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Unit
4: Research
Techniques and Considerations
 Reading
Recommendations: In-text links.
Study
Guide
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I The 4 Rs- Alternatives to
the Use of Animals
"...the humanest
possible treatment of experimental animals, far from being an obstacle
[to research], is actually a prerequisite for successful animal
experiments."
-WMS Russell and RL Burch
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Introduction:
Since the earliest
existence of man, we have competed with and used animals.
Animals vied for the same nuts and berries primitive people needed to
eat, dead animals themselves were eaten, and animals were domesticated
for a myriad of purposes. Early man utilized animals like they
utilized their tools--as a necessary part of life, without judgment of
it as right or wrong. Often, animals were worshiped and thanked
for their sacrifice to feed people.
With the development of
civilization, man began to contemplate his relationship to others,
including animals. The advances in biological knowledge
demonstrated many similarities between humans and other animals.
All vertebrates share the same basic nervous system, and it can be
assumed that stimuli that cause pain or distress in humans will cause
pain and distress in animals. Animals with "human"
attributes, such as complex social relationships and the ability to
learn difficult tasks, including sign language, are often termed
"higher" animals, as contrasted with simpler
"lower" animals, such as frogs.
As knowledge of our
relationships with animals grew, so did concern over the ethics of
animal use. Is it right or wrong for us to use animals in
experiments? If it's right for some experiments, which research is
unacceptable? What is our responsibility toward the welfare of the
animals that we use in research, for food production or as pets?
Do we, as a "higher" animal, have an obligation to protect
less "advanced" species? When the needs of people and
animals conflict, which takes precedence?
The research community,
in part impelled by the animal rights and animal welfare organizations,
believe that people not only have the right to use animals in research,
but they have the responsibility to use them wisely, humanely and only
after thoughtful consideration and rejection of all possible
alternatives. No animal use is preferred over animal use if
an alternative is available. Research is required to use animals less
and better.
The Animal Welfare Act
amendment of 1985 (Improved Standards for Laboratory Animals) requires
a researcher to provide written assurances that he or she considered
alternative techniques for painful or stressful procedures and that
alternatives to the use of animals for the research protocol have been
evaluated. Additionally,
regulations by funding agencies, such as the Public Health Service
(Health Research Extension Act of 1985) and National Institutes of
Health (Revitalization Act of 1993) also require consideration of
alternatives.
Re-evaluation of the relationship of the researcher to his animal
subjects began in 1959, when William Russell and Rex Burch published a
study of man's relationship to research animals called The Principles
of Humane Experimental Technique. In it, they defined three
criteria, replacement, reduction and refinement, as
essential to proper use of animals in biomedical research. These
three Rs have become the cornerstone of the concept of alternatives--the
humane use of fewer and "lower" animals.
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- Replacement:
Replacement means replacing "higher" animals with something
else. This includes "lower" vertebrate animals,
invertebrate animals,
nonanimal living systems and nonliving systems.
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The higher vertebrates of research are usually considered to be nonhuman
primates and carnivores, such as dogs and cats. We have invested
these animals with the greatest moral value. Lower vertebrates
include the rodents, such as the transgenic mice used for HIV studies
and preserved frogs used for dissection.
The fruit fly (Drosophila
melanogaster) is an invertebrate that has traditionally been used
for genetics and mutagenicity studies. Other invertebrate
replacement animals include cockroaches and sea slugs used for research
in neurobiology. Micro-organisms such as Salmonella typhimurium
are used to screen for carcinogenic chemicals.
Cadavers may be acquired
from the slaughterhouse or purchased for dissection from a commercial
company. These animals, however, carry their own moral dilemmas,
and many students are concerned about the killing of animals to teach
dissection.
In vitro
techniques replace animals with nonanimal living systems. Cell,
organ and tissue cultures can be used instead of animals. These
cultures minimizes nonexperimental variables, and temperature, humidity,
nutrients and pH can be standardized. Cell cultures are used for
the production of monoclonal antibodies, vaccine production, vaccine
potency testing and screening of chemicals for cytopathologic effects.
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Nonliving systems can also be used to replace animals.
Quantitative chemical analyses allow the determination of the potency of
a chemical. Substances with a pH less than 2.5 or greater than
11.5 are considered corrosive, and animal testing (including the Draize
test) need not be performed.
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Serologic testing for immune function has replaced the use of animals in
many diagnostic tests, such as the ELISA pregnancy test (rabbits no
longer need to be euthanized and have their ovaries examined).
Models can be used to
demonstrate procedures, such as stuffed mice for learning
injections. Computer-linked manniquins (crash test dummies) have
replaced live dogs in testing survivability in automobile collisions.
Computer simulations use
mathematical formulae to create "virtual" experiments and can
replace some dissections in teaching.
The primary disadvantage
of replacement is the need to validate any model, to ensure that
it is a suitable replacement for the original animal used. It is
necessary to build a data base to ensure that the research with the
alternative is valid and the results are reliable.
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Reduction:
Reduction is the use of
fewer animals in research. There are a number of ways that this can
be accomplished. Improved experimental design is an important
contributing factor. An adequate literature review will help
prevent unnecessary duplication of experiments and will supply all
available information about other researchers experience with similar
animals and protocols. Databases such as the Model
Organisms for Biomedical Research list animals that have been proven to be
acceptable models for different types of research.
A pilot study using a few animals can be run to detect possible problems
before committing a large number of animals to the protocol. A
researcher, for example, may believe that a fifty-pound Yorkshire pig is
suitable for an experiment, not realizing until after the research was
well underway that the fifty pound pig is rapidly growing and will weigh
five hundred pounds by the time the experiment is completed.
The use of the best quality animals and provision of the best husbandry
and veterinary care possible also contribute to reducing the number of
animals needed. Research results are compromised by nonexperimental
variables in the form of illness and stress in the animals.
Avoiding these complications make the results from each animal more valid.
Statistical analysis can be
conducted to ensure that the number of animals used in a study will
produce valid results. Too few animals may not yield sufficient
information and too many animals wastes time and money.
It may be possible to
utilize animals more effectively within a research facility. One
animal may be used for more than one project (for example, we use the same
dogs in a variety of veterinary technology courses at Medaille College)
and the control animals may be shared by several investigators. Many
facilities have a tissue sharing program that replaces live animals.
Phylogenetic reduction is
the replacement of "higher" vertebrates with the least advanced
species of animal. In addition, genetic engineering may produce
better-suited animals that minimize the total number needed. The
transgenic p53 mouse, for example, is used for carcinogenicity
testing. Because of its increased sensitivity to cancer-causing
chemicals, only half as many p53 mice are required when compared to other
mouse strains, such as the BALB/C.
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- Refinement:
Refinement is the refining
of experimental protocols to decrease the pain or distress experienced by
animals.
Any stress, including that
caused by pain, fear or poor husbandry, complicates interpretation of the
results of an experiment. In addition, exposing an animal to
unnecessary pain and distress is ethically and legally unacceptable in
modern biomedical research.
Identification, prevention
and relief of pain and distress are the critical components of
refinement. These criteria must be addressed in the Animal Care and
Use Plan and be approved by the Institutional Animal Care and Use
Committee. The appropriate use of tranquilizers, analgesics and
anesthetics is essential. Any procedures that produce more than
slight or momentary pain or distress must be justified and reasons for not
relieving pain or distress must be explained in detail.
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It is essential to ensure that the care and
handling of animals on and off study is optimal. Temperature,
humidity and noise must be regulated and monitored. Cages must be cleaned
routinely and food and water must be pure and uncontaminated. The
health of the animals must be monitored and they must be appropriately
conditioned prior to a study. Animal personnel must be
properly trained prior to performing any procedures.
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Decreased invasiveness will minimize the
stress associated with technical procedures. Magnetic resonance
imaging of one animal over a period of time can be used instead of
euthanasia of several animals along a time curve. Vascular access
devices, such as catheters and ports can be used to obtain repeated
samples from an animal with little or no restraint.
Severe endpoints should be
minimized. The end point of an experiment must be clearly defined in
the experimental protocol. Death as an endpoint should be avoided
whenever possible, because it is usually preceded by pain and
distress. The LD-50 (the amount of substance that kills half of the
animals) can be replaced with the HID-50, the hypothermia -inducing dose
for 50% of the animals. Mice, for example, may be humanely
euthanized when their body temperature is less than 35°
C.
Surgery must be performed
aseptically to prevent infection and proper post-surgical care, including
thermoregulation, must be followed.
There are a variety of
research techniques that are recognized for their pain-causing
potential. These include the Draize test, tumor induction, ascites
production of monoclonal antibodies and some methods of blood
collection. Refinement of these procedures must be addressed in the
Animal Care and Use Plan.
The research community,
including APHIS and the
Humane Society of the United
States, are
redefining pain, distress and the responsibilities of animal personnel
to their subjects.
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The 4th R: Responsibility:
Russell and Burch
introduced replacement, reduction and refinement in The Principles of
Humane Experimental Technique. These are not enough,
however: it is essential that the everyone in the research team is responsible
for the welfare and well-being of the animals that they use. The
animal technicians and caretakers, in particular, are the advocates for
the animals. They are the people that work directly with the animals
day-by-day, who know the individual animals in their care and who often
are the first to detect pain and distress in their charges. It is
their responsibility to bring these problems to the attention of the
laboratory animal veterinarian and to the investigators and to ensure that
these problems are addressed.
Responsibility is adoption
of the 3 Rs: it's using fewer animals, it's using them better and
it's using them more humanely. It's knowing why a particular
protocol is being used and strictly adhering to it so that the results are
valid. It's ensuring that the cages are clean and medications are
given on time. It's caring about the animals.
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Researching Alternatives:
One of the requirements of
the Animal Welfare Act amendment of 1985 is knowledge of how to identify
appropriate alternatives. Specifically, the investigator should be
able to use the database available through the Animal
Welfare Information Center (AWIC), part of the USDA's National
Agricultural Library. The AWIC newsletters address a number of
different issues, including methods for researching alternatives.
Some useful articles include:
Several other websites are available that provide readings and resources,
extensive information about available alternatives and how to perform
searches. Several of the most important sites include:
Several agencies validate (approve as valid) alternatives, including:
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