lab animal science Flashcards
Why talk about experimental design and
statistics?
- what are they
- which are the key steps
• What is science all about?
- Measuring stuff and demonstrating causes and
effects (cause → effect)
• What are experimental design & statistics all
about?
- Control of variability
- Isolating causal factors
- Demonstrating interactions between factors
• Which are the key steps in experimental design?
- Start out with a well-defined hypothesis and
research question
- Design the appropriate control group(s)
- Determine appropriate sample size.
- Randomly allocate experimental units to
treatments
- Control and reduce variability through blocking
(stratification) and factorial experiments
statistical power
- sample size, variability, and significance level affect
low power of a statistical analysis
- reduced chance of detecting a true effect
- low power reduces the likelihood that a statistically significant result reflects a true effect
Which factors determine whether our statistical test gives a
significant outcome?
B beta (); or Type-2 error (false negative) probability. (caused by biological variation) Power is calculated as (1-). Typically: 0.10 ≤ ≤ 0.20 E effect size (ES) and effect direction; one- or two-sided testing To be stated by the researcher A alpha (); or Type-1 error (false positive) probability (caused by biological variation) Conventionally: 0.01 ≤ ≤ 0.05 N n; number of experimental units (“number of animals”) To be calculated by the researcher S s; variability in measurements (standard deviation (s, ) or standard error (SE)) To be controlled by the researcher 41 NOTA BENEEE : Any effect size becomes statistically significant...when the sample size is large enough
How can you reduce variability?
Reducing variability by clever experimental design - fully randomized design (Full randomization doesn’t always work well with small
sample sizes as it can lead to segregation of treatments
or factors.)
Randomization and stratification
• If you can (and want to), fix a variable.
- e.g., use only 8-week old male mice from a single
strain.
• If you don’t fix a variable, stratify (block) it.
- e.g., use both 8-week and 12-week old male mice,
and stratify with respect to age.
• If you can neither fix nor stratify a variable,
randomize it.
B·E·A·N·S
̶ Knowing 4 parameter values, you can
calculate the 5th.
60
B – β, Type-2 error probability (1 - β = power)
E – Effect size (ES)
A – α, Type-1 error probability (significance
level)
N – Sample size (n)
S – Standard deviation (measure of
variability)
The bottom line when it comes to sample size
Sample size is proportional to the “signal-to-noise
ratio” in your population
So, sample size n can be reduced by: - Reducing noise, random variation () - Increasing the effect size (ES) • Reduction of noise through: - Standardization of protocols and measurements - Use of inbred strains - Clever experimental design (e.g. paired observations) 61 This illustrates an inverse square relationship between effect size and sample size: “To detect an effect 3 as small, sample size has to increase 32 = 9-fold.” (All else being equal.)
Size does matter! • A sample too small... - ...is difficult to replicate - ...is likely to produce a positive result that is a fluke - ...is a waste of time, money, means, and animals • Conclusions based on large samples are more reliable than those based on small samples. • But... a sample too large... - ...is also a waste of time, money, means and animals • Remember the 3 Rs: Replacement, Reduction, and Refinement. 62 Remember the inverse square relationship between effect size and sample size: “With a sample size 4 as large, you will be able to detect an effect only 4 = 2× as large.” (All else being equal.)
What are good effect sizes or effect size indices?
- Raw difference (delta) between two groups.
- Cohen’s d standardized difference.
- Pearson’s correlation coefficient, r.
- Odds ratio, OR.
- Risk ratio or relative risk, RR.
- Proportion explained variance, (ANOVA)
randomization
- why is it used
- methods
- what can be randomised
- to prevent bias (1) correct selection of experimental units (2) randomise experimental units to treatment (3) randomise order of measurements and animal housing (4) blinding and coding samples
- simple randomisation (paper or computer)
- latin square design (A Latin square is a block design with the arrangement of v Latin letters into a v×v array (a table with v rows and v columns). Latin square designs are often used in experiments where subjects are allocated treatments over a given time period where time is thought to have a major effect on the experimental response)
- blocked randomisation, stratification
- treatment groups, housing, order of treatment
Classification of animal models
Disease models
Induced (experimental) disease models (inducing obesity, tumor growth, renal damage, etc.)
Spontaneous (genetic) disease models (nude mice, hypertensive rat, etc.)
Transgenic disease models (KO, KI (i.e. introducing a crippled version of a GPCR), tissue selective, inducible)
Neutral models
Healthy animals from all species
…… inbred or outbred?
INBRED/OUTBRED
Inbred (isogenic) strains: produced by many (>20) generations of brother x sister mating. Aim:
Genetic homogeneity
The same genotype can be reproduced indefinitely, though over a period of time there may be some genetic drift due to the accumulation of new mutations.
Outbred strains: breeding colonies in which there is (certain) degree of genetic variation. Aim:
Genetically undefined outbred stocks
The amount of genetic variation depends on the breeding history of the particular colony.
inbreds will have a higher statistical reproducibility’ - Someone using an outbred stock generallyknows nothing about the genetic characteristics of individual animals, what genes they carry or how heterozygous they are. Background data on characteristics will be unreliable because they can change rapidly.
More than one strain can be used in a factorial experimental design without increasing the total number of animals
Why use INBRED
On average isogenic strains are more sensitive than outbred stocks to experimental treatments, which also increases the power of experiments which use them.
They are internationally distributed, so that work can be replicated all over the world.
Searchable lists of inbred strains of mice and rats and their characteristics are maintained by, for instance,
The fundamental principle of the controlled experiment is that treated and control groups should be identical, except for the treatment
validity of model
MODEL VALIDATION:
Best possible resemblance between model and research target
face validity
(resemblance in symptoms and signs)
predictive validity defined as the measure of how well a model can be used to predict currently unknown aspects of the disease in humans
(resemblance in reaction to current treatment - the similarity of the relation between, on the one hand, the triggering factors and the occurrence of the disease and, on the other hand, between the therapeutic agent and the disease)
construct validity
(resemblance in origin/mechanism)
what is genetic drift, vulnerability and what to do to prevent gd
constant tendency of genes t evolve even in the absence of selective forces. GD is fueled by spontaneous neural mutations that disappear or become fixed in a population at random
- single base changes, deletions, duplications, inversions in the DNA
- small colonies are more vulnerable to fix a mutation - colonies separated by 20 or more generations - phenotypic or genetic differences are discovered
- prevention: maintain pedigrees lines and detailed colony records
- watch for phenotypic changes in mutants and controls
- refresh breedes frequently (10 generations)
- avoid selection pressure
- verify genetic background with genome scanning
- cryopreserve unique strains
substrains? genetically different from parent colony
infections affect 3 things
quality of research, animal welfare and zoonoses
reccomendations for health monitoring of animal colonies
Agents • Impact on animal health and welfare • Impact on animal experiments • Zoonoses Frequency of testing • Dependent on impact and prevalence of pathogens Sample size • Reliable detection of microorganisms in a population
Mouse Parvovirus (MPV)
Host species • Mouse Prevalence • 1 – 14 % Clinical disease • Inapparent in most situations • Can only replicate in cells undergoing active division • Lymphocytotropic • Embryos/fetuses • GI tract Interference with research • Replicating in cells undergoing active division - Alters the immune system and response • Acute: Virus dispersed within lymph nodes • Persistent: Virus localized in germinal centers • Modulate lymphocyte mitogenic responses - Immunosuppression • Altered/cryptic clinical picture • Increased susceptibility to other infections (opportunistic) - Reduce transplantable tumor take rate - Suppress tumor development • Virally or chemically induced
Helicobacter spp.
Host species • Multiple • Prevalence • 3 – 20 % Most relevant species (mouse/rat) • H. hepaticus • H. bilis Clinical disease • Inapparent in many strains • Chronic proliferative hepatitis • Hepatic tumors (model) • GI-tract tumors (model) • Rectal prolapse • IBD (model)
Interference with research • Colitis in immunodeficient animals • Rectal prolapse in transgenic mice • Immune modulation • Altered hepatic metabolism of some chemical compounds • Impact on oncology studies - Hepatic lesions with karyomegaly and oval cell hyperplasia in A/JCr mice - Can cause hepatocellular carcinomas in B6C3F1 mice - Increased incidence of hepatocellular neoplasms when combined with triethanolamine
why should infections be avoided?
Difficult to predict the magnitude and type of impact –
so infections should be avoided as an uncontrolled variable.
Research is all about controlling variables and their impact on
your results
• The less control you have of variables the higher the variance →
lower statistical power → bigger group sizes (anti-3R)
• Or even worse – unreproducible / wrong results
• Infectious agents may be potent variables
Health monitoring program
• Sensitivity of the program is defined by:
• Sample type
- animal (live animal and samples
- non animal (consumables - eg diet, water bedding enrichment - environment - eg surface swabs, biological indicators, sanitary control )
• Sample size
- Microisolation housing technologies do not allow for easy dissemination of infections in the population • Resulting in lower prevalence compared with open-top caging - The sample should be representative of the population, and the agent/target should be present in the sample if present in the sampled subject • Type of sample • Test method applied • Agent tested for • The sensitivity of sentinels as a representative sample depends on • Agent monitored • Adequacy of procedures for dirty bedding transfer
- Sample collection
- Sample shipment
• Diagnostic test method
DIRECT
The organism is present in
the sample and We find the organism
examples
• Culture (Sample types: Swab (throat, skin, abscess), Feces/caecum,Environmental (swab, water,
food, mold trap)
• Direct microscopy (Primarily parasites, Sample types: Anal and skin tape Caecum and gut contents)
• Necropsy
• PCR (sample type: all , application: amplification of a specific dna-rna sequence, possible for all known agents
INDIRECT • The organism has been present in the subject • We find evidence that the organism has been there
examples
• Serology (IFA
Indirect Fluorescent Antibody test, ELISA Enzyme Linked Immunosorbent Assay, MIA Multiplex Immunofluorescent Assay)
Health standards
• Conventional
- Positive for one or more pathogens normally not accepted
• Specific Pathogen Free (SPF)
- Negative for all specified pathogens
• Specific Opportunistic Pathogen Free (SOPF)
- Negative for all specified pathogens and opportunists
- Breeder-standard for immune-deficient models
• Gnotobiotic/Defined Flora
- Completely known flora (e.g. Altered Schaedler Flora (ASF))
• Germ free/Axenic
- No microorganisms present (sterile)
Controllable barrier components
Ventilation: HEPA filtration
Water: Sterile filtered, UV light, acidification, chlorination
Housing: Animal handling procedures, cleaning procedures - open top cages, individually ventilated cages and isolators
Diet: Radiated, autoclaved
Equipment: Autoclaved, UV light, radiated, Ethylene oxide e
Uncontrollable barrier components
Personnel_Uniform, gowning procedure, pet policy, quarantine system
Animals_ Health status evaluation, quarantine procedure, testing, derivation
Cells_ Microbial evaluation, quarantine procedure, testing (host)
Risk of introducing animals and biologicals
More exchange of mouse models and biologicals than ever
• New transgenic lines are made every day
• Several PDX initiatives
• More collaborations between big pharma, biotech, and academia with
exchange of animal models and biologicals
Prevalence of agents
• Infectious agents are still relatively prevalent
• Regional differences and similarities
Health reports and certificates are historical
• What happened since last testing?
Importing animals - the right way
- Embryo transfer (w/wo IVF)
- Hysterectomy
Barrier principle - summary
Very resource demanding to implement and maintain
• High investment costs
• High maintenance costs
• Reduction in efficiency and flexibility for employees and users
Necessary to ensure the proper “function” of the animals
• Infections can impact and/or invalidate research
• More and more journals require detailed information about health
Consequences of compromized barrier
• Invalid research, loss of time, loss of resources, delays in grants
• Infections results in loss of animals, delays for all users, loss of reputation
Severity categories
Non-recovery (code 1):
Procedures which are performed entirely under general anaesthesia from which the animal shall not recover consciousness shall be classified
as ‘non-recovery’.
Mild (code 2):
Procedures on animals as a result of which the animals are likely to experience short-term mild pain, suffering or distress, as well as
procedures with no significant impairment of the well-being or general condition of the animals shall be classified as ‘mild’.
Moderate (code 3):
Procedures on animals as a result of which the animals are likely to experience short-term moderate pain, suffering or distress, or long-lasting
mild pain, suffering or distress as well as procedures that are likely to cause moderate impairment of the well-being or general condition of the
animals shall be classified as ‘moderate’.
Severe (code 4):
Procedures on animals as a result of which the animals are likely to experience severe pain, suffering or distress, or long- lasting moderate
pain, suffering or distress as well as procedures, that are likely to cause severe impairment of the well- being or general condition of the
animals shall be classified as ‘severe’.
when can you perform a procedure?
For all procedures, including anesthesia and killing: competency required
To acquire competency, the following steps are taken
- Get qualified (art 9)
- Get trained (by a competent and qualified trainer)
- Get assessed (by the AWB or a qualified assessor)
Only then are you allowed to perform a procedure
anesthesia, 1. what is it and why is it used
- common agents
- considerations
1
Definition: a loss of sensation, either local or general (then also loss of consciousness)
Used for procedures that: Are painful
and/or Require the animals to be perfectly still for a shorter (e.g. precision injection) or longer (e.g. imaging) period of time
2
Isoflurane
- For nearly all types of procedures
- Direct response, depth of anesthesia can be regulated, easy recovery, less respiratory depression
Ketamine with xylazine or ketamine with (dex-)medetomidine
- If isoflurane is not an option (e.g. pregnant operator, - if isoflurane influences experimental outcome)
Pentobarbital
- For perfusion or as a method of euthanasia
- Not suitable for non-terminal anesthesia
3.
Considerations when selecting an agent for anesthesia
Characteristics of the animal
E.g. species, strain, age, weight, health status
Characteristics of the procedure
E.g. type of procedure, duration, possible complications
Requirements of the experiment
E.g. effects of agents on outcome parameters
anesthesia operative care
PRE OPERATIVE
Habituation period
Housing, food and drinking water comparable to what they will be after surgery
Suitable analgesia in case of painful procedures – start in time
Premedication if needed
Health check
INTRA OPERATIVE
Injectable analgesia directly before the start of a (painful) procedure
Local anesthesia if applicable
e.g. lidocaine before drilling the skull, lidocaine gel for ear bars
Provide oxygen (oxygen/air mixture)
POST OPERATIVE Suitable space to recover Keep the animal warm E.g. incubator or heating pad Monitor recovery E.g. direct monitoring until fully awake, postoperative monitoring of weight, grimace scales Provide adequate analgesia Make sure of food and water intake E.g. (wet) food on the floor, gel pads Sufficient period of recovery Duration depending on the nature of the procedure
EUTHANASIA COMMON METHODS AND CONSIDERATIONS
Cervical dislocation
-Rapid, inert, training needed, only for mice and young rat pups, may cause bleeding
CO2
-Gradual fill, not for neonates, can be done in home cage
Overdose of pentobarbital
- Rapid, suitable for perfusion, may influence phyiological or other outcome parameters
Decapitation
- Neonates, adults ONLY under anesthesia unless in project license, inert
Bleeding under anesthesia
- Terminal blood collection for large volumes, followed by cervical dislocation
Considerations when choosing a method of killing
Animal characteristics
E.g. species, age, weight, undergone procedures
Influence on outcome parameters
E.g. physiological parameters, gene expression, histology of organs
Experimental needs
E.g. blood collection, perfusion, extremely fresh tissue, intact brains
