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Environmental factors Environmental factors and laboratory animalsand laboratory animalsEnvironmental factors Environmental factors and laboratory animalsand laboratory animals

Dag EideDag Eide

Norwegian Institute of Public HealthNorwegian Institute of Public Health

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The experimental animal

– a living instrument that requires:

• controlled conditions to be calibrated

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Do you record or control all factors affecting your animals?

– variability in observations

The experimenter is only one among many factors affecting the animal

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Neuroendocrine interactions

hypo- thalamusanterior pituitary

sympathetic

Interleukin-1

somatotrophin

ThyroxinT-cells

Thymic hormones

insulin, sex hormones

Interleukin-4

sensory pathways

Cathecholamines, corticoids

islets, gonads

thymus

thyroid

adrenals

Lymphoid tissue

sympathetic

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Standardize:

• The experimental animal

• +

• The animal environment

• =

• Increased Reproducibility

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Reproducibility

• The experiments should be possible to repeat

• by yourself and others

• Ideal conditions: Only the experimenter should introduce variation - and record it!

• Everything else should be kept constant• A controlled environment -> more clear-

cut results• IN AN IDEAL WORLD - our vision!

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Environmental factors• Climate• Light• Sound & Noise• Barometer pressure• Air quality• How do we dress ?• Bedding in cages• Crowding & isolation• Feed and water

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Temperature

In general: kept constantIn general: kept constant• max: ± 2°Cmax: ± 2°C

– i.e. within technical limitsi.e. within technical limits

The comfortable temperature for us, the The comfortable temperature for us, the caretakers and scientistscaretakers and scientists– Which is probably OK?Which is probably OK?

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Temperature affects toxicity

• Berti & Cima (1955)Berti & Cima (1955)

Impact of temperature on LD50 for chlorpromazine,

mice.

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40Room temperature, °C

Mg

chlo

rpro

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Is there an ideal temperature?

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Pigeon

Mouse

Guinea pig

Rat

Sheep

Dog

Chimpansee

Chicken

Humans

Rabbit

Goat

Squirrel

Malamute

-30 -20 -10 0 10 20 30 40

Neutral EC US

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Humidity

Relative humidityRelative humidity Keep: RH = 55±5% (UFAW)Keep: RH = 55±5% (UFAW)

– RH = the most difficult factor to control?RH = the most difficult factor to control?• Cold wintersCold winters• Humid, hot summerHumid, hot summer

– Eur Conv: between 40% and 60%Eur Conv: between 40% and 60%• Limits should not be exceeded for “Limits should not be exceeded for “longer periods longer periods

of timeof time””

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Too dry conditions

< 35% RH:< 35% RH:• dry skindry skin• tracheitis, bronchitistracheitis, bronchitis• increased dust formationincreased dust formation• increased static electricityincreased static electricity

– lowered fertilitylowered fertility– cannibalismcannibalism

• ringtail in the ratringtail in the rat

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Too humid conditions

– hardly any problem at our latitudes?hardly any problem at our latitudes?

RH > 80%:RH > 80%:• disturbing metabolismdisturbing metabolism• poor hygienepoor hygiene• increased growth of microorganismsincreased growth of microorganisms

Surveillance and controlSurveillance and control– automated data recording automated data recording – Humidity and temperature must be continually Humidity and temperature must be continually

monitoredmonitored– important part of GLP, AAALAC accreditationimportant part of GLP, AAALAC accreditation

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Light– Nocturnal animals prefer low light conditions

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Albino rats’ preference, max (Schlingmann & al. 1993c)

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340 Work light 1 m above floor, recommended (UFAW handbook, 1987)

Lower limit for work (Schlingmann & al. 1993c)

Max light preferred by rats (Bloom & al 1993)

Max to avoid retinal damage in albinos (Schlingmann & al. 1993b)

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Lux

Rats prefer gloomy conditionsRats prefer gloomy conditions

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Light varies within the room

– Impossible to achieve homogenous light conditions

• Cages in racks receive different levels of light

• rotating cages - an alternative?

– ca 200 lux could do for the caretakers, but too much for the animals

• keep the animals behind a curtain

• …in a cabinet

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Light cycle

diurnal, seasonal, annual– Biological clock evolutionary persistent

Electric lightning the only alternative• no windows in animal rooms (except at equator?)

• Experimental animals: 12 h/12 h cycle

• Breeding: 14h light/10h dark

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Biological clock evolutionary persistent

• Strain differences (The Jackson Labs):

How can they sense the spring?

Seasonal variations in inbred mice

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an

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ps p

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Nocturnal animals 2

– activity inverse of light intensity• Small animals sleep less

– reverse light cycle is used by some:• Lights off during the day, on during the night

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Light lowers fecundity• Fisher/344 - rats in a breeding colony where the automatic light

switch did not work:

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Permanent lightning

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Sound & Noise - Frequencies

1 10 100 1000 10000 1E+05 1E+06

Mouse

Rat

Rabbit

Guinea pig

Human

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ecie

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Frequency, Hz

Upper Range

Most sensitive

Lower range

deaf

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Sensitive ears

– 3dB = 2 X intensity (Milligan, Sales & Khirnykh, 1993)

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Ferrets

Rabbits

Chimpanzees

Marmosets

Mice

Cats

Dogs

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Threshold for humans

~140 dB = pain threshold

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Sources of noise• < 55 dB could be accepted, assuming no

fluctuations • electrical supplies• dimmers!• Other animals• Pigs, dogs, chicken• Acoustic shocks: Alarms, drills,

– Noise can provoke• convulsive seizures• reduced fertility (C57BL/6?)• abortions• cannibalism

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Air pressureAir pressure

• differences induced in animal units• Negative pressure: protects the animal workers• Positive pressure: Protects the animals

– Fluctuations in barometer pressure may affect experiments

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Air qualityAir quality

• Gases– Fresh air in the cages?

• CO2 carbon dioxide: – irritates mucosal surfaces– affects respiration, hyperventilation– might lead to acidosis

• NH3 ammonia:– mucosal surfaces: eyes, respiratory tract– facilitates infection– indicator of ventilation and hygiene

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Air quality : OdorsAir quality : Odors• Predators

» Cats, dogs,

• Pheromones• Perfumes ≈ pheromones?• Noradrenaline (norepinephrine) release

– Wang & Tsai 1991

• Pavlovian effects– Odors can stimulate the immune system

» E.g.: NK-cells mobilized by odor in sensitized mice» Solvason & al. 1991

– diesel, smoke, fumes

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A genetically altered male A genetically altered male mouse that seemingly cannot mouse that seemingly cannot distinguish the sex of other distinguish the sex of other

mice, a task normally mice, a task normally accomplished in rodents accomplished in rodents

through the detection of sex-through the detection of sex-specific pheromones. Without specific pheromones. Without

TRP2, an ion channel TRP2, an ion channel necessary for activation of the necessary for activation of the

vomeronasal organ by vomeronasal organ by pheromones, such mice pheromones, such mice display unusual lack of display unusual lack of

aggression toward other male aggression toward other male mice and initiate mating mice and initiate mating

behavior with both males and behavior with both males and females.females.

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Air exchangeAir exchange

• Ventilation systems should be effective– animal rooms

• 15-20 air changes / hour required

– ventilated cages• ≈ 40 changes/hour optimal

– Isolators

• avoid draft

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Bedding in cagesBedding in cages• Materials used from different trees

– different contents of oils, – Induction of hepatic enzymes (see Figure)– dust formation differs

• Contamination?

Pine, Greece

Bedding 1, NZPine, Russia

Pine, AUS

Bedding 2, NZBedding 3, NZ

Shredded paper, AUS

Aspen, SFSugar cane, CU

Corn cobs, US

Vermiculite, SA

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Toxic index, hepatocyte toxicity test

Pelkonen & Hänninen, 1994

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Air and bedding quality:Air and bedding quality:• How often do we change cages?

– Standard: weekly– Breeders with litter may go 2 weeks– Do not change cages too often..

• Scent marking makes mice feel at home, a new cage with new bedding is a new home.

• Large differences between mouse and rat strains, between sexes, different ages.

– Males like their own scent• Females to his place, if you want good breeding results

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Crowding & isolation

• Stress induced changes: isolation:

Adrenal size Thyroidea size hepatic enzyme activity Never alone in a cage?

Crowding may increase phagocytosis neoplastic growth (corticoid effect?)

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Caging - Crowding

• Thigmotropism : social animals like to touch each other

Piling up

• Male mice get aggressive 7-8 w olds often fight Strain differences

A/J versus SJL

• Male rats usually don’t get aggressive, until they get old

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Never alone in a cage?

• - no probably not!Even male mice like to be social

• Statistical problem?What is the experimental unit?Cage or animal?

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Immunization experiment 1:

PestivaxImmunizak

controls• What is N in this experiment?

• Observations within cage not independent - i.e. correlated in some way.

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Immunization experiment 2:

• What is N in this case?

• BUT you can not use this set-up in all experiments.. When animals contaminate/coinfect each other..(infections, toxicity)

Pestivax

Immunizak

Pestivax

Immunizak

Pestivax

Immunizak

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How to analyze experiments where independence is violated ?

Independence is assumed in ANOVA, Regression, correlation (linear models)Some methods could correct for the correlation structure in the data.The Refinement is: You could use all individual data from groups of animals that have a correlation structure– (otherwise you should use the group means as

one data point, loosing much information)

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How to analyze experiments with such a covariance structure ?

SAS: use Proc mixed, specify subject and covariance structure:– Proc mixed data:mouse;

• Class cage drug;• Model bloodpressure = drug;• Random intercept / subject = cage structure=vc;• Run;

S-plus: use grouping in the spec of your model (pull down menu)(for details, look in relevant literature on mixed models, available for SAS and S-plus)

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Feed and water– Nutrient contents– Contaminants

• Microorganisms: sterilization• Acidified drinking water preserves water quality

– pH between 2.5 and 3.2 optimal (Pseudomonas)

• Pesticides• moulds• insects & mites

– Feeding procedures• ad libitum feeding

• overfeeding common - boredom?• kidney failure

• restricted feeding leads to a longer life• group hierarchy• rabbits

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Handling

Dedicated caretakersRats remember their caretakers for 6

months!Handling is AS IMPORTANT as other

environmental factorsNot everyone is suited for animal handlingIdeally: The same person should handle the

animals during all proceduresEspecially important in breeding colonies

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Clothing in the animal room ?Paper A (Crabbe & al 1999)

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Even more “outta control”?

• Lunar phases

• Magnetic fields

• Background radiation

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Acclimatization

Animals need to acclimatize in a new facilitytransport: short or long distances

leads to changed• feeding• temperature• humidity• ventilation• light conditions• sound/noise level

Combine with quarantene?

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Acclimatization

Time to acclimatize depends on the duration and distance travelled??

No acclimatization - low reproducibility

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Be patientAcclimatization (acclimation) times increase with size of the animal

Species # of days(minimum)

Primates 40 - 60

Dogs 20 - 30

Cats 20 - 30

Rabbits 20 - 30

Smallrodents

5 - 15

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one example to illustrate– Concanavallin A LPT

response in cattle– > 2 weeks to reach some

baseline for cellular activity

– Red line is start of experiment

– Lines = different origins, affect the whole experiment

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What are you responsible for?

• The experimenter should• take care when working with the animals• thorough planning• record all relevant data• What do you write in M&M?

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Appropriate controls - the most important issue

• If you can, do the experiment all at the same time– Historical controls may be invalid

• Robust animals may be less sensitive to environmental change– F1 hybrids robust and isogenic– Outbred animals robust, but no genetic

information– Inbreds more sensitive– Genetically modified/mutants may be

extremely sensitive

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Sources of information

• The UFAW Handbook on the care & management of laboratory animals

• Available free:– European convention– US Guide for the care and use….– Canadian Guide for the care and use….