Laboratory Animals & their managing Techniques: a key approach for animal screening Dr. S. P. Pattanayak M.Ph.(P.col.), IDCTCR, Ph.D., Post Doc.(HU), PBC Awardee Division of Pharmacology Birla Institute of Technology (BIT), Mesra, Ranchi - 835215

Controversy regarding lab. animals for virtually every medical achievement in 20th and 21st century involved animals in some way each of us is a consumer of some of the many outcomes which came from animal research that humans have obligations to ourselves that they do not have to animals against animal research is cruel and unjustifiable even when providing benefit for humans since they can be seen as yet another of many species inhabiting this world with no superior moral rights we could probably benefit the same way from experiments performed by Nazis on humans all living organisms have the same rights in the nature

History of the use of animals in medicine until first half of 20th century dead animals (mainly domestic or stray animals = dissection) Greeks (Corpus Hippocraticum -400 př. n. l.) Galen of Pergamon renaissance live animals (anaesthesia = functions in vivo) W. Harvey, M. Malpighi, Ch. Darwin, R. Koch, L. Pasteur, F. Banting & G. Best, ... C. Bernard (1867) “Introduction to the experimental medicine” after second half of the 20th century experimental animals on its own specialised breeding of laboratory animals inbreed and outbreed strains development of new strains alternative exp. models from 80th of the 20th cent. transgenic

Research use of animals – role of experiment Evidence-based medicine knowledge/learning  empiricism  science Observation and experiment are tools of understanding observation looking at nature as we find it the more sophisticated understanding the more variables can be looked concurrently we can measure but we can’t reproduce experiment controlling nature and observing how it response to stimuli the more sophisticated understanding the more variables can be controlled concurrently can be reproduced

Use of animals in education we don’t perform experiments to prove already proven but to: understand principles of experimental work be able to critically evaluate experimental results of others event. be able to conduct experiments ourselves in the future

Ethics – “3R” concept Replacement Reduction Refinement use of alternative methods whenever possible Reduction minimal number necessary Refinement treat animals as “humanly” as possible

Alternative methods

Genetics of lab. animals outbred every specimen is genetically unique inbred (=syngenic) breeding between related subjects (over 20-x and more between given sibling pair) in order to increase homogeneity congenic different in solely 1 locus (mutation) recombinant cross-breeding between 2 inbred strains genetically manipulated transgenic knock-out

Microbiologic classification

The most important species of laboratory animals Mouse – most frequently used. Pharmacology, genetics of mammals, virology, models of human diseases (mutant strains, transgenic and knock-out mice) Rat – physiology of cognitive processes, behaviour, models of diabetes Rabbit – serology, insulin quantification, pyrogens quantification, tests of irritable effect of chemical substances on the cornea Cat – study of CNS and respiratory system Dog – e.g. beagle, use in electrophysiology, neurophysiology Guinea-pig – in microbiology and serology, physiology of the auditory system Hamster - genetics Pig – training of surgical techniques, temporary covering of burns with porcine skin Primates – rhesus monkey, baboon, chimpanzee – use in neurology, virology, behaviour Frog – physiology of blood circulation, electrophysiology Fish, molluscs, insects...

GENETICS OF LABORATORY ANIMALS 1. Isogenic = genetically defined strains (isogenicity= genetic uniformity of all individuals) 2. Non-isogenic = genetically undefined strains 3. Genetically semi-defined strains

Isogenic strains - 1 Inbred strains - obtained by close breeding for more than 20 generations (brother + sister or offspring + one of the parents) - homozygosity higher than 98 % (Degree of homozygosity is expressed as a coefficient of inbreeding.) - features: isogenicity, phenotype uniformity (low variability of reactivity), usually low fertility, disposition to diseases - advantages of use in experiment: homogenous statistical set, lower number of individuals is sufficient - disadvantages of use in experiment: a risk, that the findings are strain-specific and are not valid for other strains, problematic generalization of the results Coisogenic strains (mutant strains) - differ from the original strain only in one gene, in which a mutation occurred Congenic strains - strains originated by cross-breeding of two strains and following back-cross- breeding with one of the original strains (at least 10 times, selection of some specific feature) - presence of specific genes of one strain on genetical background of the second strain

Isogenic strains - 2 Recombinant-inbred strains - crossing of 2 strains, the hybrids give origin of new lines which are then crossed brother x sister, what leads to establishing of a new strain Rekombinant-congenic strains - crossing of 2 strains followed with 3 back-crosses to one of the original strains and inbreeding with crossing brother x sister (at least 14 times) Consomic strains - a complete chromosome of one strain is transferred on the background of the second strain with back-crosses (similarly as for individual genes in congenic strains, but the process is more complicated)

Non-isogenic strains Outbred lines - genetically heterogeneous population without crossing with individuals coming from different, in the frame of the population close crossbreeding is avoided so that the coefficient of inbreeding remains as low as possible - features: some level of phenotype variability (higher variability of reactivity), higher fertility and resistance to diseases - advantages of use in experiment: cheaper and easier production, the findings have more general validity - disadvantages of use in experiment: less homogeneous set, higher number of animals is necessary Genetically heterogeneous lines - originate by crossing of several inbred strains followed with breeding according to principles of outbred population Outbred selected lines - in an outbred population given phenotype feature is selected  

Animal models of diseases Mutant animals Transgenic animals Knock-out animals   Nude Mice Nude Mice SCID Mice

GNOTOBIOLOGY OF LABORATORY ANIMALS Conventional animals - undefined microflora - open breeding facility complying basic hygienic conditions SPF animals = specified pathogen free - microflora of the animals certainly does not contain specified pathogens. - barrier breeding facility Gnotobiotic animals - breeding isolators 1. Axenic animals = germ free - without any microbes - pups obtained with sterile hysterectomy or hysterotomy into sterile atmosphere of the isolator 2. Asociated animals - derived from axenic animals colonising them artificially with one or more species of microorganisms - monoxenic, dixenic, polyxenic

Laboratory Animal Ethical Use

Ideas To comply with the Animal Welfare Ordinance and avoid mishandling of animal in research To provide basic concepts of animal handling technique to new animal user

Laboratory Animal Handling Technique - Rat A. Intraperitoneal injection B. Blood collection from tail vein C. Blood collection from cardiac puncture D. Oral feeding E. Sexing

Intraperitoneal Injection in Rat The most common method of administering drugs to rats

Tools for intraperitoneal injection in rat 75% alcohol cotton ball for surface disinfection medium size towel for restraining the rat 25G 5/8” needle with 1cc syringe for injection

Let the rat relaxes on the top of the lid.

Stretch the body of the rat by pulling up it’s tail and then cover the rat with a towel by your left hand

Fold the skirts of towel under the rat from all directions

Grasp up the left hindlimb of the rat to expose the abdomen

The injection site should be in the lower left quadrant of the abdomen because vital organs are absent from this area

Only the tip of the needle should penetrate the abdominal wall to prevent injection into the intestines.

Collection of blood from tail vein in rat General anesthesia needed small amount: 0.1-1 ml

Tools for collection of blood from tail vein 75% alcohol cotton ball for surface disinfection 27G1/2” needle with 1 ml syringe for blood withdrawal a vial for blood collection

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

Disinfect the tail with 75% alcoholic cotton ball

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

Pull back the plugger by the ring finger to withdraw blood from the tail vein

Using a scalpel to make a small wound on the tail is also an option for collecting blood from tail vein

Blood can be collected b using a pipetteman after then

Collection of Blood from Cardiac puncture in Rat General anesthesia needed Large amount: up to 3% of body weight

Tools for collection of blood from cardiac puncture 75% alcohol cotton ball for surface disinfection 24G needle with 10cc syringe for blood withdraw 15 cc centrifuge tube for blood collection

Disinfect the left thorax with 75% alcoholic cotton ball

Search for the point of maximum heart beat

Insert the needle straight on the selected point and withdraw blood by your left hand

Oral Feeding in Rat Feeding amount limited to 1% of body weight

Tools for oral feeding in rat 16 G ball-tipped feeding needle with syring Leather glove

Restrain the shoulders of the rat by your thumb and index finger, then support the lower limbs with your right hand

Restrain the tail of the rat in between your ring finger and little finger

Let the rat lying on your left palm and introduce the ball-tipped feeding needle from the pharynx in to the esophagus when the rat is in the act of swallowing

Sexing Rat- the distance between the anal and genital orifices is greater in the male (left) compared to the female (right)

CPCSEA GUIDELINES FOR LABORATORY ANIMAL FACILITY

Contents ; Need of CPCSEA Governing body of CPCSEA Guidelines of CPCSEA 3 R’s for animal testing Alternative to animal testing

CPCSEA ;

Need of CPCSEA To ensure that lab animals are well maintained and experiments are conducted according to ethical norms. To promote humane care of animals used in biomedical and behavioral research. To enhance animal well being and quality . To improve laboratory animal facility To enhance biological knowledge that is relevant to humans and animals.

Governing Body MS. Maneka Gandhi- Chairperson of CPCSEA. It is established under the provision of Prevention of cruelty act of 1960. The Experiments on animals amendment rules(1998) and Breeding of and experiments on animals(1998) gave powers to the CPCSEA. To make rules in relation to the conduct of experiments on animals. To authorize any of its officers to inspect any place at any time. To prohibit a person or an institute from carrying out experiments on animals.

GUIDELINES Veterinary care Animal procurement Quarantine Daily observation of animals. Reviewing protocols and proposals Establishment of appropriate policies and procedures for animal husbandry. To maintain zoonosis control programmes. Animal procurement Animals are procured from CPCSEA registered breeders. Quarantine Quarantine is the separation of newly received animals from those already in the animal facility. It determines microbial status of newly received animals. Minimizes the chance for introduction of pathogens into an established colony. Duration- small animals – 1 week Large animals- 6 week.

Stabilization and separation Physiological, psychological and nutritional stabilization is needed. Separation is based on species to prevent diseases and transmission. Animal care and technical personnel Animal care programmes should be conducted. People trained in animal science should be appointed. Personal hygiene Showers, changing rooms, footwares. Clothing. Use disposable wear such as gloves ,masks, head covers, coats, shoe covers. Avoid eat, drink and smoke in animal facility.

Animal experimentation involving hazardous agents Procedures and facilities involving hazardous agents should be reviewed by, Institutional Bio-safety Committee. Institutional Animal Ethics Committee(IAEC). Multiple surgical procedures on single animal No animal should be used for experimentation for more than 3 years unless adequate justification is provided. Physical restraint for examination , collection of samples and surgical operations. Accomplished by manually or by devices. Devices should be suitable in size design for the animal being held and operated properly to minimize stress and avoid injury to the human. Period of restraint should be minimum. Less restrictive systems should be used.

Animal facility` Functional areas Separated from personnel areas such as offices and most laboratories. Located as far away from human habitations as possible. Not exposed to dust ,smoke, noise , wild rodents, insects and birds. Sharp fluctuations in temperature, humidity, light, sound and ventilation should be avoided. Functional areas Separate areas for food, bedding, surgery ,treatment, storage, washing. Area for repairing cages and equipment. Specialized laboratories.

Environment Animal husbandry Social environment Temperature - 18-29O C . Humidity- 30-70 %. Ventilation- air conditioning systems. Power and lighting- fluorescent lights, emergency power. Animal husbandry Adequate space, maintenance of body temp ,urination, defecation and reproduction. Keep the animals dry and clean. Polypropylene, polycarbonate and stainless steel cages should be used. Easy for inspection. Social environment Population density can affect reproduction, metabolism, immune responses and behaviour.

Emergency weekend and holiday care Personnel and training Food Palatable, non contaminated and nutritionally adequate. Should contain crude fiber crude protein, essential vitamins, minerals ,fat and carbohydrates for providing appropriate nutrition. Bedding Absorbent, free of toxic chemicals. Change the bedding twice a week. Optimizes normal animal behaviour. Water fresh, potable, uncontaminated. Pest control Pest control programmes. Emergency weekend and holiday care Everyday care is essential Personnel and training Animal facility staff is critical component in the management.

Sanitation and cleanliness Cages should be sanitized before the animal is placed. Cages and water bottles can be disinfected by rinsing at a temperature of 82.20 C or by chemical agents such as hypochlorite to destroy pathogenic microbes. Autoclave or gas sterilizers can be used. Record keeping Animal house plans. Staff record. Standard operating procedures(SOP) Health records. Breeding, stock, purchase and sales records. Death record Records of experiments on animals. Clinical record of sick animals. Waste analysis report.

Guidelines for Use of Anaesthesia Useful for painful procedures. At no stage of experiment the animal is conscious to perceive the pain. If any irrepairable injury occurs , the animal should be sacrificed. Overnight fasting. Use pre-anaesthetics were ever applicable. Local or general anaesthetics may be used. Side effects such as excessive salivation, convulsions, excitation and disorientation.

Euthanasia Painless killing. Animal is required to sacrificed on termination of an experiment or otherwise for ethical reasons, death without causing anxiety, pain with minimum time lag phase. Location should be separated from animal rooms. Tranquillizers should be given for larger animals such as monkeys, dogs, cats before an euthanasia procedure.

Three R’s for animal testing

Use of living system in testing- a)In-vitro system b) non animal organism; -Micro organism -Invertebrates

-Micro-organism; Advantage; ●org -Micro-organism; Advantage; ●org. can cultivated much more Easily & quickly than most animal Or human cell ●genetic make up is simple than Animal or human.

Fungal system used in mutagenecity Fungal system used in mutagenecity. Cilia protozoa respond to smoke or phenols as do the cilia in the human bronchial tube. Various protozoans used in toxicity testing of cigarette smoke, mutagenesis,reproductive toxicity.

4) Use of non living system in testing; a)chemical system- Animal replaced with analytical chemistry. Determine potency, conc. of drug such as vaccines, anti-cancer drug & vitamins.

Mathematical & computer :

Skin and eye irritation; a- chick embryo test; Chick Embryo Chorioallantoic Membrane Assay ; . Fertile eggs are incubated at 37’’ C. A window is cut at the top, and 1.5 to 2 millilitres of albumin is removed with a needle and discarded. The Chorioallantoic membrane forms on the floor of the air space, on top of the embryo. The window is taped.

Day 14. A test sample is placed on the embryonic membrane and contained within a plastic ring. Day 17. The chorioallantoic membrane is evaluated for its response to the test substance, and the embryo is discarded.

Repeated – dose toxicity tests; a) Hepatotoxicity b) Neurotoxicity Mutagenicity & carcinogenicity; Ames test-

Thank you for caring; thanks to the animals