface. Water is commonly used to
remove surface dirt. Its efficiency is affected by:
the temperature it is used at, being more effective at high
temperatures;
its physical formliquid, spray or steam;
the contact time, soaking in water often releases
stubborn dirt;
its method of application immersion, hosing or high
pressure hosing;
the addition of agitation or scrubbing.
Chemical cleaning agents can be added to water to enhance its
efficiency, for instance soaps and detergents break up fatty
materials so that water can wash them away.
Cleaning Methods
Methods employed within animal units for achieving cleanliness with
some of their principal advantages and disadvantages are listed in
table 6.1
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Table 6.1 Basic Cleaning methods
Method
Principal Advantages
Principal
Disadvantages
Dusting
Quick, looks good
Transfers dirt from
one place to another
Sweeping
Quick, looks good
Creates dust
Vacuuming
Removes dirt from the
environment
Noisy unless the
motor is outside the
animal room
Wet Mopping
Contains dust by wetting
Leaves smears of dirt,
raises RH and creates
slippery floors
Hosing
Removes dirt to the drain
Raises RH, takes a
long time to dry,
requires drains
High pressure
hosing
(Fig.6.1)
Removes stubborn dirt
As above plus greater
splashback and
aerosols, may spread
dirt. Can injure user
High pressure
hosing with
steam
As above, heat energy kills
some organisms in situ
As above, must not be
used near animals,
dangerous to operator
(scalding)
Washing items
in sinks or
tubs
Inexpensive equipment
Labour intensive
Washing
machines
Efficient, wide range of cycles
Machinery purchase
and maintenance is
expensive
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Fig. 6.1 High pressure hosing apparatus
Cleaning Cages and Pens
How often cages are changed and bedding is cleaned out or
replenished is determined by many interrelated factors which
include:
the cage (site, size and design. IVCs need cleaning less often
than open cages);
the animals (numbers, species, whether stock or breeding and
behaviour of individuals);
the type of diet and the methods of presentation of food and
water;
the amount and type of bedding used;
the likelihood of the presence of pests and parasites;
the environmental conditions (temperature and ventilation);
the experimental requirements;
the cost of materials and labour;
the interference caused to the animal’s environment and
behaviour.
The frequency of cage cleaning selected is a compromise between
the factors above. For instance, it is important to provide a clean
environment for animals and to ensure they are not exposed to high
levels of ammonia in the cage. However changing cages too
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frequently disturbs the animals and they take some time to settle
down once they have been put into new cages.
Once animals are removed from a cage the soiled bedding must be
discarded before the cage can be washed. In breeding colonies, it is
common practice for some nesting material from the old cage to be
transferred into the new cage so that pheromones that dams use to
identify their young are retained. Both removing animals and
emptying bedding from a cage causes a release of allergens and
other contaminants into the atmosphere creating a risk to animals
and staff.
Work practices must be designed to eliminate, as far as possible,
circulating allergens. This can be done if animals are transferred to
clean cages in cage cleaning stations. The dirty cages can then be
covered and transferred to the cage-wash room on pallets.
The air flow in cage cleaning stations (Fig. 6.2) is designed to allow
the animals to be handled and the cages to be serviced without
contaminants from the cage leaving the station or contaminants
from the outside reaching the animal. Fig. 6.3 shows the pattern of
air flow.
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Fig. 6.2 Cage cleaning station
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Fig. 6.3 Airflow through a cage cleaning station
It is essential to use cage cleaning stations when servicing animals
housed in IVCs as the purpose of these units is to separate animals
from the external environment. The cage cleaning station should be
disinfected between each cage serviced to prevent cross
contamination.
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In large facilities robotic systems may be used to handle the dirty
cages (Fig. 6.4). Robots lift the cage from pallet, invert it over a
waste shute to remove the soil and then place it on the conveyor of
a tunnel washer. At the other end of the washer the clean, dry cage
is held under a hopper to receive a measured amount of bedding
and then replaced on a pallet to be taken to an animal room.
With these systems people can be excluded from the areas where
there is likely to be high levels of allergens.
Fig. 6.4 Robot working in the cage cleaning room
Where robotic systems are not available or when pens are being
cleaned soil has to be removed manually but vacuum extraction
systems reduce the airborne allergens. People in the area must
wear appropriate protective clothing including face masks or
respirators.
Cage Washing Machines
Machines are available for washing animal cages and ancillary
equipment; they are usually either tunnel or rack washers.
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Tunnel Washers
A tunnel washer consists of a grid-type conveyor belt which carries
cages through the tunnel past jets which spray hot washing
solutions followed by rinse water onto all surfaces of the cages. The
washing solutions include detergents and descalers. Cages are
loaded at the ‘dirty end’ and are removed from the ‘clean end’ clean
and dry. There is a physical barrier between the clean and dirty end
to prevent cross contamination. The clean cages are unloaded from
the other end of the machine (Fig. 6.5).
Fig. 6.5 Robot loading cages onto a tunnel washer
Rack Washers
Cages are loaded onto special racks that are placed into the
machine (Fig.6.6). The cages are washed by spray from moving jets
on spinners or sliding batons. To ensure efficient washing, materials
must be loaded into the washer correctly.
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Fig. 6.6 Cage and Rack Washer
Bottle Washers
Systems are available that remove the caps from bottles, empty
them, wash them, refill and recap them and then load them onto
trollies so they can be taken back to the animal room or to the
autoclave.
These systems not only save time but also limit the risk of repetitive
strain injury from removing bottle tops and back injury from lifting
racks of full water bottles.
Sterilisation and Disinfection
Definitions
Sterilisation is a process that results in the complete destruction
or removal of all living organisms. Sterility is an absolute state; an
area or object is either sterile or it is not; it cannot be nearly sterile.
In order to ensure sterility has been achieved the process must be
validated (that is tests must be carried out to ensure sterility has
been achieved).
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Disinfection is a process that removes the causes of infectious
disease. Disinfection does not destroy all forms of pathogenic
organisms; it cannot be relied on to kill bacterial spores. The
intention is to reduce the numbers of organisms to levels where
there are too few to cause disease.
Pasteurisation is a form of disinfection that kills vegetative forms
of bacteria (that is those that are growing, not spores) by rapidly
raising the temperature to 70 - 80
o
C followed by rapid cooling. The
process is used to treat food because the times and temperatures
used are effective in reducing micro-organisms but do not damage
the nutrient quality. Pasteurisation was originally introduced to kill
tubercle bacilli in milk. The pelleting process for laboratory animal
diets achieves pasteurisation, reducing the numbers of viable micro-
organisms in the diet from 5 x 10
6
/g in raw ingredients to as little
as 1 x 10
3
/g in expanded pellets.
Fumigation is the use of gases or vapours in order to achieve
disinfection. It is used to treat rooms and other spaces to clean
them of potentially harmful organisms. Formaldehyde and hydrogen
peroxide are commonly used agents and both are dangerous and
must only be used by trained personnel in accordance with an
established protocol.
Fogging is the process whereby a fine mist of disinfectant is
sprayed around an area to decontaminate it. The mist is produced
by forcing the disinfectant solution through the nozzles of a fogging
machine (Fig.6.7). The nozzle rotates so the whole area is covered.
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Fig. 6.7 Fogger
Methods used to Achieve Sterilisation
Sterilisation can be achieved by the use of heat, chemicals, filtration
and radiation.
Heat
Dry heat and wet heat are used.
Dry heat kills by coagulating or oxidising proteins. To ensure
sterility materials must be exposed to very high temperatures for
long periods. Dry heat has limited use in an animal facility because
the material being treated is often damaged by the high
temperatures needed.
Dry Heat Methods
Flaming - in this method the material to be treated is exposed to a
naked flame, either from a Bunsen burner or a flame gun,
temperatures of 500
o
C are reached with these techniques. There
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are obvious dangers with this method and, although it has some use
in microbiology, it has little use in laboratory animal technology.
Hot Air Ovens - Metal or glass can be sterilised by exposure to
temperatures of 160 - 180
o
C for one hour in a hot air cabinet. The
whole process takes much longer than one hour because the cabinet
will take time to reach the required temperature and then must be
allowed to cool down afterwards. In hot air ovens the items being
sterilised must be wrapped in an impervious material, such as
aluminium foil, so they remain sterile until they are used.
Incinerators - A useful dry heat method to render material sterile in
animal facilities is incineration. Incinerators are fuelled by gas or oil
and are built to reach temperatures in the region of 1000
o
C. At
these temperatures a wide range of materials e.g. carcasses,
bedding and sharps are reduced to an inert ash. Even highly
infective materials are rendered sterile, although care must be
taken when transporting infective material to the incinerator.
Incinerators are expensive to install and their installation and use
are strictly regulated.
Wet Heat
Wet heat destroys micro-organisms by denaturing proteins.
Although steam produced at atmospheric pressure (when water
boils at 100
o
C) is a useful disinfectant to achieve sterilisation, steam
must be produced above atmospheric pressure so water boils at 121
or 134
o
C. Steam at these temperatures is an effective killing agent
because of its specific heat of vaporisation. It is not the temperature
that kills but the release of energy from the steam as it condenses
on the surface of objects exposed to it. Steam also penetrates loads
efficiently. In order to maintain steam under pressure a metal
chamber called an autoclave is used.
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Autoclaves
Autoclaves are pressure vessels in which objects can be exposed to
steam produced at above atmospheric pressure. To work efficiently
the steam must come into contact with micro-organisms. This
means all air must be removed from the chamber and the objects
being treated must be physically clean. If materials are not to be
used immediately after sterilisation they can be wrapped in
autoclave bags so they remain sterile after the autoclave is opened.
These bags are permeable to steam when they are heated but
become impermeable to micro-organisms when they cool.
Autoclaves have a door at each end of the chamber, one for loading
the non-sterile items (the dirty side) and the other, opening into a
separate room, for emptying the sterile goods (the clean side).
Once the chamber has been loaded and the door closed the air is
removed and steam is introduced. This is done in a series of pulses
(some air is sucked out and some steam is introduced, then more
air is pumped out etc.) until all the air is out and the chamber is full
of steam. The vacuum ensures even penetration of steam
throughout the chamber including between the particles of porous
materials like bedding. At the end of the sterilisation cycle another
vacuum is pulled which removes the steam. This has the effect of
drying the load.
Autoclaves are electronically controlled in order to make them more
efficient and to prevent accidents due to human error. Electronic
sensors enable the cycle to be monitored throughout the cycle. This
monitoring information can be viewed on screens and/or printouts
on the front of the autoclave (Fig.6.8). A wide variety of
temperature, pressure and time cycles are available and the cycle
should be selected that suits the type of material loaded in the
chamber.
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Fig. 6.8 Autoclave for introducing goods into a full barrier unit showing
temperature and pressure gauges on the front and a computer print out
providing a record of the cycle.
Smaller autoclaves, suitable for light loads like surgical instruments,
are available. They are usually of the downward or upward
displacement type, which means instead of being fitted with a
vacuum to extract air, air is introduced at the top or bottom of the
chamber and it pushes the air out of an exhaust valve at the
opposite end of the chamber. When a heat sensor in the exhaust
registers the temperature of steam the valve is closed. It is
assumed all the air has been pushed out and the chamber is full of
steam (Fig 6.9).
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Fig. 6.9. Small air displacement Autoclave for sterilising small
items such as surgical instruments.
Each type of autoclave needs to be operated according to specific
instructions and it is vital that these are strictly adhered to. It is
also essential to load them properly to ensure even distribution of
steam.
Monitoring Autoclaves
Autoclaves have internal monitoring systems as mentioned but
other methods of monitoring are often used. These are placed
around the load being sterilised and are read once the load is
opened. Three commonly used monitors are mentioned here.
1) Autoclave tape (Fig. 6.10). This is masking tape
impregnated with strips of a chemical that changes from
clear to black/brown when exposed to steam. This tape can
be used to seal autoclave bags. The result is easy to read
but it is only an indicator that the items have been exposed
to steam; it does not guarantee they have been exposed to
the required temperature or for the required time.
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Fig. 6.10 Autoclave Indicator Tape.
Top appearance before the cycle,
bottom appearance after a successful cycle.
2) Autoclave indicator strips. Fig.6.11 illustrates one of the
many types of strip available. These can be placed in
different locations around the load to ensure the steam
reaches everywhere. They are more accurate than the tape
because they only change colour when exposed for the
required amount of time. Different strips are available for
different cycles.
Fig. 6.11 Indicator Strip. The yellow dot changes to blue after the
temperature has been reached for the required amount of time.
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3) Spore strips. These are strips of paper impregnated with
bacterial spores from a species that do not cause human or
animal disease (Bacillus stearothermophillus). This species
of bacteria can tolerate high temperatures. The spore strips
are arranged around the load and once the cycle has ended
they are sent, with a control strip that has not been in the
autoclave, to a microbiological laboratory where they are
cultured (that is placed in conditions where they should
grow). If the autoclave cycle has been successful, the
control strip should grow but the ones that had been in the
autoclave would not. The culture takes a few days so the
test is most useful if the sterilised items are packed in
autoclave bags and are not going to be used immediately.
It is also a useful periodic test for any autoclave to check it
is working well.
Chemical Sterilants
Very few chemicals can be relied on to achieve sterilisation. The few
that can are toxic and should only be used in controlled conditions
by trained staff. They usually need long exposure times to kill
bacterial spores. Examples of chemical sterilants are Peracetic acid
and Alcide that are used as surface sterilants for isolators and their
supplies. Other agents are considered suitable for sterilising surgical
instruments.
Filtration
Air filtration is commonly used in laboratory animal facilities and
filters can remove up to 99.97% of airborne particles. Liquids can
also be sterilised by filtration.
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Radiation
Ionising radiation emitted in the form of gamma rays (usually from
a cobalt
60
source) is an effective sterilising agent. Exposure has to
be done at specialist units because the process is very dangerous.
The rays destroy organisms by damaging DNA. Many materials used
in animal units are treated with gamma radiation e.g. food,
bedding, hypodermic syringes.
Ultra-violet radiation will also achieve sterilisation but as these rays
have very poor penetration ability they can only be used for
surfaces or very thin layers of material. UV light can also be used to
sterilise the air in air locks, cage cleaning stations and safety
cabinets, although it is important to make sure the source is
switched off when people are using them.
Disinfection
Methods used to achieve Disinfection
An ideal disinfectant should be:
lethal to the undesirable micro-organisms and should not
permit the establishment of resistant forms;
harmless to animals and to man (e.g. non-toxic, non-irritant,
non-allergenic, non-corrosive);
harmless to materials (e.g. non-corrosive, non staining);
not inactivated by the presence of materials especially organic
matter;
quick to work at a wide range of temperatures;
compatible with other chemicals, e.g. detergents;
suitable for a wide range of applications;
easily stored and have a long shelf life;
reasonably priced at use dilution.
No disinfectant has all of the above properties, for instance
disinfectants tend to kill certain micro-organisms but may do little
harm to others. It is possible that a disinfectant will leave an area
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sterile because that will depend on what organisms were there in
the first place.
Disinfection can be achieved by the application of heat and chemical
means.
Heat
Boiling water or steam at atmospheric pressure (free steam) can be
used to reduce the numbers of micro-organisms on materials.
Immersion in boiling water exposes materials to temperatures not
exceeding 100
o
C. It is useful to treat water and heat resistant
materials like surgical instruments.
Steam Lances - can be used to treat exposed surfaces.
Steam Chests - caging and equipment can be treated in a steam
chest that is a cabinet into which steam is supplied. Exposure times
of approximately 1 hour are necessary to achieve adequate
disinfection.
Chemical Disinfectants
Routine chemical disinfection is used in all animal units but it is
important to be aware of the limitations of disinfectants.
Disinfectants must be freshly made up when used and old
disinfectant should not be topped up with new. All utensils used to
apply the disinfectant should be cleaned after use, preferably with a
different type of disinfectant method. This prevents the
establishment of resistant strains of micro-organisms (that is strains
of micro-organisms that are able to survive exposure to the
disinfectant).
As we have said before it is important to remove all dirt, particularly
organic matter from the surface being disinfected, as this will both
protect the micro-organism and inactivate the disinfectant.
The activity of disinfectants can be affected by a number of other
Page | 101
factors such as mixing with certain other disinfectants and
detergents, acid or alkaline conditions, humidity and temperature.
Time is another important factor in ensuring disinfection is effective;
some micro-organisms are killed very quickly while others need to
be exposed for much longer.
By definition chemical disinfectants are designed to destroy living
organisms so must be used with care and disposed of safely.
Appropriate protective clothing must be used when making up and
using these agents.
Manufacturers instructions will detail all of this information together
with the range of activity, hazards, safe use and disposal of their
product.
Disinfectants must be used in accordance with local rules and SOPs
The properties and use of some of the major groups of disinfectants
is given below. It should only be used as an illustration of the
different agents available. Within most groups there are many
disinfectants each with a different range of activity. Before using
any agent the manufacturers literature should be consulted.
Page | 102
Properties and Uses
Alcohols
Examples: Ethanol
Properties: Alcohols damage the cell membrane and denature
proteins. Ethanol will quickly kill bacteria and will destroy fungi and
viruses but will not destroy bacterial spores. Alcohols are readily
inactivated by organic material and oil. Alcohol is used between 70
90% concentration; higher concentrations are less effective
because water is necessary to enable it to work.
Alcohol activity is reduced in the presence of organic matter. It will
affect rubber and some plastics and is flammable and so must be
used with great care.
Uses: They are used to treat clinical thermometers and clean
surgical instruments. They are particularly useful for wiping clean
surfaces and sensitive equipment, e.g. electronic balances and hair
clippers, as the vaporisation brings about rapid drying. They are
incorporated in detergents for skin disinfection.
Aldehydes
Example: Formaldehyde
Properties: This agent is bactericidal, bacteriostatic, sporicidal,
fungicidal and virucidal, being more active in hot solution. Use
dilution can vary from 1 to 40% depending on method of use but
the stability is poor. It is a highly toxic chemical and should only be
used for fumigation in controlled conditions.
Uses: Fumigation (used at 40% W/V solution in water).
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Halogens
Halogens include disinfectants whose active ingredients are either
chlorine or iodine.
Examples: Sodium hypochlorite, Iodophors
Sodium Hypochlorite
Properties: The efficiency of this agent is measured in terms of the
parts per million of available chlorine (ppm). It will kill vegetative
bacteria, fungi and viruses at low concentrations (up to 500 ppm);
at this concentration it has low toxicity. It will rapidly kill bacterial
spores at very high concentrations (2,500 ppm) but this
concentration is both corrosive and an irritant to mucus membranes
and skin so should only be used in controlled circumstances.
Activity is reduced by a factor of 10 for every unit of pH above 7. It
is inactivated by light and some metals.
Uses: Water treatment, feeding and watering utensils, animal cages
and ancillary equipment when combined with a compatible
detergent. Chlorinated detergents can be used in cage washing
machines.
Iodophors
Properties: Iodophors are effective for a wide range of bacteria,
fungi and viruses. They will kill spores but need a long contact time.
Activity is enhanced by an acid pH and heat. They have low toxicity.
Uses: Skin disinfection, footbaths and dunk tanks, clean surfaces.
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Oxidizing Agents
Examples: Hydrogen peroxide, Virkon S
Properties: These agents destroy pathological agents by oxidising
their proteins and fats.
Hydrogen peroxide
This agent will kill bacteria, fungi and at high concentrations it will
kill spores. It destroys some types of viruses (non-enveloped
viruses) but its effect on enveloped viruses is variable as is its effect
on mycobacteria.
It can be used in liquid form or as a mist where it can be used as an
alternative to formalin for fumigating rooms or in air locks or safety
cabinets.
Virkon
Virkon is reported to be active against bacteria, including spores,
viruses and fungi. It can be irritant and is corrosive to metals if
exposed for long periods.
Uses: It can be used in a 1% solution to disinfect solid surfaces and
is used as part of the decontamination process in safety cabinets.
Phenols and Derivatives
Properties: These agents are bactericidal, fungicidal and destroy
some types of viruses (enveloped viruses) but have no effect on
bacterial spores. Their activity is reduced by the presence of organic
material and oils. They are more active in an acid pH and also when
in hot solution. They are stable at use dilution, which is usually 1 to
2%, and have fair rinsing properties. Phenolic compounds are toxic,
corrosive and have an unpleasant smell. They should not be used in
areas housing animals, cats and dogs as they are particularly
sensitive to them.
Uses: Application to surfaces, e.g. walls and floors, following the
Page | 105
removal of gross soiling.
Quaternary Ammonium Compounds
Properties: Selectively bactericidal and bacteriostatic. Some
compounds are also fungicidal and virucidal. They are good
detergents but as they are cationic detergents their activity is
reduced if they are mixed with anionic or non-ionic detergents.
Their activity is also diminished by hard water, rubber and plastics,
oil and some metals. Activity is enhanced by an alkaline pH and
heat. They are stable at use dilution, which is usually 1 to 2%. They
have low toxicity and poor rinsing properties due to surface
adsorption. Numerous different types of quaternary ammonium
compounds are commercially available for use as disinfectants and
the level of activity will vary considerably between different types.
Uses: Treating surfaces, e.g. walls, floors and bench tops following
the removal of gross soiling. Feeding and watering utensils followed
by thorough rinsing. Combined with compatible detergent in cage
washing machines. Skin disinfection at low concentration.
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Summary
Hygiene is concerned with the maintenance e of health and is
achieved by routine cleaning, sterilisation and disinfection with
the aim of minimising disease causing agents and reducing
exposure to circulating allergens;
Cleaning routines differ according to the species, type of
animal housing and other circumstances;
Equipment such as cage cleaning stations can be used to
protect animals from the risk of cross contamination;
Cage cleaning stations, robotic systems, cage and bottle
washers help to minimise exposure to laboratory animal
allergens and to prevent repetitive strain and other muscular
injury;
Sterilisation methods aim to render the treated object free of
all living organisms; the sterile state can be achieved by using
appropriate techniques e.g. autoclaving, radiation, filtration;
Disinfection seeks to reduce the level of microorganisms to a
level where they are unlikely to be able to cause disease;
disinfection can be achieved by the use of chemicals or heat;
Sterilisation and disinfection methods will only be effective if
they are applied correctly.
Page | 107
Page | 108
Chapter 7
FEEDING AND WATERING
Food
In nature animals manage to fulfil their nutritional requirements by
eating a variety of foodstuffs. In the animal facility they only have
the diets provided for them. In theory animals eat to satisfy their
energy requirements, which implies that when energy needs are
satisfied the animals stop eating even if they have not taken in
enough of the other nutrients. It is, therefore, necessary to ensure
that laboratory animal diets are balanced so they contain the
nutrients the animals require in the proportions that will satisfy their
needs.
The nutrients that must be present in the diet of an animal are:
proteins, fats, energy sources (which can come from carbohydrates,
proteins or fats), vitamins and minerals. In addition to these
nutrients food often contains undigestible fibre that provides no
nutrient value but is a necessary aid for efficient digestion. Water is
also essential for animals and this will be considered later in this
chapter.
The amount of each nutrient required by animals depends on
several factors:
physiological condition (growing, pregnant and lactating
animals have a higher demand for nutrients);
activity (active working animals require more energy than
inactive);
age (older animals need different amounts of nutrients than
younger ones);
experimental demands (e.g. animals that are regularly bled
may need supplements to their diet to replace those being
removed);
Page | 109
species (some species have a specific requirement for a
particular nutrient e.g. primates and guinea pigs for Vitamin
C, cats for the amino acid Taurine).
The amount of food consumed by the animal is affected by all of the
above. The following factors will also have an influence:
palatability
physical form (hard, soft, powder)
boredom (a few species, such as cats and primates, may
get bored with a diet and may stop eating it; many species
eat more because they are bored and have nothing else to
do).
Diets are available to satisfy nutritional requirements of individual
species in the various stages of their lives. The two main types of
diet are maintenance diets that aim to keep the animals in a steady
state, neither gaining nor losing weight and breeding diets that
provide the extra nutrients required for lactation and growth. Diets
are also available for more specific needs e.g.
enriched diets for weak or sick animals;
experimental diets (formulated to meet individual scientist
requirements);
autoclavable diets - include supplements to make up for
those lost in the high temperatures of an autoclave.
Page | 110
Presentation of Diet
In captivity food is usually presented in containers. This keeps it
clean, minimises waste and is convenient. A variety of containers
are used to accommodate the animals feeding behaviour and the
type of food they eat.
Bowls are used for animals that are likely to eat a whole ration
immediately after it is presented (e.g. dogs), where mashes or
seeds are fed or when animals are housed in pens. It may also be
appropriate to present food in an open container on the floor to
animals that have difficulty reaching a hopper or basket, for
instance sick animals or some genetically altered strains. Baskets or
hoppers are used where the animal eats little and often and when
the food is intended to last for several days (e.g. rabbits and
rodents). The shape and size of the food container used will be
influenced by the species, the size and age of the animal (and
therefore the amount they consume) and the number of animals in
the cage or pen. Where more than one animal is housed in a cage
food should be presented over a wide area; animals are competitive
feeders and some dominant individuals will try to prevent others in
the cage from feeding. The wider the area of food presentation the
less successful they will be (Figs. 7.1, 7.2, 7.3).
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Fig. 7.1 Mouse cage showing food basket
Fig. 7.2 Rabbit hopper and water bottle
Page | 112
Fig. 7.3 Food bowl in floor pen
Although it is usual to present diet in containers it provides
environmental enrichment for some species if they have to work for
their food. For instance, fruit may be presented to primates over the
floor and they will amuse themselves searching for it. Similarly,
pellets can be provided in forage boxes.
The Latin term ad libitum (ad lib) which means "at ones pleasure" is
used to describe the practice where food or water is freely available
to an animal at all times. The term restricted feeding is used when
food is only available for part of the day or where only measured
amounts are fed.
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Forms of Diet
Food is presented in different physical forms to suit the
requirements of the animal and experimental demands.
Pelleted Diet
Diet is pelleted to reduce waste and to make them easier to handle.
The ingredients are ground and thoroughly mixed during
manufacture to produce a homogenous mixture so animals cannot
selectively eat individual components.
Two main types of pellet are produced, traditional and expanded
(Fig. 7.4).
Fig. 7.4 Traditional pellets (left) and expanded pellets (right)
Traditional pellets are produced by grinding and mixing raw
materials with steam. The mixture is then passed through a
pelleting machine, under mechanical pressure, and emerges from
the die face as pencil shaped pellets.
Raw materials for expanded pellets are mixed in the same way but
additional steam is introduced into the pelleting machine (called an
expander) as the mixture is passing through. This causes a build up
of temperature and pressure within the expander. When the pellets
emerge from the die into atmospheric pressure the compressed
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steam they contain expands producing the larger pellet. The higher
temperature reached in expanders “cooks” the ingredients
producing a more homogenous mix with a harder biscuit like
texture. The extra heat also reduces the microbial load to a greater
degree than in the traditional pelleting process and this results in a
longer shelf life (six months rather than the three usually quoted for
traditional pellets).
Expanded diet is thought to be more attractive to animals and cause
less waste because it is less likely to crumble but it does take up
more space in the hopper. The smaller size of traditional pellets
means they are more likely to be used in isolators.
Powdered Diet
Powdered diet consists of finely ground components. The usual
method of manufacture is to pass diet that has already been
pelleted through a grinder. This ensures that the quality of the diet
is similar to the "normal" pellets and that the particle size is
consistent. Powdered diet is especially useful when experimental
work requires test substances to be added to the diet.
Mash
Mash consists of pelleted diet mixed with water. The diet is readily
eaten when in this form. However, the increased water content
encourages rancidity and mould growth so fresh mash must be
made up immediately before feeding and changed at least daily. In
view of this and the fact that the containers in which the mash is
fed must be frequently cleaned, feeding mash is very labour
intensive. Mash can be used to encourage sick or weak animals to
eat and is a particularly useful method to supply food to some
transgenic strains. Commercial diets in the form of doughs and gels
are available from diet suppliers. These are a cleaner and more
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convenient replacement for mashes.
Fresh Food
Fruit and vegetables
Given the choice, many species would prefer to eat fresh food in the
form of fruit and vegetables but in most cases it is not possible to
provide it for them. Fresh food is a vector for infectious organisms
and could introduce disease into the facility so cannot be used in
barrier units. It has an unknown nutrient content and that content
drops as the food deteriorates with age.
Where it is possible to feed fresh food, however, it should be
encouraged. Primates, for instance, gain great benefit from being
fed fruit and vegetables. It provides variation in the diet relieving
boredom from eating the same diet all the time. It can also supply
some of the Vitamin C needs of the primates. A common practice is
to feed pellets in the morning and fresh food in the afternoon.
Manufactured fruit flavoured foods or dry fruits are available to
provide enrichment for animals that cannot be fed fresh fruit. These
manufactured foods can be autoclaved or irradiated and do not
present an infection risk to the health of the animals. Some can be
purchased with an analysis of possible chemical contaminants.
Hay
Herbivorous animals such as rabbits and guinea pigs benefit from
the addition of hay to their diet. It provides some nutrients but also
aids digestion. Hay is available in several forms, for instance as
baled hay or as hay blocks. It is essential that the hay is irradiated
or autoclaved before it is presented to the animals.
Supplementary Feeding
Some species have a need for a specific nutrient and it is important
to ensure that this nutrient is supplied in their diet (e.g. Vitamin C
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for primates and guinea pigs, Vitamin D3 for New World primates
and the amino acid Taurine for cats). Diet obtained from specialist
laboratory animal diet manufacturers can be relied on to have all
the nutrients required by the animals they are designed for but diet
can deteriorate and levels of some ingredients can reduce if they
are not stored properly or they exceed their use by date.
Ascorbic Acid (Vitamin C)
Ascorbic acid provides an illustration of a specific nutrient
requirement.
Most common laboratory animal species can synthesise ascorbic
acid in their tissues. Primates (including humans) and guinea pigs
cannot, it must be included in their diet. As it is a water-soluble
vitamin it is not stored well in the body so it must be fed every day.
A deficiency of Vitamin C (commonly known as Scurvy) in the
guinea pig will first show as a staring coat and reluctance to move.
A severe case might cause loose teeth, bleeding gums,
haemorrhages under the skin and/or stiff and swollen joints. The
long bones may break easily and death may follow.
The deficiency signs in primates are similar and include weakness,
reluctance to move, bruising, weight loss and anaemia.
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Methods of Administration of Vitamin C
Diet manufactured for guinea pigs and primates contains enough
ascorbic acid for the animalsneeds, however it is easily converted
into an inactive chemical when exposed to oxygen and this
oxidation is speeded up if the ambient temperature increases. To
ensure the diet contains sufficient Vitamin C for the animals need it
must be stored correctly and must not be fed after its use by date.
Feeding pellets enriched with ascorbic acid is the only practical way
of ensuring that the animals get an adequate supply. As we have
said before fresh fruit will contain some of the vitamin but it will be
a variable and unknown amount. Fruit and vegetables are better
seen as environmental enrichment rather than a provider of
nutrients. In emergencies or for experimental reasons ascorbic acid
can be fed in the drinking water but it will need to be changed every
day.
Pelleted Diet for Laboratory Animals
Manufacturers of laboratory animal diet produce feeds that provide
for all of the animals nutritional needs. Samples of each batch of
food produced are analysed to ensure levels of nutrients are as they
should be and tables of nutritional content are made available to
purchasers. The ingredients, however, are commercially confidential
therefore it is only possible to give a typical list of ingredients and
not a recipe. Manufacturers do supply detailed nutritional
information of their diet on their websites. The ingredients below
are for healthy, adult animals.
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Mice, Rats and Syrian Hamsters
There is a variety of rodent diet commercially available. A typical
Rodent Maintenance diet could contain
Wheat, Barley, Wheatfeed
Extracted Soya protein, Whey Powder
Soya Oil
Supplements: Vitamins, Major and Trace Minerals, Amino
Acids
Rodents are normally fed pelleted diet in baskets incorporated into
the lid of the cage. Food is usually made available ad lib, however
in some cases restricted feeding may be appropriate, for instance
rats that are kept into old age remain healthier and live longer if
they are fed on diet with a lower nutrient value or have restricted
access to diet.
Forage mixes and other supplements are available for rodents that
can be added to the cage as a form of enrichment. However, it is
important that dietary additions do not interfere with experimental
work or unbalance the normal diet.
Guinea Pigs and Rabbits
A typical guinea pig diet could contain:
Wheat, Oats, Barley, Wheatfeed, Bran, Grass meal
Linseed cake, Extracted Soya protein, Potato Protein
Supplements: Vitamins, Major and Trace Minerals, Amino
acids
A typical rabbit diet could contain:
Barley, Wheatfeed, Oats, Bran, Grass meal
Extracted Soya protein, Whey powder
Supplements: Vitamins, Major and Trace minerals, Amino
acids
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Guinea pigs and rabbits are herbivores and their diet contains a
higher fibre content than other small laboratory animals. Their large
intestines are populated with micro-organisms that can digest fibre
and make the products of the digestion available to them. Both
benefit from the presence of hay in their cages. Guinea pigs must
have Vitamin C supplied in the diet. If they are housed in cages
they are fed from hoppers but if they are in floor pens bowls may be
used.
Ferrets and Cats
A typical dry cat or ferret diet could contain:
Maize, Wheat, Wheatgerm
Extracted Soya protein, Whey powder, Fishmeal
Soya oil, Sugar beet pulp, Yeast
Supplements: Vitamins, Major and Trace minerals, Amino
acids
Ferrets and cats survive well in laboratories when fed on dry diet
provided they have a plentiful supply of water (not milk). Pellets for
these animals have a fat coating to increase palatability. Tinned cat
meat can also be fed. These animals are usually fed from open
bowls.
Dogs
A typical dog diet could contain:
Maize, Rice, Wheat, Wheatfeed
Poultry meat meal, Fishmeal, Dried egg
Linseed oil, Soya oil
Supplements: Vitamins, Major and Trace minerals, Amino
acids, Fructo-oligosaccharide
A complete dry diet may be fed ad libitum or as a daily measured
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ration in accordance with the recommendations of the
manufacturer. Like cat and ferret diet, dog pellets are coated with
fat to increase palatability. The hard expanded pellet helps to keep
the dogsteeth and gums in good order. This will constitute a
balanced diet if fed together with unrestricted water. Dogs may also
be fed traditional dog biscuits with cooked or tinned meat. It is
usual to feed dogs from open bowls.
New World Primates
A typical New World primate diet could contain:
Wheat, Wheatfeed, Maize
Extracted Soya protein, Poultry meat meal, Whey powder
Soya oil, Yeast
Supplements: Vitamins, Major and trace minerals, amino
acids
As well as Vitamin C, New World primates (e.g. Marmosets and
Tamarins) need Vitamin D3 because they cannot utilise Vitamin D2.
Although this diet is balanced and should provide all the animals
need, it is still recommended that the diet is supplemented with
small amounts of fresh fruit and/or vegetables as the animals
obviously enjoy consuming natural foods and the variety reduces
the boredom that can occur with the same pelleted diet and
provides environmental enrichment.
Old World Primate Diet
A typical Old World primate diet could contain:
Wheat, Wheatfeed, Maize
Extracted Soya protein, Whey powder, Yeast
Soya oil
Supplements: Vitamins, Major and trace minerals, Amino
acids
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Although this diet will provide for the animalsnutritional needs, for
the reasons mentioned in the section above, the animals benefit
from the addition of small amounts of fruit and vegetables.
Contribution of the ingredients to the nutrient value of the
diet.
Cereals such as wheat, oats, barley and maize, contribute
carbohydrate, protein, some fats and some vitamins to the diet. The
amino acids that make up cereal protein are the same as those
found in animal tissues but animals require some of them in greater
quantities than are found in cereals. Plants such as soya and linseed
and animal products such as poultry, fishmeal and whey are added
to supply the deficient amino acids. Oil based plants such as soya
oil make sure the levels of essential fatty acids are achieved.
Although raw ingredients should contain all the nutrients that the
animals require, they may be variable in quantity so synthetic
supplements of vitamins, minerals and specific amino acids are
added.
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Table to show the Average Amounts of Dry Diet and Water
that you might expect to offer a Fit, Adult Male per Day
DIET (g)
WATER (cm
3
)
Mouse
5
6
Rat
15
35
Syrian Hamster
10
8
Guinea Pig (800g)
40
100
Rabbit (3kg)
150
500
Ferret
65
45
Cat (3kg)
200
500
Dog (12kg)
400
1500
Macaque (per kg body weight)
50
75
Note: The above figures are given as a guide only. Many factors influence the
amount an animal eats and drinks. Some animals play with food and water, which
is wasteful, and can lead to them running out so they must be checked and
topped up regularly. It is essential to keep a close watch on the body condition of
each animal to ensure it is getting an adequate diet.
Diet Quality
Diet quality depends on the ingredients used, the manufacturing
process and the conditions the diet is kept in between manufacture
and being eaten by the animal.
Diet that has been properly manufactured, cooled and packaged
may still be contaminated during manufacturers’ storage, delivery
and in diet storerooms and bins on user’s premises. Prior to use,
diet should be kept in cool, dark, dry, well-ventilated conditions that
are insect and vermin-proofed in order to protect it from chemical
deterioration, spoilage and contamination by organisms.
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Chemical Deterioration
Natural interactions between the chemical components of the
foodstuffs and oxygen occur at a rate that depends largely on the
temperature. An ambient temperature of less than 15
o
C will reduce
this problem to an acceptable level. Sunlight causes a rise in
temperature and increases the rate of oxidation of the food.
Vitamins and fats are particularly susceptible to deterioration. Fats
become rancid causing a reduction in the palatability and nutritional
value of the diet.
Spoilage and Contamination by Organisms
Temperatures above 15
o
C increase the metabolic and proliferation
rates of organisms that may spoil the food. All such organisms
require a source of water and their activities are limited by low
relative humidity. Both temperature and humidity are limited by
adequate ventilation. Dark conditions tend to discourage many of
the flying insects. Details of some of the organisms that cause the
deterioration of diet are given later in this section.
If spoilt or contaminated food is fed to animals, there is a danger
that it will no longer meet their nutritional requirements and
contamination from outside sources could give rise to poisoning or
infectious diseases.
Storage of Diet
Diet stores should be cool, dry, dark, well ventilated, insect and
vermin-proofed and easily cleaned.
The diet should be stacked on pallets or duck boards and away from
walls to ensure good circulation of air and to avoid condensation
(Fig. 7.5). Walls of storerooms should be free from cracks and
crevices and should not be penetrated by pipework so there are no
places for insects to hide. Un-insulated hot water or steam pipes
should not pass through diet stores. Rodent barriers should be fitted
to doors and fly screens should be fitted to openings. The use of
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insect electrocutors will help to reduce flying insects (Fig. 7.6).
Fig. 7.5 Diet stored on a pallet
Fig. 7.6 Electrical insect killer
Food Store Management
Food stores should contain only food. Pelleted diet and fresh foods
should be stored separately.
On receipt, all bags should be checked and any showing signs of
dampness, damage or discoloration should be rejected. Open bags
should not be kept in the diet store. Care should be taken when
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handling diet bags within the store so that the risk of damage to
bags and the diet within them is minimised. Any spillage should be
cleaned up immediately. Diet should be stacked to facilitate use in
strict rotation, i.e. oldest diet first. Diet must be used before its
expiry date.
The amount of diet taken from the store should be strictly limited to
the immediate requirements as the warmer temperatures in the
animal room speeds up the deterioration of the diet. Diet bins or
other food containers should be routinely emptied and cleaned. Bins
and feeding hoppers should not be continually topped up as the diet
at the bottom may remain for long periods and deteriorate. Empty
diet bags should be removed from the area as the food dust they
contain can attract food pests.
Food stores should be regularly cleaned and inspected for signs of
infestations.
Organisms Causing Deterioration of Diet
Although detail of the life history of food pests is included here for
interest, the most important requirement is to be able to recognise
the signs of infestation.
The species described below represent a small sample of the pests
that could be found in a food store.
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Moulds
Moulds are a type of fungi. The spores of moulds are ubiquitous.
They grow quickly in warm, moist conditions (Fig. 7.7). Many types
may affect foodstuffs, e.g. pin mould and mildews. The presence of
moulds can be seen clearly if they are well established. Even if they
cannot be seen they may be recognised by a musty smell. If diet is
stored in dry conditions from manufacture to use, moulds should
not be a problem.
Most moulds not only cause food spoilage but some may also cause
allergic respiratory disease in animals and humans if inhaled.
Fig 7.7 Life cycle of typical mould
Clockwise from top: a) growing mould with hyphae growing into the food
and aerial hyphae bearing spores; b) developing spores; c) ripe spores;
d) spores being released; e) released spores germinating.
Fig 7.8 Flour mite
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Arachnids
The only arachnids that infest foodstuffs are mites one example of
which is the flour mite.
Flour Mite
Adult females are 0.5mm long whilst the males are slightly smaller
at 0.4mm (Fig. 7.8). The body is translucent, white and sparsely
covered with hair; the legs are pale violet. The adult mites have 4
pairs of legs but the larvae have only 3 pairs.
Mass infestation by flour mites only occurs in damp conditions, but
the damp need only be in a small area. The female lays about 20
eggs that hatch into white larvae about 0.15mm in length. These
six-legged larvae then pass through two eight-legged nymph stages
before becoming adults.
As the adults are so small it is difficult to detect them in the diet. In
the case of a severe infestation the mites will appear as a moving
layer of dust. Infestation by mites leads to damage caused by their
feeding, produces a bad smell and causes rapid deterioration of the
foodstuffs.
Insects
Many insect pests may infest or contaminate foods, including flies,
cockroaches, crickets, moths, silverfish, beetles and weevils. Insects
do not necessarily harm the nutritional value of the diet but they
are vectors of pathogens and present a risk to the health of the
animals.
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Flour Moth
The body of the adult moth is approximately l3mm long with a
wingspan of 20 to 25mm (Fig. 7.9, 7.10). The forewings are blue-
grey with dark wavy transverse bars and a row of dark spots at the
tip. The hind wings are pale grey. During the day the moths usually
cling to ceilings and walls and they usually fly about at dusk. Mating
takes place immediately after the adults emerge from the cocoon
and up to 350 eggs are laid during the first 4 days. The eggs are
laid in a sticky secretion in or near the diet and hatch 3 to 17 days
later. The caterpillars are whitish with a reddish-brown head. A
silken thread is produced from a gland near the mouth and trails of
this thread are left behind in the diet giving the appearance of
webbing. After 3 to 5 moults the caterpillars are full-grown and 15
to l0mm long. They eventually find a corner or crevice in which they
spin cocoons and pupate for 7 to 16 days. These moths overwinter
as caterpillars but, in contrast to other species, usually remain in
the foodstuff.
As with most moths, it is the caterpillars that do the damage. The
food is contaminated with excreta and silk thread.
Fig 7.9 Flour moth Fig 7.10 Flour moth with closed wings
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Grain Weevil
The adults are 3 to 5mm long and dark