CPCSEA GUIDELINES FOR LABORATORY ANIMALS:

CONTENTS:
CONTENTS INTRODUCTION OBJECTIVE ANIMAL PROCUREMENT TRANSPORT
OF ANIMALS PERSONNEL & TRAINING LABORATORY ANIMAL HUSBANDRY
ANESTHESIA & EUTHANASIA BREEDING LABORATORY ANIMAL ETHICS
CONCLUSION REFERENCES

INTRODUCTION:

WHY ANIMALS ARE USED IN RESEARCH ?:

There are 4 reasons The principles of anatomy and physiology are true for humans and
animals, especially mammals. Certain strains or breeds of animals get the same diseases or
conditions as humans. With animals we can control their environment (temperature,
humidity, etc.), and shield them from diseases or conditions not related to the research
(control their health). We can use scientifically-valid numbers of animals.

BACKGROUND

ANIMAL USE IN RESEARCH:

Several hundred years : animals placed an important role in biomedical research 19 th
century: development of anesthetics opened door to physiological and pharmacological
studies in animals that would not have been possible in conscious animals

Nearly every medical advance was made possible by such studies Note worthy examples 19
th century : breakthroughs that led to the germ theory of disease Early 20 th century :
screening to identify compounds effective as antiepileptic agent 1950s: development of the
polio vaccine

Opposition to use of animals:

Steps to more tightly controlled use of animal subjects were only a beginning

Documented brutal conditions and lack of care by suppliers of dogs to research laboratories
Beginning of increased regulation of animal use in research Within the year , first Animal
welfare Act written and approved

Animal right movements :

Movement gained strength of two philosophers Peter singer and Tom Royan

Within the next year -government approved more guidelines and regulations to reduce risk
that privilege of working with animal subjects would be abused

GUIDELINES:
GUIDELINES MINIMISE PAIN AND DESTRESS-
If privilege to conduct research is to continue then the goal of scientist must be to minimize
the potential pain and distress To accomplish this goal , Russel and Burch proposed three
specific strategies Replacement Reduction Refinement

The three Rs:

Reduction : to obtain comparable levels of information from fewer animals or to obtain more
information from same number of animals. Replacement : preferred use of non animal
methods over animal methods whenever it is possible to achieve the same scientific aim.
Refinement : alleviate or minimize potential pain , suffering and enhance animal welfare for
the animal still used . The three Rs

:
C OMMITTEE FOR THE PURPOSE O F CONTROL AND S UPERVISION ON
EXPERIMENTS ON ANIMALS (CPCSEA) - under the provisions of prevention of cruelty
to Animal act 1960

OBJECTIVES:
To promote the humane care of animals To avoid unnecessary pain before , during and after
experiment To provide guidelines for -Housing ,care ,breeding and maintenance -Sources of
experimental animals -Acceptable experimental procedures for anesthesia and euthanasia

ANIMAL PROCUREMENT:
Acquired lawfully From recognized facilities Transportation Consignment- inspected for
compliance with procurement specifications

QUARANTINE:
Minimizes the introduction of pathogens Minimum duration small animals-1 week large
animals-6 weeks For non human primates- limit exposure of humans to zoonotic infections

Physiological, psychological &nutritional stabilization :

Length of time - duration of transportation -species -intended use of animals

Physical separation Housing different species in separate rooms -to prevent inter species
disease -to eliminate anxiety -to eliminate physiological and behavioral changes Different
species in same room-similar pathogen status and behaviorally compatible

Veterinary care:
Adequate veterinary care should be provided Observed regularly for signs of illness, injury or
abnormal behaviour – reported to ensure timely delivery of of veterinary care

More frequent observations required during -post operative recovery When animals are ill
Have a physical deficit Contagious disease – isolated from healthy animals
TRANSPORT OF LABORATORY ANIMALS:
Mode of transport-road, rail or air Animal density in cages Food & water during transport
Protection from transit infection and injuries

Requirements for transport of laboratory animals:

mouse rat hamster G. pig rabbit cat dog monkey Maximum No. of animals per cage 25 25 25
12 2 1 or 2 1 or 2 1 Materials used on transport box Metal cardboard, synthetic material
Metal cardboard, synthetic material Metal cardboard, synthetic material Metal cardboard,
synthetic material Metal cardboard, synthetic material metal metal Metal/ bamboo/ wood
Space per animal (cm2) 20-25 80-100 80-100 160-100 1000-1200 1400-1500 3000 3000-
4000 Requirements for transport of laboratory animals

PERSONAL HYGEINE:
Protective clothing- Face masks Gloves Lab coats Eyeglasses Bouffant hair caps (microbial
isolators) Regular medical check up Vaccination- against tetanus & other zoonotic diseases

Training:
Handling of animals Personnel hygiene Emergency situations

Animal User Protection:

Health risks associated with animal research include Zoonotic disease Allergies Bites &
Scratches Needle Sticks Chemical or Biohazard exposure Environmental/Laboratory hazards
Exercise CAUTION at all times when in the animal facilities Report any indications of
illness, respiratory problems, or physical trauma (ie. bites or scratches) to the nurse
immediately

LABORATORY ANIMAL HUSBANDRY & MANAGEMENT:

ANIMAL HOUSE:

What is an animal facility ?:

An animal facility is a specialized laboratory exclusively for the purpose of holding &/or
breeding specific animals for scientific purpose

Laboratory animals are very sensitive to their living conditions -housed in an isolated
building Provide natural behavior and social structure of the study species

Environmental enrichment:
Little things can make a great difference Essential for animal health and happiness Can
improve results : ensures natural behavior expressed

Achieved by providing:
Social opportunities sufficient space and facilities, nesting material Nutritional rewards Other
species specific requirements
Social environment:
Group housing – for communal animals In grouping animals consider -population density -
ability to disperse -familiarity among animals Age , sex Group composition – held stable

Animal Husbandry :
Weekend and holiday care for the animals is essential In the event of a disaster (including
loss of power), make sure you are aware of what needs to be done to care for the animals.
Make sure you know all species specific handling procedures Be careful to watch food and
bedding supplies and order in advance. Some bedding materials are ordered on an annual
basis.

PHYSICAL FACILITIES:
Building material –Durable, moisture proof, fire resistant Doors – rust, vermin &dust proof,
rodent barriers Walls & barriers – free of cracks Floors – smooth, non absorbent & skid proof

Corridor – wide enough Exterior window – not recommended for small animal facilities
Floor drains – to prevent high humidity Storage area – designed for feed , bedding , cages and
materials not in use Experimental area - for small animals experimental procedures carried
out in separate area -for large animals separate area for surgical procedures and supportive
treatment

FUNCTIONANAL AREAS:
Animal rooms-50-60% Stores – 8-10% Washing – 8-10% Machine rooms – 4-5% Quarantine
& corridors – 12-15%

TEMPERATURE , HUMIDITY & VENTILATION:

Constant temperature Alteration - change in biological response - fertility and lactation
Ammonia built up Relative humidity- 30%-70% Effective ventilation-10-12 air changes per
hour of 100% air

Lighting:
Time controlled lighting system All animals are to be kept on a 12-14 hour photoperiod
NOISE CONTROL Minimize all disturbances and noise to the animal facility Concrete walls

ANIMAL CAGE:

Designed carefully to facilitate:

Designed carefully to facilitate Animal well being Meet research requirements Minimize
experimental variables

The housing should provide -adequate space -Comfortable environment -An escape proof
enclosure -Easy access to food and water -Meet the biological needs of animals -Keep the
animals dry and clean -Adequate ventilation
Metal-stainless steel, galvanized iron sheets Synthetic- polypropylene, polycarbonate Mice &
rat –shoebox cages Smooth and impervious surface - for easy cleaning Eliminate sharp edges
, & broken wires – to prevent injuries to animals

IDENTIFICATION :
Cage cards- sex, number, principal investigator Temporary identification- pen marks, hair
clipping, dyeing of fur

Outdoor housing -Protection from extremes in temperature or harsh whether conditions -

adequate protective and escape mechanism -sufficient ventilation -prevent building up of
waste materials and excessive moistue -Ground level surface – absorbent bedding , sand ,
gravel or grass

Animal Husbandry Requirements: Bedding:

Bedding Absorbent Free of toxic chemicals Sterilizable Non palatable Ammonia binding
Keep dry and clean

Bedding should remain dry between cage changes Contaminated bedding should not come in
contact with food or water sources Bedding must be changed at appropriate intervals (varies
from daily to weekly) All used bedding and wastes should be disposed of immediately Newly
delivered pups- nesting materials (eg. Tissue paper and cotton)

Animal Husbandry Requirements: Food :

Food All animals must be fed palatable, non-contaminated, nutritionally adequate food daily
Immediately dispose of moldy, insect infested, or otherwise contaminated food Food should
be stored in sealed containers and kept off the floor Animal foods generally have a shelf-life
of 6 months

No drug, hormone or antibiotic should be added Rodents- pelleted natural ingredient diet
composed of cereal grains

Animal Husbandry Requirements: Water:

All animals must have access to potable, uncontaminated drinking water Immediately dispose
of contaminated water When using water bottles, it is better to replace than to refill Watering
devices-examined for proper orientations Water and food levels should be checked daily

SANITATION:
SANITATION Animal rooms, corridors, storage space-cleaned with detergents and
disinfectants Animal waste is removed by hosting Cages should be sanitized before animals
are placed in them Cages , water bottles , stoppers-rinsing at a temp. of 82.2 C or chemical
agents
Extra cages available at all times Rabbit and guinea pig – urine with high conc. Of proteins
and minerals –so treated with acid solution before washing If pathogenic organisms present –
autoclaving or gas sterilization essential Frequency of cage washing and bedding changes –
concentration of ammonia , appearance of cage , condition of bedding - Number and size of
animals housed in the cage

Monitoring of sanitation Done by visual inspection of materials and microbial monitoring .

Waste disposal:
Waste disposal Waste should be removed frequently Waste disposal – incineration Storage of
waste – separated area cold storage – prevent decomposition of biological waste

RECORD KEEPING:
Animal house plans Animal house staff record Health records All SOPs relevant to animals
Breeding, stock, purchase records Minutes of IAEC Meetings Records of experiments Death
record Clinical record of sick animals

SOPs:
SOPs Maintain SOPs regard to animal husbandry , maintenance , breeding , animal house
microbial analysis and experimentation .

DISPOSAL :
Reused- ensured that animal does not experience repeated painful procedures Suffer severe
pain or distress- Euthanized Ensure clinically dead

ANIMAL EXPERIMENTS:
Duration of exp.- Not more than three years Physical restraint - period should be minimum -
provide training Exp. Involving hazardous agents-procedures used reviewed by both IBC and
IAEC

Physical sampling - consider non invasive sampling Invasive method - minimize pain - use
anesthetics or analgesics Repeated blood sampling- implant indwelling catheters

Surgery-under anesthesia -carried out by trained personal Infection-studied in infected

animals Nutritional restriction-below maintenance requirement avoided as possible

:
Selective breeding -need special justification -affect animals integrity -increase mortality rate
Deprivation- severity and duration minimized Previous experience of animal- trained in
advance to reduce fear associated with procedures

LABORATORY ANIMAL ETICS:

Constitution of AEC –for rational use of animals
ETHICAL GUIDELINES FOR LABORATORY ANIMALS:
Advancement of knowledge Appropriate species & quality Minimum no. of animals Treat
with kindness

Invasive methods-under sedation, analgesia or anesthesia Suffer sever pain- euthanized Best
possible living condition & veterinary care Qualified personnel Alternate methods- In vitro
methods

ANAESTHESIA:
Anaesthesia = without sensation an = without, aestos = sensation

For painful procedures that cause pain in humans cause pain in animals Anesthetic agent
generally affect cardiovascular , respiratory & thermoregulatory mechanism in addition to
CNS

Inspection before anaesthesia:

Inspection before anaesthesia Check animals before starting Do not anaesthetise sick animals,
they are unsuitable for experiments anyway Pay attention to symptoms of infections

Preanaesthetic treatments:
Preanaesthetic treatments Pigs , cats , dogs & primates – fasted for 8-12 hours -Minimize
vomiting Rabbits and rodents – do not vomit - Do not require fasting

Access to drinking water Weighed – dosage calculation Pre treatments – anticholinergics ,

tranquilizers , sedatives

Anaesthetic depth:
Anaesthetic depth Mouse respiratory rate, cornea tail pinch and pedal reflex pedal best Rat
respiratory rate, tail pinch pedal reflex and ear pinch ear pinch best

Pedal reflex:
Pedal reflex Tail pinch

Anaesthetic depth:
Guinea-pig palpebral reflex and ear pinch ear pinch best may move 1-2 times, is not
lightening of anaesthesia Rabbit light surgical - pedal reflex medium depth - palpebral reflex
& ear pinch corneal reflex - dangerously deep

Ear pinch:
Ear pinch

Local anesthesia & general anesthesia Local anesthesia – for minor and rapid procedures
General anesthesia-inhalants , im , iv Given for full duration of exp. Side effects- excessive
salivation , convulsions , excitements &disorientation prevented and controlled
Commonly used anesthetic drugs in laboratory animals:
DRUGS (mg/kg) MOUSE RAT HAMSTER G.PIG RABBIT CAT DOG MONKEY
Ketamine HCl (I /m) 22- 24 22-24 - 22-24 22-24 30 30 15-40 Pentobarbitone sodium (i/ v) 35
25 35 30 30 25 20-30 35 Thiopentone sodium (I /v) 25 20 20 20 - - - - Urethane sodium (i/ v)
0.75 1.5 1 1.25 1 1 Commonly used anesthetic drugs in laboratory animals

EUTHANASIA-easy death:
eu = good thanatos = death

PURPOSE:
End of experiment , to provide tissue for scientific purpose Free the animal of pain and
suffering Diseased animal or animals in bad condition Animals unsuitable to breed Too many
animals in a stock

The method should meet the following requirements - Death without causing anxiety , pain &
distress with minimum lag phase - minimum physiogical and psychological disturbance -
Compatible with the purpose of study -minimum emotional effect on operator - location-
separate from animal rooms Larger species - tranquilizers should be administered

Causes of death : 1.hypoxia 2. direct depression of vital neurons heart or respiratory failure 3.
physical destruction of brain activity -brain death • methods: chemical or physical • death
must be confirmed

Approved: CO2 or overdose of anesthetics Not approved: cervical dislocation or other

physical means without anesthesia Do not perform euthanasia, or any other procedure on an
animal until a person experienced with the procedure has trained you, and you feel confident
performing the technique. When possible, two mechanisms of euthanasia should always be
performed on each animal – make sure you verify death Do NOT perform euthanasia in the
presence of other animals

EUTHANASIA OF LABORATORY ANIMALS :

PHYSICAL METHODS - ELECTROCUTION - EXANGUINATION - DECAPITATION -
CERVICAL DISLOCATION

DRUG ADMINISTRATION - BARBITURATE OVER DOSE - CHLORAL HYDRATE

OVER DOSE - KETAMINE OVER DOSE - SODIUM PENTOTHOL OVER DOSE

Barbiturate euthanizing doses:

Barbiturate euthanizing doses Species IV route (mg/kg) IP route (mg/kg) Mouse 150 150 Rat
150 150 Rabbit 100 150 Guinea pig 120 150 Hamster 150 150 Dog 80 80

INHALATION OF GASES - CARBON MONOXIDE - CARBON DIOXIDE - CARBON

DIOXIDE PLUS CHLOROFORM
Species Mouse Rat Hamster G.pig Rabbit dog Physical methods .Electrocution
.Exsanguinations .Decapitation .Cervical dislocation NR A A A NR A A A NR A NR A NR
A NR NR NR A NR NR NR NR NR NR Inhalation of gases Carbon monoxide Carbon
dioxide Carbon dioxide +chloroform A A A A A A A A A A A A A A A A NR NR Drug
administration Barbiturate overdose Chloral hydrate overdose Ketamine overdose Sodium
pentothol overdose A NR A A A NR A A A NR A A A NR A A A A A A A A A A

PowerPoint Presentation:
Methods not acceptable for any species of animals a) Physical methods –decompression -
stunning b) Inhalation of gases – Nitrogen flushing - Argon flushing c) Drug administration –
curariform drugs -nicotine sulphate - magnesium sulphate - potassium chloride

BREEDING:
Breeding stocks – From CPCSEA registered breeders Of known genetic make up and health
status Inbred strain- characters and number of generations must be known

BREEDING TYPES:
BREEDING TYPES Inbred strains -To reduce the number of heterozygotes and to increase
the number of homozygotes F1 hybrid- First generation of cross between two highly inbred
strains Random breeding- Mated together without regarding relationships Selective breeding-
Choosing parents according to characters that is desired to change

BREEDING METHODS:
Hand mating – Rabbit and hamsters Pair mating – Mice and rat Trio mating Harem mating –
Guinea pig

TRANSGENIC/ KNOCK OUT ANIMALS:

Transgenic animals- into whose germ line foreign gene have been engineered Knockout
animals- specific gene have been disrupted leading to loss of function

USES:
To study biological functions of specific gene To develop animal models of diseases To
produce therapeutic products

CONCLUSION:

REFERENCES:
INDIAN JOURNAL OF PHARMACOLOGY 2003 ; 35 :257-274 FUNDAMENTALS OF
EXPERIMENTAL PHARMACOLOGY BY GHOSH, 2 ND EDITION , PAGE NO.1-2
DRUG DISCOVERY AND EVALUATION BY H.GERHARD VOGEL , SECOND
EDITION , PAGE NO:1369-1382 http://www.google.com

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