Replacement
Reduction and Refinement:
Increasing
the Humaneness of Animal Experimentation
The
concept of increasing the humaneness
of animal experimentation through
“replacement, reduction, and
refinement” was developed by
W. M. S. Russell and R. L. Burch in
The Principles of Humane Experimental
Technique (Charles C. Thomas, 1959).
Much replacement, reduction and refinement
have taken place in the natural course
of scientific development in the ensuing
years. Nevertheless, the ideas are
still relevant 30 years later and
the principles afford opportunities
for the improvement of well-being
of experimental animals and the quality
of experiments.
Replacement - Humaneness of animal experimentation
can be increased by replacement of
animals with non-sentient material.
Models, videotapes and slide-tape
presentations should be developed
and used in training programs where
the quality of learning does not depend
on animal usage. While an intact animal
may be required to answer some research
questions, tissue culture or other
in vitro techniques, including computer
or mathematical modeling, should be
used when their use will produce equivalent
or better experimental results than
the use of animals.
Reduction - Much attention has been given to
the concept of reducing the numbers
of animals in experimentation. Too
often this defeats the intended purpose
by preventing collection of sufficient
data for meaningful statistical analysis,
thus requiring repetition of the experiment.
A more relevant goal than reduction
of numbers is to optimize the productivity
of the animals used. Ways in which
this can be accomplished include:
avoiding unnecessary repetition of
experiments; use of appropriate experimental
design; control of unwanted animal
and environmental variability, and
sharing of animals and tissues.
A
thorough and thoughtful review of
applicable literature may avoid experimentation
already conducted by others, provide
insight to design better animal experiments,
and help determine the most efficient
animal model to answer a particular
research question. The MU library,
through its collection and the interlibrary
loan system, has access to literature
concerning all aspects of animal experimentation.
The OAR has files and literature searches
of general information on laboratory
animal husbandry and health matters
and on experimental techniques. Specific
information on animal experimentation
and techniques may be sought using
a variety of databases including AGRICOLA;
which is maintained by the National Agricultural Library.
For
optimal results, the design of animal
experiments requires careful attention.
Use of an appropriate sample size
and good experimental design increases
the probability of achieving statistically
significant results with a minimal
animal and resource cost. Control
of unwanted viability also ensures
experimental productivity and efficient
use of the investigator’s resources. The Division
of Biostatistics (phone 882-6376)
is available to assist in experimental
design and sample size selection.
Animal
disease and environmental factors
may contribute significant variability
to animal experiments. While the initial
cost of disease-free animals may be
higher, the long-term benefits usually
outweigh this. It makes little sense
to inject a $20 rabbit carrying Pasteurella
multocida, an organism that can produce
significant disease or death in rabbits,
with an antigen that costs $1,000
to prepare. The most treacherous diseases
are those that are sub-clinical and
which alter biologic responses that
are interpreted as experimental treatment
effects. In shared facilities, failure
of one animal user to insist on healthy
animals may put the experiments of
all investigators at risk.
Environmental
variables such as: ventilation, temperature,
light cycles, relative humidity, noise
levels, ammonia levels, cage type,
and population density, influence
biologic responses of animals to experimental
treatments. Optima are not known for
most of the environmental factors,
therefore making it impossible to
determine in advance the ideal set
point for each. It is possible in
most experiments to assure that environmental
factors are set within limits known
to be suitable for a given species;
to assure that the environment is
the same for all animals in the experiment,
and to monitor and document major
environmental factors. Doing so puts
environmental factors in the controlled
variable category.
Shared
use of animals or tissues can reduce
the number of animals required and
the expense to animal users. Difficulties
include scheduling and assuring compatibility
of use. The OAR has a list of animal
projects pending and in progress for
the Medical School and College of
Veterinary Medicine and can facilitate
arrangements for sharing. The OAR
can arrange to hold animals as donors
for blood or other biologic samples
if there are enough users to cover
the expenses. Organs, tissues, antibodies,
etc., from sources serving many investigators
may be commercially available at less
cost than the purchase of animals
as donors for one investigator’s
use.
Refinement - Refinement, as presented by Russell
and Burch, simply means to reduce
to a minimum the amount of distress
imposed on experimental animals. They
divided animal experiments into stressful
and neutral investigations. Stressful
studies are those in which the mechanisms
of pain and distress, and/or their
autonomic and endocrine sequelae,
are subjects of interest. Neutral
experiments are those in which pain
and distress are not topics of central
interest, and in which the introduction
of pain and distress causes “a
source of confusion, which will find
its ultimate expression in terms of
cost, error and wasted effort.”
Refinement
is the addition or selection of procedures
that reduce pain and distress in a
given experiment. Use of anesthetics,
analgesics, tranquilizers in neutral
experiments, selection of the most
appropriate animal model, the use
of telemetry or tethers in data collection,
and the application of clinical procedures
to determine an endpoint other than
death, all are examples of the addition
of procedures that may reduce pain
and distress.
Specific
Issues in Increasing the Humaneness
of Animal Experiment
Prolonged
Physical Restraint -
Prolonged physical restraint may be
stressful to animals and should be
avoided unless essential to the research
objectives. All physical restraint
for periods longer than one hour should
be specifically justified in the protocol
review form for consideration and
approval by the ACUC.
Convenience alone is not adequate
justification for the use of prolonged
physical restraint.
If
prolonged physical restraint is required,
animals should be trained by gradually
increasing times of restraint until
the required length of time is reached.
The period of restraint must be limited
to the minimum required to accomplish
the research objectives. For comfort
and safety of the animal, certain
kinds of restraint equipment, such
as slings for dogs and chairs for
monkeys, require that the animals
be attended throughout the period
of restraint. When reasonable alternatives
to prolonged restraint is not possible.
Animals
should be removed from restraint devices
for exercise periods as long and as
frequently as possible.
Attention
must be given to the possible development
of lesions or illnesses associated
with restraint, including contusions,
decubital ulcers, dependent edema,
anorexia, and weight loss. If these
or other problems occur, prompt veterinary
care must be provided. If, in the
opinion of the attending veterinarian,
the restraint is threatening the well-being
of the animal, then the animal’s
welfare must take priority over continuation
of the experiment.
Immunization
of Research Animals - There are diverse
opinions on humaneness of techniques
for animal immunization. The following
are considered, by the ACUC,
to be general guidelines for animal
immunization. Documentation on the
need for these procedures should be
provided in review forms.
Complete
Freund’s Adjuvant (CFA) causes
local inflammation and, often, chronic
pain. When draining skin granulomas
form and tissue is sloughed, the antigen-adjuvant
emulsion may be lost. CFA should be used only
in primary injections. It should be
prepared as a 1:1 suspension with
the antigen solution and injected
aseptically at scattered sites. Subcutaneous
or deep intramuscular injections,
rather than intradermal, should be
used. For subcutaneous administration,
the inoculum containing the adjuvant
should be divided into fractions so
that no more than 0.05 ml is injected
per site. Injection sites should be
widely separated to ensure continued
blood supply to adjacent areas of
skin and subcutis. Intramuscular inoculum
should be of a volume no greater than
0.5 ml for rabbits (proportionately
less for smaller species), administered
by a single injection into a deep
muscle mass. Laboratory personnel
using CFA should be cautioned that inadvertent
self-injection of CFA on needle tips
has resulted in painful and chronic
inflammation in humans.
Previously
sensitized animals should not be injected
with CFA because it will cause hypersensitivity
reactions that may be painful. After
a suitable priming period, booster
doses may be given as antigen in Incomplete
Freund’s Adjuvant (IFA) or an aqueous vehicle such as saline.
Ethylene-vinyl
acetate copolymer (EVAC),
the Ribi Adjuvant
System (RAS),
muramyl dipeptide, and liposomes have
proven to be effective alternatives
to CFA for some studies. Their use should
be given serious consideration.
Footpad
injections are not generally acceptable
and must be approved by the ACUC. CFA inoculated
into the footpad produces swelling,
ulceration, and necrosis. This route
should be used only when justification
for scientific reasons is documented.
Adjuvants should only be inoculated
into one foot; on rodents, a hind
foot should be used.
Post-injection
care of animals given antigens after
sensitization should include observation
for signs of anaphylactic shock and
administration of appropriate treatment
if such a reaction occurs. Inflammatory
reactions at injection sites should
be reported to a veterinarian for
examination and treatment if indicated.
Ascites
Production - High
doses of CFA or pristane injected
intraperitoneally to induce peritoneal
fluid production are associated with
weight loss, hunched appearance, and
lethargy. When these clinical signs
are observed, the volume injected
in subsequent animals should be reduced
to the minimum necessary to produce
ascites. The rate of ascites fluid
production is extremely variable.
Animals must be observed daily and
the peritoneal fluid drained as necessary
to prevent excessive accumulation
and resultant pain and distress. The
following general guidelines should
be followed: the ascites fluid should
be collected when the abdomen is approximately
the proportion of a near-term pregnant
animal; collections should be made
using a 21g or smaller gauge needle,
preferably on an anesthetized animal,
and animals should be euthanatized
if their condition begins to deteriorate
(rough hair coats, thin, hunched,
lethargic or have difficulty moving,
etc.). Administration of saline subcutaneously
or intraperitoneally after collection
of ascites may help prevent shock.
REFERENCES:
Guidelines
for Use of Freunds Adjuvant in Laboratory
Animals, NIH (Laboratory Animal Review
Board).
Amyx,
H. Control of Animal Pain and Distress
in Antibody Production and Infectious Disease
Studies. JAm Vet Med Assn 1987; 191:1287.
Bomford,
R. The Comparative Selectivity of
Adjuvants for Humoral and Cell-Mediated
Immunity.
I. Effect on Antibody Response
to Bovine Serum Albumin and Sheep
Red Blood Cells of Freund~s Complete and
Incomplete Adjuvants, Alhydrojel,
Corynebacterium
parvum, Bordete/la pertussis, Muramyl
Dipeptide and Saponin. Clin Exp Immunol
1980;
39:426.
Edelman,
R. Vaccine Adjuvants. Rev Infect Dis
1980; 2:370.
Pearson, C M, Waksman, B H, and Sharp,
J T. Studies of Arthritis and Other
Lesions Induced in Rats by Injection
of Mycobacterial Adjuvant. J Exp Med
1961; 11 3:485.
Tal,
C and Laufer, A. Amyloidosis in Mice
Following Injection with Freund’s
Adjuvant, Its Components Separately and Homologous
Liver-Adjuvant Mixture. Br J Exp Pathol
1960;
41:605.
Niemi,
S V, Fox, J G, Brown, L R, and Layer,
R. Evaluation of Ethylene-Vinyl Acetate
Copolymer as a Non-Inflammatory Alternative
to Freund’s Complete Adjuvant
in Rabbits.
Lab An Sci 1985; 35:609.
Sidgel,
R A and Langer, R S. Controlled Release
of Polypeptides and Other Macromolecules.
Pharm Res 1984; 1:1.
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