Lab.

animals

Presented by Dr. Kush Pathak

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

 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. mice, rats, birds, Number of Invertebrates 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.

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.

History
Earliest animal testing are found in the writings of the Greeks in the 2nd and 4th centuries B.C. Aristotle (384322 B.C) and Erasistratus (304258 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 8 method of testing surgical procedures before applying them to human patients.

 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. 11

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 transparent.

The worm's body is completely

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 19

Specific pathogen free (SPF)

1. Animals that are particular pathogens. guaranteed free of

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. b. c. d. e. f. Out bred Inbred Hybrid Mutant Transgenic 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 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 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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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 Mice Weight (grams) <10 1010-15 1515-25 >25 <100 100100-200 200200-300 300300-400 400400-500 >500 Floor area/animal (inches) 6.0 8.0 12.0 15.0 17.0 23.0 29.0 40.0 60.0 70.0 Height (inches) 5 5 5 5 7 7 7 7 7 7
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Rats

Animal

Weight (grams)

Floor area/animal (inch2)

Height (inches)

Hamsters

<60 60-80 80-100 >100 350 >350

10.0 13.0 16.0 19.0 60.0 101.0

6 6 6 6 7 7

Guinea pig

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Animal

Weight (kg)

Floor area/animal (ft2) 1.5 3.0 4.0 5.0 3.0 4.0 8.0 12.1 24.0

Height (inches) 14 14 14 14 24 24

Rabbits

<2 2-4 4-5.4 >5.4 4 >4 <15 1515-30 >30

Cats

Dogs

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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 nonhuman primates.

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ANIMAL SPECIFICATION
Normal Special
a. b. c. d. Pregnancy, Suckling, Old, 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 : Humidity : Ventilation : LightLight-dark circles : 25 25C 2C throughout the year 4545-55% throughout the year 1616-20 fresh air changes/hour 12 hours light and 12 hours dark or 14 hours light and 10 hours dark. 300300-400 lux one metre above the floor level Fluorescent tube lights fixed in such a way to facilitate uniform distribution of light. Should be less than 65 decibels (should avoid all high frequency sound)
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LightLight-intensity : Light source :

Noise level :

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 Roasted Bengal gram flour Skim milk powder Casein Refined groundnut oil Salt mixture Vitamin mixture 22.5% 60.0% 5.0% 4.0% 4.0% 4.0% 0.5%

Scale of Diet Rat Mouse Hamster Monkey Rabbit Guinea pig 1515-20 g/day 5 g/day 15 g/day 150 g/day 5050-100 g/day 25 g/day

Diet Composition for Monkeys, Rabbit & Guinea pigs Wheat flour Roasted Bengal gram Casein Refined groundnut oil Salt mixture Vitamin mixture Vitamin C 61.3% 28.2% 1.0% 5.0% 4.0% 0.5% 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

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Hanging cage

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Metabolic cage

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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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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.
Feeder chamber

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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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. chip,
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E.g. Wood shaving, corncob, paper

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

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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 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.
75% alcohol cotton ball 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 study evolution and genetics. to

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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 suffer animal species that naturally 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 83 implanting pig insulin secreting cells into diabetics did reduce these people's need for

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, or subcutaneously. intramuscularly,

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. 85

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 intravenously, when introduced orally, 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 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 and regulations, policies and guidelines, and applicable institutions policies and procedures concerning animal activities.
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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 of IACUC 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 Procedures
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 Necropsy Physical Plant Upkeep Equipment Maintenance Scientific/Clinical Input on IACUC Review IACUCs 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 husbandry or experimental procedures minimize pain and distress of to
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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=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 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): 6376. Americans for medical progress www.amprogress.org Foundation for biomedical research www.fbresearch.org

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Thank You
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