Environmental factors and laboratory animals

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Environmental factors and laboratory animals

• Dag Eide
• Norwegian 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
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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 -gt 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 constant
• max 2C
• i.e. within technical limits
• The comfortable temperature for us, the
  caretakers and scientists
• Which is probably OK?

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Temperature affects toxicity
Mg chlorpromazine
Room temperature, C

• Berti Cima (1955)

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Is there an ideal temperature?
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Humidity

• Relative humidity
• Keep RH 555 (UFAW)
• RH the most difficult factor to control?
• Cold winters
• Humid, hot summer
• Eur Conv between 40 and 60
• Limits should not be exceeded for longer periods
  of time

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Too dry conditions

• lt 35 RH
• dry skin
• tracheitis, bronchitis
• increased dust formation
• increased static electricity
• lowered fertility
• cannibalism
• ringtail in the rat

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Too humid conditions

• hardly any problem at our latitudes?
• RH gt 80
• disturbing metabolism
• poor hygiene
• increased growth of microorganisms
• Surveillance and control
• automated data recording
• Humidity and temperature must be continually
  monitored
• important part of GLP, AAALAC accreditation

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Light

• Nocturnal animals prefer low light 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?

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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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Sound Noise - Frequencies
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Sensitive ears

• 3dB 2 X intensity (Milligan, Sales Khirnykh,
  1993)

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Sources of noise

• lt 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 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 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 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 mouse that seemingly
cannot distinguish the sex of other mice, a task
normally accomplished in rodents through the
detection of sex-specific pheromones. Without
TRP2, an ion channel necessary for activation of
the vomeronasal organ by pheromones, such mice
display unusual lack of aggression toward other
male mice and initiate mating behavior with both
males and females.
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Air 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 cages

• Materials used from different trees
• different contents of oils,
• Induction of hepatic enzymes (see Figure)
• dust formation differs
• Contamination?

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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 dont 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

• 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)

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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 datamouse
• Class cage drug
• Model bloodpressure drug
• Random intercept / subject cage structurevc
• 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 caretakers
• Rats remember their caretakers for 6 months!
• Handling is AS IMPORTANT as other environmental
  factors
• Not everyone is suited for animal handling
• Ideally The same person should handle the
  animals during all procedures
• Especially 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 facility
• transport 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 patient

• Acclimatization (acclimation) times increase with
  size of the animal

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one example to illustrate

• Concanavallin A LPT response in cattle
• gt 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 MM?

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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.