Local anaesthesia 1.Affects only a specific part of the body2.The animal remains conscious.3.Induce analgesia in the affected part of the body

Application:1.Surface 2.Local infiltration/deep3.Local nerve block/nerve supply4.Regional nerve block

BLS 211 LABORATORY ANIMAL SCIENCE

Prevention/reduction of pain,disconfort, dystress

Examples of local anaesthetics

1. Lidocaine

2. Procaine

3. Bupivacaine

4. Prilocaine

BLS 211 LABORATORY ANIMAL SCIENCE

Analgesia

1.Loss of sensory function (analgesia)

2.Animal remain conscious

3.No relaxation of skeletal muscles

4.Reflex activities not suppressed

BLS 211 LABORATORY ANIMAL SCIENCE

Example of analgesics

Buprenophine Is the most widely used injectable drug to induce general analgesia for a short procedure such as skin biopsy in rodents

Lignocain, a local anaethesia can also induce local analgesia in feline, canine, and other large animals

BLS 211 LABORATORY ANIMAL SCIENCE

CAUTIONWhen anaesthetic or analgesics have to be used it is necessary to consult the person with enough expertise so that

1.Correct anaesthetic for the particular species is used 2.Correct dosage to achieve the required level of anaesthesia and or analgesia.

Anaesthetic overdose can kill

Anaesthetic drugs are dangerous, must be used in accordance with the National laws

BLS 211 LABORATORY ANIMAL SCIENCE

Laws related to laboratory animals

The Animal Welfare Act, 2008 - (Act No. 19/08)Provide for the humane treatment of animals,establishment of the Animal Welfare advisoryCouncil, monitoring and mitigation of animal abuse,promoting awareness on the importance of animalwelfare and other related matters. .

Other laws1. Food, drugs and cosmetics act (2003)2.Veterinary act (2003)3. Animal disease act (2005)

BLS 211 LABORATORY ANIMAL SCIENCE

When laboratory animals are killed for any reason-To end animal suffering, end of exp’ment ….

method used to kill them must 1.be humane2.be painless 3.be reliable4. be economical 5. be safe for the laboratory personnel. 6. be done by a trained person.

Animals should not be killed in presence of others

Humane end point

BLS 211 LABORATORY ANIMAL SCIENCE

Euthenasia

Laboratory animals should not be kept alive after the experiment if pain and distress are likely to be experienced.

when they are diagnosed to have an advanced terminal disease any condition causing suffering to the animal.

BLS 211 LABORATORY ANIMAL SCIENCE

Methods

1.100% CO2

2. overdose of general anaesthesia 3.Decapitation4.cervical dislocation.

Larger animals such as pigs, ruminants, sheep, goats, and horses can be rendered unconscious by captive bolt pistols, and then killed by exsanguinations from a cut through the carotid arteries.

BLS 211 LABORATORY ANIMAL SCIENCE

STANDARDIZATION OF ANIMAL EXPERIMENTS

Defining the properties of any given animal/population and environmentKeeping the properties & environment constant or regulating them

The aim of standardization Reduces variation1.Increase comparability of results between laboratories2.Reduce variation in measured values between identical animals within a given experiment 3.Lower number of animals needed per experiment 4.Increase reproducibility

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Variation in measured values

Can occur in two levels

1.Between apparently identical experiments known as between experiment variation

2.Between apparently identical animals within a given experiment known as within experiment variation

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WITHIN EXPERIMENT VARIATION

Variation between identical animals in a given experiment can be observed in two levels

1. Intrinsic intra-individual variation

2. Intrinsic inter-individual variation

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Intrinsic inter-individual variation Variation caused by contribution of each individual

animal to the measured value

• Intrinsic contribution will differ per animal and will be independent of type of treatment subjected to the animal.

• Inter-individual variation can increase due to analytical errors and ‘’time effects’’ at the time the measurements were made

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Intrinsic inter-individual variationIf inter individual variation (standard variation) is large the precision of Various Results Will be low and the experiment will lack ‘Statistical POWER’’

Statistical POWER : Ability to detect a true treatment effect however small it might be

If statistical power is to remain constant given an increasing inter individual Variation in a measured Value

The number of Animals required per experiment will increase.This have economical, practical and ethical implications

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Intrinsic intra-individual variation

Suppose that body weight is determined daily in a fully grown rat.

• The recorded weight will vary depending on small errors in reading the balance or by rounding number with decimals

• Body weight will also vary depending on whether the animal has recently eaten, drunks, defaecated, or urinated

• Daily weighing will result into small fluctuations which will be in addition to intrinsic real body weight differences between individuals

EXAMPLE

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Intra-individual variations and value of multiple measurements

To minimize the level of intra-individual and analytical variations• making measurements in duplicate or triplicate . • Applying uniform experimental procedures in all animals.

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VARIATION BETWEEN EXPERIMENT Repeating a given experiment with the same or different

group of animals will result in varied group mean of measured values

Treatment effect which refers to differences between the group average for control and test group will vary between experiments

This between experiment variation is subject to two fluctuating components

1. Variation in measurement values between individual animals

2. Differences in experimental conditions

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SOURCES OF VARIATIONS WITHIN AND BETWEEN EXPERIMENTS

• An important source of variations is the animal itself. Differences between animals in one treatment group or between two groups could involve

• differences in sex, age, genotype, microbiological quality, diet, experimental conditions, and differences in analytical procedures

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SUMMARY

Sources of between and within experiment variations

1.Genetic

2.Microbiological

3.Treatment/analytical procedures

4.Environmental conditions

5.Diet

6.Age

7.Sex

8.Weight

9.Spp

10.Husbandry

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MINIMIZING WITHIN AND BETWEEN EXPERIMENT VARIATIONCan be done through:

i.Genetic standardization

ii.Microbiological standardization

iii.Controlling experimental conditions

iv.Uniform analytical procedures

v.Standardized diet

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GENETIC STANDARDIZATION

Genetic variability in animal experiments may be acceptable

In some experiments eg when results are to be extrapolated in various spp: Example in toxicity tests.

In most other experiments requires more uniform animalPopulation. However results should be reproducible in all cases

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• Example: Use of Monozygotic animals • (i) 9-banded armadillo (Dasypus novemcintus) used in

Leprosy experiments

(ii) Genetically identical twins

Usually littermates have different genotypesHowever certain spp produce only genetically uniform offspring (from a single fertilized egg)

Genetic uniformity

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Inbred strain

Strain developed by inbreeding or crossing of closely related animals to increase homozygosity.

Homozygosity increases with increasing generations

An ibred strain can be obtained after 20 (brother x sister) successive breedings

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Co-isogenic strainDiffers from the established inbred strain by only one differentiating gene

Name must consit: Full strain & substrain followed by a hyphen and a symbol of a mutant gene e.g BALB/cRij-nunu

F1 hybrids Resulting from a cross between two inbred strains

All F1 hybrids are genetically uniform

Are heterozygous for all genes for which the two parents differ24

Congenic strainInbred strain in which a genetic trait has been introduced by repeatedly backcrossing up to 10 times

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Consomic strains A chromosome is exchanged by a homologous chromosome of another inbred strain

Recombinant inbred strainProduced b x s mating of individuals from F2 generation of a cross between two unrelated inbred strains progenitor strains Strain A x Strain B

F1 (A x B) F2N=20

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Transgenic animals

carrying additional genes

Existing genes have been altered can be passed to offspring

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The gene is present on part of the bodyChimera

Knock out and Knock in animals

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Nomenclature: An inbred strain is designated by :

A code that consist of 1-4 capital letter (s), example A; WAG; DBA.

Exceptions: names widely accepted before this rule example: C3H; C57BL; 129

READING ASSIGNMENT: Nomenclature Rules for rats & mice at: http://www.informatics.jax.org/mgihome/nomen/index.shtml

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Infections to experimental animalsInterfere with the experimentsZoonoses

Classification of laboratory animals based on microbial status

Conventional animals (CV) Colonization resistance flora (CRF) Gnotobiotic animals Specified Pathogen Free (SPF) animals

Microbiological standardization

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Germ free animals (GF)

Specified Pathogen Free (SPF)

Rederivation

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Quality Barrier system

Conventional (CV) animals Open system

Rederivation

Germ Free (GF) animals Absolute isolator

Intestinal flora

Colonization resistant flora (CRF) Absolute isolator

Specified Pathogen Free (SPF)

Microbiological examination

Classical barrier

SPF animals (Experiment) Modified Classical barrier32

DIET STANDARDIZATIONIdeally a standard diet for laboratory animals does not exist because of :

Continuous changes in ingredients, source and quality of ingredients

Some researches may require specific diets

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DIET STANDARDIZATION

• In most cases researchers tend to avoid specifying the diet and composition of diets in laboratory animals.

• Sometimes they just give trade names

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DIET STANDARDIZATION Practical approach to ensure uniform diet during the experiment: Initially researchers should analyse the diet to establish the

concentration of components which have been identified and these components must be kept constant throughout the experiment

Depending upon the parameter being investigated, the results in

many experiments may not be affected by small changes in case of dietary components

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• Various feeding regimes can be applied to laboratory animals depending on practical and scientific criteria. The most common regimes are:

1.Ad libitum feeding regime2.Meal feeding regime3.Restricted feeding regime4.Paired feeding regime

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Feeding regimes

Ad libitum feeding

Free access to food any time of the day or night (rodents consume most of their food during dark phase of the day)

Meal feeding

Animals are fed during fixed time periods. During this period(s) per day, animals are allowed to consume as much food as they can

Restricted feeding

Limiting food intake or underfeeding to the level that animals will not suffer malnutrition or deficiency signs. This regime can be used to equalize feed intake between different groups or between control and treatment group

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Paired feeding

• Is a specific form of restricted feeding involving measurement of amount of food consumed by animals with depressed intake eg treatment group while giving the same amount to the control

• In this regime, each animal in the treatment group must have a counterpart in the control group. The pair is given same amount of food. The amount given to the treatment group is given to the counterpart in the control group one day later.

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Variations in food intake

• Reduced feed inatake can be due to• Intoxication with the compound under study• Compound under study is not/less palatable• Compound under study induce anorexia The difference in feed intake between the

treatment and control group will imply that the treatment effect is not reliable

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