1. 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

2. 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

3. 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

4. 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

5. 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

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

7. Alternative methods

8. 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

9. Microbiologic classification

10. 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...

11. 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

12. 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

13. 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)

14. 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

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

16. 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

17. Laboratory Animal Ethical Use

18. 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

19. 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

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

21. 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

22. Let the rat relaxes on the top of the lid.

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

24. Fold the skirts of towel under the rat from all directions

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

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

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

28. Collection of blood from tail vein in rat
General anesthesia needed
small amount: ml

29. 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

30. 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

31. Disinfect the tail with 75% alcoholic cotton ball

32. 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

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

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

35. Blood can be collected b using a pipetteman after then

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

37. 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

38. Disinfect the left thorax with 75% alcoholic cotton ball

39. Search for the point of maximum heart beat

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

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

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

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

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

45. 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

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

47. CPCSEA GUIDELINES FOR LABORATORY ANIMAL FACILITY

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

49. CPCSEA ;

50. 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.

51. Governing Body
MS. Maneka Gandhi- Chairperson of CPCSEA. It is established under the provision of Prevention of cruelty act of 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.

52. 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.

53. 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.

54. 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.

55. 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.

56. Environment Animal husbandry Social environment
Temperature O C .
Humidity %.
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.

57. 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.

58. 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 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.

59. 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.

60. 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.

61. Three R’s for animal testing

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

65. -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.

66. 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.

67. 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.

68. Mathematical & computer :

69. 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.

70. 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.

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

73. Thank you for caring; thanks to the animals