Lab.

animals

Presented by –
Dr. Kush Pathak 1
 Content
• 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
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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 the last 100 years
(as well as advances in veterinary medicine) has depended on
research with animals. Animal studies have provided the
scientific knowledge that allows health care providers 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 animal
experimentation, animal research, and in vivo testing, is the
use of non-human animals in experiments.

• Number of vertebrate —ranges from the tens of millions to

more than 100 million used annually.

• Number of Invertebrates - 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 between countries and
species; most animals are purpose-bred, while others are
caught in the wild or supplied by dealers.

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 Definition

• "vivisection" means the "cutting up" of a living animal.

• Animal testing, also known as animal

experimentation, animal research, and in vivo testing, is the
use of non-human animals in experiments.

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 History
• Earliest animal testing are found in the writings of
the Greeks in the 2nd and 4th centuries B.C.
 
• Aristotle (384–322 B.C) and Erasistratus (304–258 B.C) were
among the first to perform experiments 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 before applying them to human patients.
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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 of many 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
research
Organs 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 bones that contain marrow and produce blood
cells.

3. Same hormones that aid digestion and regulate the reproductive cycle.

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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 variables to 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 are largely unregulated by law.

ii. Most used invertebrate species are Drosophila melanogaster,

a fruit fly, and Caenorhabditis elegans, 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 quality
I. Health quality

a. Conventional
b. Specific pathogen free (SPF)
c. Germ free

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Conventional - An animal is colonized by
the burden of resident microorganisms which is
normally associated with its particular species.

Germ-free animal –
1. Animals that have no microorganisms living in or on it.

2. Such animals are raised within germ-free 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
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Specific pathogen free (SPF) –
1. Animals that are guaranteed free of 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 bred
b. 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.

Wistar Rat Sprague Dawley Rat Syrian Hamster

Dunkin Hartley New Zealand White

Guinea-pig Rabbit 22
 Inbred animals

• Individuals of a particular species which are nearly identical

to each other in genotype due to long 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 also available

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DBA/2J BALB/cA

C3H/Hen C57Bl/6J
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 Hybrid

BCB6F1 ( BALB/cA + C57BL/6J)

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 Mutant

Nude(BALB/c- nu )

Result from an instance of mutation, which is a base-pair sequence change

within the DNA of a gene or chromosome, resulting in the creation of a new
character or trait not found in the wild type.

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 Transgenic
 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 DNA

technology.

 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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28
 Knockout animals

129 wild type 129 iNOS(knockout gene)

A knockout mouse is a genetically engineered mouse in which researchers have
inactivated, or "knocked out," an existing gene by replacing it or disrupting it with an
artificial piece of DNA. 

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 Weight (grams) Floor area/animal Height
(inches) (inches)

Mice <10 6.0 5

10-15 8.0 5
15-25 12.0 5
>25 15.0 5

Rats <100 17.0 7

100-200 23.0 7
200-300 29.0 7
300-400 40.0 7
400-500 60.0 7
>500 70.0 7 35
Animal Weight (grams) Floor area/animal Height
(inch2) (inches)

Hamsters <60 10.0 6

60-80 13.0 6
80-100 16.0 6
>100 19.0 6

Guinea pig ≤350 60.0 7

>350 101.0 7

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 Animal Weight (kg) Floor area/animal Height
(ft2) (inches)

Rabbits <2 1.5 14

2-4 3.0 14
4-5.4 4.0 14
>5.4 5.0 14

Cats ≤4 3.0 24
>4 4.0 24

Dogs <15 8.0

15-30 12.1
>30 24.0

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Activity
Provision should be made for
animals with specialized loco motor
pattern to express these patterns,
especially when the animals are
held for long 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

Animal room Wash room Research room

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 Environment control –
 Temperature and humidity affect metabolism and behavior.
 Air conditioning is an effective means of regulating these
environmental parameters for laboratory animals.

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/hour appears to provide adequate ventilation
for animal facilities.

 Ventilation also provides an odor free environment.

Illumination
Light can affect the physiology and behavior of 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 of 350-400 lux one metre above the floor level is ideal.
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 Sound

 Separation of human from animal areas is the best way to

minimize disturbances.

 Continuous exposure to acoustical levels above 85 dB can

have 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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 ENVIRONMENTAL CONDITIONS REQUIRED FOR
LABORATORY ANIMALS
Temperature : 25°C± 2°C throughout the year
Humidity : 45-55% throughout the year
Ventilation : 16-20 fresh air changes/hour

Light-dark circles : 12 hours light and 12 hours dark or 14 hours

light and 10 hours dark.

Light-intensity : 300-400 lux one metre above the floor level

Light source : Fluorescent tube lights fixed in such a way

to facilitate uniform distribution of light.

Noise level : Should be less than 65 decibels (should

avoid all high frequency sound)
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food

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Animals should be fed palatable,
non-contaminated, and
nutritionally adequate food daily
unless 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,
Mice & Hamsters)
Wheat flour 22.5%
Roasted Bengal gram flour 60.0%
Skim milk powder 5.0%
Casein 4.0%
Refined groundnut oil 4.0%
Salt mixture 4.0%
Vitamin mixture 0.5%
Diet Composition for Monkeys, Rabbit
Scale of Diet & Guinea pigs
Rat 15-20 g/day Wheat flour 61.3%
Mouse 5 g/day Roasted Bengal gram 28.2%
Hamster 15 g/day Casein 1.0%
Monkey 150 g/day Refined groundnut oil 5.0%
Salt mixture 4.0%
Rabbit 50-100 g/day Vitamin mixture 0.5%
Guinea pig 25 g/day Vitamin C 50mg/100g diet
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Equipment preparation –
a. Cages – shoe box, filter top, IVC, MICE, Hanging, metabolic

Shoe box habitat

Squirrels habitat
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Filter top cage habitat

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IVC - Individually ventilated cages
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MICE – MICROENVIORNMENT – ISOLATION – COTAINMENT - ERICHMENT 51
Hanging cage

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Metabolic cage 53
The metabolic cage main features are:

• Upper chamber - made of PC 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 five
sizes.

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 to

dismantle cage or disturb animal.

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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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58
 Water
Tap, RO, chlorinated, acidified, sterile

Bedding
 Bedding should be absorbent, free
of toxic chemicals or other
substances that could injure animals
or personnel, and of a type not
readily eaten by animals.

 Bedding should be removed and

replaced with fresh materials as
often as necessary to keep the
animals clean and dry.

E.g. Wood shaving, chip, corncob,

paper 59
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

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

General anesthesia needed small amount: 0.1-1 ml

75% alcohol cotton ball for
surface disinfection.

27 G1/2” needle with 1 ml

syringe for blood withdrawal

A vial for blood collection

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Optimal site of blood withdrawal is around the
distal one-third of the tail since this part of tail
gives better visualization of the veins
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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 slowly until
blood get into the dead space of the needle head 70
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 vein
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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.cotton ball
75% alcohol
for surface disinfection.

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 hand
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 Research
Classification –
• Pure research

• Applied Research – Xenotransplantation

• Toxicology Testing – Cosmetic 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.

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• E.g. Studies on embryogenesis and developmental biology
where mutants are created by adding 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 to study 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 diseases or

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.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.

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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 used in 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 insulin-secreting
cells into diabetics did reduce these people's need for insulin

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Toxicology testing – (safety testing)
 Conducted by pharmaceutical companies testing drugs, or by
contract animal testing facilities.

 Tests are conducted without anesthesia, because interactions

between drugs can affect how animals detoxify chemicals,
and may interfere with the results.

 Used to examine finished products such

as pesticides, medications, food additives, packing materials,
and air freshener.

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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 an inhalation
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 to evaluate the

toxicity of a substance by determining the dose required to
kill 50% of the test animal population.

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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
triggered by ultraviolet light) and mutagenicity.

 Cosmetics testing is banned in the Netherlands, Belgium, and

the UK.

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Drug testing –
 Metabolic tests - how drugs are
absorbed, metabolized and excreted by the body when
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 by the 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.

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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
Use Committee)
 Is an institutional body.

 Responsibility for review and oversight of the

institution’s 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 and regulations, policies and
guidelines, and applicable institution’s policies and
procedures concerning animal activities.

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 IACUC Charge
• Review at least once every six months institution’s animal
care facilities, laboratories, use program and areas where
animals are used.

• Prepare and submit reports of IACUC program evaluations

and facility inspections to the IO.

• Review and investigate concerns involving the care and use

of animals in accordance with institution’s 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 Procedures
 
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• Necropsy

• Physical Plant Upkeep

 
• Equipment Maintenance
 
• Scientific/Clinical Input on IACUC Review
 
• IACUC’s “eyes and ears” in the Field

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 Records

OLAW (office of laboratory Animal welfare )

 Annual Report/Renewal of Assurance
 Update program changes
 For-cause site visits
 Will waive not-for-cause site visits if AAALAC accredited

USDA (U.S. Department of Agriculture)

 Annual Report/Numbers of Animals Used
 For-cause site visits
 Not-for-cause on-site inspections 2x/annually

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• AAALAC (Association for assessment & accreditation of
laboratory animal care ) –
 Annual Report
 Update program changes
 On-site visits every 3 years

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 AAALAC (Association for assessment
& 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 of Humane Experimental
Technique
• In 1959, Russell and Burch proposed the concept of the Three
Rs.

 REPLACEMENT refers to methods which avoid or replace the

use of animals in an area where animals would otherwise
have been used.

 REDUCTION refers to any strategy that will result in fewer

animals being used

 REFINEMENT refers to the modification of husbandry or

experimental procedures to minimize pain and distress
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• These Rs are widely accepted ethical principles.

• Now embedded in the conduct of animal-based science in

Canada and throughout many countries in the world.

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 Conclusion

With the use, study & proper care of lab animals &
the knowledge gained through research, we can
continue improving the lives & life standard of not
only humans, but our pets, wildlife & other animals
too.

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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=animal_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

• 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

experimental technique; ALTEX 20 (Suppl 1): 63–76.

• Americans for medical progress – www.amprogress.org

• Foundation for biomedical research – www.fbresearch.org

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Thank You
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