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Lab. animals
Presented by Dr. Kush Pathak 1
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Introduction
Definition
History
Reasons for use of animals
Animals used in research
Importance of animals in biomedical research
Animal Selection
Facility Preparation Animal care through experiment
Collection from Animals
2
Content
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Research
Important bodies which enforce standards
Records
The Principles of Humane Experimental Technique
Conclusion
References
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Virtually every major medical advance of thelast 100 years (as well as advances in veterinary
medicine) has depended on research with animals.
Animal studies have provided the scientific
kno
wledge that allo
ws health care pro
viders to
improve the quality of life for humans and
animals by preventing and treating diseases and
disorders, and by easing pain and suffering.
Foundation for Biomedical Research,
Understanding the Use of Animals in Biomedical
Research
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INTRODUCTION
Animal testing, also known as animalexperimentation, animal research, and in
vivo testing, is the use of nonhuman animals inexperiments.
Number of vertebrate ranges from the tens ofmillions to more than 100 million used annually.
Number
of I
nvertebrates mice, rats, birds,fish, frogs. estimate of mice and rats used in
the United States alone in 2001 was 80 million.
Most animals are euthanized after being used in
an experiment.
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Sources of laboratory animals vary betweencountries and species; most animals are purpose
bred, while others are caught in the wild orsupplied by dealers.
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Definition
"vivisection" means the "cutting up" of a living
animal.
Animal testing, als
o
kno
wn as animalexperimentation, animal research, and in
vivo testing, is the use of nonhuman animals in
experiments.
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History
Earliest animal testing are found in the writingsof the Greeks in the 2nd and 4th centuries B.C.
Aristotle (384322 B.C) and Erasistratus (304258
B.C) were among the first to performexperiments on living animals.
Galen, a physician in 2nd-century Rome, dissected
pigs and goats, and is known as the "father of
vivisection.
Moorish Spain who also practiced dissection,introduced animal testing as an experimental
method of testing surgical procedures before8
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1946 Post WWII boom in public funding of science.
1950 Chicago Five Animal Care Panel
Louis Pasteur convincingly demonstrated
the germ theory of medicine by
inducing anthrax in sheep.
Ivan Pavlov famously used dogs to
describe classical conditioning. On November 3,
1957, a Russian dog, Laika, became the first ofmany animals to orbit the earth.
1963 Guide for Lab Animal Facilities and Care.
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1966 Lab Animal Welfare Act.
In the 1970s, antibiotic treatments and vaccines
for leprosy were developed using
armadillos, then given to humans.
1973 PHS Policy.
1985 PHS (Public Health Service Policy) Act.
1991 PHS Act Amended.
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Reasons for use of Animals in
researchOrgans and body systems similar to humans and other animals
1. Similar anatomies
2. Even though they may be different sizes and
shapes, humans and animals both have bonesthat contain marrow and produce blood cells.
3. Same hormones that aid digestion and regulate
the reproductive cycle. 11
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Susceptible to same diseases that affect humans
1. Cancers, diabetes, and heart diseases. Eg. dog
whose cardiovascular system closely resembles
that of the human.
2. Many successful medical breakthroughs that
can be attributed to the dog and that are now
commonly applied to humans includes Heart
transplantation, Coronary bypass surgery,
Artificial heart valves, Pacemaker implantation,Angioplasty
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Short life span allows animals to be studied throughout their
entire life
1. Rodents such as rats and mice
Environment easily controllable to keep experimental variablesto minimum
1. Under stress, the research is not reliable.
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Animals used in research
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Invertebrates
i. Although many more invertebrates than
vertebrates are used, these experiments arelargely unregulated by law.
ii. Most used invertebrate species are Drosophila
melanogaster, a fruit fly, and Caenorhabditiselegans, a nematode worm.
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C. elegans The worm's body is completely
transparent.
Studies in the fly D. melanogaster can use an
amazing array of genetic tools
Vertebrates
Mice
Rats
Hamsters
Dogs, cats, rabbits, farm animals, fish, frogs,
birds, nonhuman primates
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Animal Selection
Animal qualityI. Health quality
a. Conventional
b. Specific pathogen free (SPF)
c. Germ free
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Conventional - An animal is colonized bythe burden of resident microorganisms which is
normally ass
ociated with its particular species.
Germ-free animal 1. Animals that have no microorganisms living in or
on it.
2. Such animals are raised within germfree isolators
in order to control their exposure to viral,
bacterial or parasitic agents
3. When known strains of bacteria or micro biota are
introduced to a germ-free animal, it usually
referred to as a gnotobiotic animal.
4. Used in the study of probiotic research 19
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Specific pathogen free (SPF)
1. Animals that are guaranteedf
reeof
particular pathogens.
2. It is always accompanied by a list of the absent
pathogens.
3. Use of SPF animals ensures that specified
diseases do not interfere with an experiment
4. For example, absence of respiratory pathogens
such as influenza is desirable when investigating
a drug's effect on lung function.
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II. Genetic quality
a. Out bredb. Inbred
c. Hybrid
d. Mutant
e. Transgenic
f. Knock out
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Out bred animals
Animals that are generated from breeding two
genetically dissimilar strains of the same species.
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Wistar Rat Syrian HamsterSprague Dawley Rat
Dunkin Hartley
Guinea-pig
New Zealand White
Rabbit
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Inbred animals
Individuals of a particular species which are
nearly identical to each other in genotype due tolong inbreeding.
Frequently used in laboratories.
For some experiments, genetic diversity in the
test population may be desired. Thus out bred
strains of most laboratory animals are alsoavailable
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DBA/2J
C57Bl/6J
BALB/cA
C3H/Hen
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Hybrid
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BCB6F1 ( BALB/cA + C57BL/6J)
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Mutant
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Nude(BALB/c- nu )
Result from an instance ofmutation, which is a base-pair sequence change
within the DNA of a gene orchromosome, resulting in the creation of a new
character ortrait not found in the wild type.
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Transgenic
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Refers to an animal in which there has been a deliberate
modification of the genome (material responsible for inherited
characteristics) - in contrast to spontaneous mutation (FELASA, 1995 -
Federation for Laboratory Animal Science Associations)
Foreign DNA is introduced into the animal, using recombinant DNAtechnology.
Must be transmitted through the germ line so that every cell, including
germ cells, of the animal contain the same modified genetic material.
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Knockout animals
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129 iNOS(knockout gene)129 wild type
A knockout mouse is a genetically engineered mouse in which researchers haveinactivated, or "knocked out," an existing gene by replacing it or disrupting it with an
artificial piece ofDNA.
Causes changes in a mouse's phenotype, which includes appearance, behavior & other
observable physical and biochemical characteristics
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The importance of animals in
biomedical research
Allergies
Arthritis
Asthma
Birth defects Cancer
Tuberculosis
Epilepsy
Heart disease
kidney disease
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Lye disease
Ulcers
Measles Influenza
Hypertension
Glaucoma
Diabetes Bronchitis
Leukemia
Deafness
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HOUSING
It should
Provide adequate space & a comfortable environment.
Provide an escape proof enclosure that confines animals safely.
Provide easy access to food and water.
Provide adequate ventilation.
Keep the animals dry and clean.
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Should be constructed of sturdy, durable
materials and designed to minimize cross
infection between adjoining units.
Attention must be given to
eliminating sharp edges
and broken wires,
keeping cage in good
condition.
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Social environment
The social environment includes all interactions
among individuals of a group or among those able
to communicate.
Population density can affect reproduction,
metabolism, immune responses and behavior.
Should enrich the environment as appropriate to
the species, especially when animals will be held
for long periods.
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SPACE RECOMMENDATIONS FOR
LABORATORY ANIMALS
Animal Animal Weight (grams)Weight (grams) Floor area/animalFloor area/animal
(inches)(inches)
HeightHeight(inches)(inches)
MiceMice
RatsRats
25
500
6.06.0
8.08.012.012.0
15.015.0
17.017.0
23.023.0
29.029.0
40.040.0
60.060.0
70.070.0
55
5555
55
77
77
77
77
77
7735
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Animal Animal Weight (grams)Weight (grams) Floor area/animalFloor area/animal
(inch(inch22
))
HeightHeight
(inches)(inches)
HamstersHamsters
Guinea pigGuinea pig
100
350
>350
10.0
13.0
16.019.0
60.0
101.0
66
66
6666
77
77
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Animal Animal Weight (kg)Weight (kg) Floor area/animalFloor area/animal
(ft(ft22))
HeightHeight(inches)(inches)
RabbitsRabbits
CatsCats
DogsDogs
5.4
44
>4>4
30
1.51.5
3.03.0
4.04.0
5.05.0
3.03.0
4.04.0
8.08.0
12.112.1
24.024.0
1414
1414
1414
1414
2424
2424
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ACTIVITY
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Provision should be made for animals with specialized loco motor
pattern to express these patterns,
especially when the animals are
held forlong periods.
e.g. ropes, bars, and perches are
appropriate for branching non-
human primates.
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ANIMAL SPECIFICATION
Normal
Special a. Pregnancy,
b. Suckling,
c. Old,
d. Animal with some treatment
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Facility Preparation
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Animal room Research roomWash room
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Environment control
Temperature and humidity affect metabolism and behavior.
Air conditioning is an effective means of regulating these
environmentalparameters for laboratory animals.
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VENTILATION
The purpose of ventilation is to supply adequate oxygen;
Remove thermal loads caused by animal respiration,
Lights and equipment;
Dilute gaseous and particulate contaminants;
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16-20 fresh air changes/hourappears to provide adequate ventilation
for animal facilities.
Ventilation also provides an odor free environment.
42
ILLUMINATION
Light can affect thephysiology andbehaviorof various animals.
Lighting should be uniformly diffused throughout animals facilities
and provide sufficient illumination.
Fluorescent tube lights fixed in such a way facilitate uniform
distribution of light.
Light intensity of350-400 lux one metre above the floor level is ideal.
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Separation of human from animal areas is the best way to
minimize disturbances.
Continuous exposure to acoustical levels above 85 dB canhave both auditory and no auditory effects, including
Eosinopenia and increased adrenal weights in rodents.
Reduced fertility in rodents.
Increased blood pressure in nonhuman primates.
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SOUND
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ENVIRONMENTAL CONDITIONS REQUIRED FORENVIRONMENTAL CONDITIONS REQUIRED FOR
LABORATORY ANIMALSLABORATORY ANIMALS
Temperature :Temperature :Humidity :Humidity :
Ventilation :Ventilation :
LightLight--dark circles :dark circles :
LightLight--intensity :intensity :
Light source :Light source :
Noise level :Noise level :
2525CC 22C throughout the yearC throughout the year4545--55% throughout the year55% throughout the year
1616--20 fresh air changes/hour20 fresh air changes/hour
12 hours light and 12 hours dark or 14 hours12 hours light and 12 hours dark or 14 hours
light and 10 hours dark.light and 10 hours dark.
300300--400400 luxlux oneone metremetre above the floor levelabove the floor level
Fluorescent tube lights fixed in such a wayFluorescent tube lights fixed in such a way
to facilitate uniform distribution of light.to facilitate uniform distribution of light.
Should be less than 65 decibels (shouldShould be less than 65 decibels (should
avoid all high frequency sound)avoid all high frequency sound)
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FOOD
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Animals should be fedpalatable,
non-contaminated, and
nutritionally adequate food dailyunless the experimental protocol
requires otherwise.
Diet should be free from heavy
metals (e.g., lead, arsenic, cadmium,
nickel, mercury), naturally
occurring toxins , microbial,chemical and other contaminants.
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Diet Composition for Rodents (Rats,Diet Composition for Rodents (Rats,
Mice & Hamsters)Mice & Hamsters)
Wheat flourWheat flourRoasted Bengal gram flourRoasted Bengal gram flour
Skim milk powderSkim milk powder
CaseinCasein
Refined groundnut oilRefined groundnut oil
Salt mixtureSalt mixture
Vitamin mixtureVitamin mixture
22.5%22.5%60.0%60.0%
5.0%5.0%
4.0%4.0%
4.0%4.0%
4.0%4.0%
0.5%0.5%
Diet Composition for Monkeys, RabbitDiet Composition for Monkeys, Rabbit
& Guinea pigs& Guinea pigs
Wheat flourWheat flour
Roasted Bengal gramRoasted Bengal gram
CaseinCasein
Refined groundnut oilRefined groundnut oil
Salt mixtureSalt mixture
Vitamin mixtureVitamin mixture
Vitamin CVitamin C
61.3%61.3%
28.2%28.2%
1.0%1.0%
5.0%5.0%
4.0%4.0%
0.5%0.5%
50mg/100g diet50mg/100g diet
Scale of DietScale of Diet
RatRat
MouseMouse
HamsterHamster
MonkeyMonkey
RabbitRabbit
Guinea pigGuinea pig
1515--20 g/day20 g/day
5 g/day5 g/day
15 g/day15 g/day
150 g/day150 g/day
5050--100 g/day100 g/day
25 g/day25 g/day
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Equipment preparation a. Cages shoe box, filter top, IVC, MICE,
Hanging, metabolic
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Shoe box habitat
Squirrels habitat
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Filter top cage habitat
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IVC - Individually ventilated cages50
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MI
CE MI
CROENVI
ORNM
ENT ISOLAT
ION COTA
INM
ENT - ERICHM
ENT 51
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Hanging cage
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Metabolic cage 53
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The metabolic cage main features are:
Upper chamber - made ofPC Surface; is smooth, gnaw-proof,
silent.
Two models are available: - for mice and rats up to 300 g, with a
surface of 320 cm2 and a height of 14 cm; - for rats over 300 g, with a
surface of 450 cm2 and a height of 18 cm.
Surface and height are in compliance with the current U.S.A.* rules
and regulations.
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Feeder chamber -
Two-part feeder chamber is located outside cage. Front chamber
catches spilled food so faeces won't be contaminated. Feeder sizes
prevent rodent from nesting or sleeping inside. Available in fivesizes.
Drawer slides out of feeder chamber for easy filling , without
disturbing animal.
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Collection funnel and separating cone - Calibrated to accurately
measure intake. Drain diverts overflow into collection tube so
water can't contaminate urine.
Urine flows along the inside surface of the collection funnel and is
directed by the urine ring directly into the urine collection tube.
A simple twist unlocks either faeces or urine tube. No need todismantle cage or disturb animal.
S t id SS id l t t th h th id l
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Support grid - SS grid lets excreta; pass through the widely
spaced bars; mouse cage includes mouse-sized grid. Handles
permit easy transport of cage without stand.
Water Bottle - Calibrated PC bottle with SS cap and sipper tube.
Water Spillage Tube - Collects any water that animals
spills. Facilitates accurate collection of fluid intake data.
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W t
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Water
Tap, RO, chlorinated, acidified, sterile
Bedding
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Bedding should be absorbent, free
of toxic chemicals or other
substances that could injure animals
or personnel, and of a type notreadily eaten by animals.
Bedding should be removed and
replaced with fresh materials as
often as necessary to keep theanimals clean and dry.
E.g. Wood shaving, chip,
corncob, paper
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Hygiene control 1. Personal
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2. Equipment
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3 Cleaning schedule
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Animal care through experiment
Animal acceptation
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Decontamination Identification
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Animal quarantine
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Animal during experiment
Stress
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Animal after experiment -
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Collection from Animals
Collection of blood from tail vein in rat
67
General anesthesia needed small amount: 0.11 ml
75% alcohol cotton ball
for surface
disinfection.
27 G1/2 needle with 1
ml syringe for blood
withdrawal
A vial for blood
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Optimal site of blood withdrawal is
around the distal onethird of the tail
since this part of tail gives better
visualizatio
nof
the veins 68
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Disinfect the tail with 75% alcoholic
cotton ball
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When the needle penetrates the epithelium of
the tail, pull back the plugger a bit to create
negative pressure inside the syringe, then
push the needle in the vein slo
wly until bloo
det into the ea s ace of the nee le hea70
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Pull back the plugger by the ring finger to
withdraw blood from the tail vein
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Using a scalpel to make a small wound on the
tail is also an option for collecting blood
from tail vein72
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Blood can be collected b using a pipette,
after then
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Collection of Blood from Cardiac puncture in Rat
General anesthesia needed in large amount: up to
3% of body weight.
74
75% alcohol cotton
ball for surfacedisinfection.
24G needle with
10cc syringe for
blood withdrawal.
15 cc centrifuge
tube for blood
collection
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Disinfect the left thorax with 75% alcoholic
cotton ball
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Search for the point of maximum heart
beat
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Insert the needle straight on the
selected point and withdraw blood by
your left hand77
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Research
Classification Pure research
Applied Research Xenotransplantation
Toxicology TestingCosmetic testing
Drug Testing
Education, Breeding & Defense
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Pure Research (Basic research)
a. Investigates how organisms behave, develop,and function.
b. Uses larger numbers and a greater variety of
animals than applied research Fruit flies,nematode worms, mice and rats together
account for the vast majority, though small
numbers of other species are used, ranging
from sea slugs to armadillos.
c. By this, scientists aim to understand both how
organisms normally develop, and what can go
wrong in this process.79
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E.g. Studies on embryogenesis and developmental biology
where mutants are created byadding transposons into their genomes, or
specific genes are deleted by gene targeting.
Experiments into behavior, to understand how
organisms detect and interact with each other
and their environment (fruit flies, worms, mice,
and rats).
Breeding experiments tostudy evolution and genetics.
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Applied Research a. Applied research aims to solve specific and
practical problems.
b. They are usually carried out in the pharmaceutical
industry, or by universities in commercial
partnerships.
c. May involve the use of animal models of diseasesor conditions, which are often discovered or
generated by pure research programs.
d. Such applied studies may be an early stage in
the drug discovery process.
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E
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E.g..
Genetic modification of animals to study disease.
Transgenic animals have specific genes inserted,
modified or removed, to mimic specific
conditions such as single gene disorder, such
as Huntington's disease
Studies on models of naturally occurring disease
and condition.
Cats are used as a model to develop
immunodeficiency virus vaccines and to
study leukemia because of their natural
predisposition to FELINE LEUKEMIA VIRUS.82
A dill d h l f
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Armadillos and humans are among only a few
animal species that naturally suffer
from leprosy; as the bacteria responsible for
this disease cannot yet be grown in culture,armadillos are the primary source of bacilli usedin leprosy vaccines.
Xenotransplantation
Involves transplanting tissues or organs from
one species to another, as a way to overcome the
shortage of human organs for use in organ
transplants.
Although transplant rejection remains a
problem, recent clinical trials that involved
implanting pig insulinsecreting cells into83
T (
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Toxicology testing (safety testing)
Conducted by pharmaceutical companies testingdrugs, or by contract animal testing facilities.
Tests are conducted without anesthesia,
because interactions between drugs can affecthow animals detoxify chemicals, and may
interfere with the results.
Used t
o
examinef
inished pro
ducts suchas pesticides, medications, food additives,
packing materials, and air freshener.
84
The substances are applied to the skin or dripped
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The substances are applied to the skin or dripped
into the eyes.
Injected intravenously, intramuscularly,
or subcutaneously.
Inhaled either by placing a mask over the animals
and restraining them, or by placing them in aninhalation chamber; or administered orally,
through a tube into the stomach, or simply in
the animal's food.
E.g. The LD50 ("Lethal Dose 50%") test is used toevaluate the toxicity of a substance by
determining the dose required to kill 50% of the
test animal population.85
C
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Cosmetics testing
These are particularly controversial
Tests, which are still conducted in the U.S.,
involve general toxicity, eye and skin irritancy,
photo toxicity (toxicity triggeredby ultraviolet light) and mutagenicity.
Cosmetics testing is banned in the Netherlands,
Belgium, and theU
K.
86
D
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Drug testing Metabolic tests how drugs are
absorbed, metabolized and excreted by the bodywhen introduced orally, intravenously,
intraperitoneally, intramuscularly,
or transdermally.
Toxicology tests, which gauge acute, sub-acute,
and chronic toxicity.
Efficacy studies, which test whether
experimental drugs work by inducing the
appropriate illness in animals.
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Education, breeding & defense Animals are also used for education and training;
are bred for use in laboratories; and are used bythe military to develop weapons, vaccines,
battlefield surgical techniques, and defensive
clothing.
Pigs to study the effects of improvised
explosive device explosions on internal organs,
especially the brain.
88
Important bodies
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Important bodies
IACUC
USDA
OLAW
AAALAC
Institutional policy
Attending veterinarian / Institutional Animal
care personnel
State statues & local ordinances
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IACUC (Institutional Animal Care and
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Use Committee)
Is an institutional body.
Responsibility for review and oversight of
the institutions program for the humane care
and use of animals.
Supports, facilitates, and promotes ethical
and humane use of animals by upholding the
standards set forth in the federal statutes andregulations, policies and guidelines, and
applicable institutions policies and procedures
concerning animal activities.
90
IACUC Charge
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IACUC Charge
Review at least once every six months
institutions animal care facilities, laboratories,use program and areas where animals are used.
Prepare and submit reports ofIACUC program
evaluations and facility inspections to the IO.
Review and investigate concerns involving the
care and use of animals in accordance with
institutions Policy.
Suspend animal activities that are not being
conducted in accordance with applicable federal
regulations and guidelines.
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Animal care duties
Housing
Daily Health Checks
Pathogen Control
Feed, Bedding
Transport
Monitoring of Surgery/Other Procedures92
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Necropsy
Physical Plant Upkeep
Equipment Maintenance
Scientific/Clinical Input on IACUC Review
IACUCs eyes and ears in the Field
93
Records
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Records
OLAW (office oflaboratory Animalwelfare )
Annual Report/Renewal of Assurance
Update program changes
Forcause site visits
Will waive notforcause site visits if AAALAC
accredited
U
SDA(U.S. DepartmentofAgriculture)
Annual Report/Numbers of Animals Used
Forcause site visits
Notforcause onsite inspections 2x/annually
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AAALAC(Associationfor assessment & accreditation of
laboratory animalcare )
Annual Report
Update program changes
Onsite visits every 3 years
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AAALAC (Association for assessment
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(
& accreditation of laboratory animal
care )
Is a private, nonprofit organization.
Promotes the humane treatment of animals in
science through a voluntary accreditation
program.
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Functions
Evaluate all aspects of an animal care and use
program.
Evaluate use of an animal in research, education,
testing or breeding.
Basic components of a program that are
evaluated include IACUC policies, animal
husbandry, veterinary care.
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The Principles ofHumane Experimental
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Technique
In 1959, Russell and Burch proposed the conceptof the Three Rs.
REPLACEMENT refers to methods which avoid or
replace the use of animals in an area whereanimals would otherwise have been used.
REDUCTION refers to any strategy that will
result inf
ewer animals being used
REFINEMENT refers to the modification ofhusbandry or experimental procedures to
minimize pain and distress 98
These Rs are widely accepted ethical principles.
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These Rs are widely accepted ethical principles.
Now embedded in the conduct of animalbased
science in Canada and throughout many
countries in the world.
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Conclusion
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Conclusion
With the use, study & proper care of lab animals &
the knowledge gained throu gh research, we can
continue improving the lives & life standard ofnot
only humans, but our pets, wildlife & other animalstoo.
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References
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References
http://en.wikipedia.org/wiki/Animal_testing#Definitions
www. iacuc.org; ALAT Training Manual, page 10
www.fbresearch.org/education/laws.htm
http://www.usda.gov/wps/portal/usda/usdahome?navid=anima
l_production&navtype=RT&parentnav=agriculture
The humane care and treatment of laboratory
animals National Association of Biomedical Research.
Retrieved February 8, 2008.
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www CanadiancouncilofAnimal care com
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www.CanadiancouncilofAnimalcare.com
Ramaswamy et al. ; The Ethics of research involving animals;Nuffield Council on Bioethics; (27)2008: 46 49
W. m. s Russel & R. L. Burch; The principals of humane
experimentaltechnique; ALTEX20 (Suppl1): 6376.
Americans for medical progress www.amprogress.org
Foundation for biomedicalresearch www.fbresearch.org
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Thank You