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Laboratory Animal Care and Use Program and Facilities
Assistant Laboratory Animal Technician (ALAT) Certification Class: Birds, Amphibians, Fish
Matthew K. Hogan BVetMed, MRCVS ULAR Resident Veterinarian
Laboratory Animal Care and Use Program and Facilities
Agenda
• ALAT Training Manual – Chapter 29-Birds – Chapter 30-Amphibians – Chapter 31-Fish
Laboratory Animal Care and Use Program and Facilities
Birds
Laboratory Animal Care and Use Program and Facilities
Uses in Research
• Neurobiology • Behavior • Endocrinology • Nutrition • Microbiology • Embryology
• NOT covered under AWA if bred for research
• COVERED under PHS
policy • GUIDELINES for care in the
Guide and Ag Guide
Laboratory Animal Care and Use Program and Facilities
Taxonomy: Aves
• Order: Galliformes – Poultry
• Turkey • Chicken (Gallus
gallus domesticus) – Robust immunity – Eggs used in Ab-
production flu vaccines
• Quail
Laboratory Animal Care and Use Program and Facilities
Taxonomy: Aves
• Order: Columbiformes – Pigeon
• White Carneau (Columba livia)
– Atherosclerosis – Learning & memory
– Dove
Laboratory Animal Care and Use Program and Facilities
Taxonomy: Aves
• Order: Passeriformes – Common name: passerines
• Sparrow • Crow • Canary • Finch
– Zebrafinches (Taeniopygia guttata) » Learning, memory, language development
models (songs)
Laboratory Animal Care and Use Program and Facilities
Taxonomy: Aves
• Order: Psittaciformes – Common name: psittacines
• Parrots • Parakeets/budgies • Cockatoos
Laboratory Animal Care and Use Program and Facilities
Physical Characteristics
• Adapted for flight – Forelimbs wings – Fused & hollow bones strength, lightness
• Lightness = fragility injury during handling
Laboratory Animal Care and Use Program and Facilities
Unique Biological & Physical Features
• Spur – Male galliformes nail not
associated with a toe – On lower limbs, points towards
the tail – Fighting, defense, aggression
• Can trim, remove
• High body temperature (e.g. chicken ~107oF)
• No sweat glands thermoregulation via panting
Laboratory Animal Care and Use Program and Facilities
Feathers
• Feature not shared with any other vertebrate
• Functions: – Flight – Insulation – Water-proofing
preen/uropygial gland • Molting ~1/year
– Can occur due to stress, improper lighting, poor nutrition
Laboratory Animal Care and Use Program and Facilities
Digestive Anatomy • Crop
– Expansible esophageal outpouching – Stores food – Crop milk protein, fat, Ab rich material for hatchlings
• Gizzard – Grinds up food (seeds)
• Cloaca – One chamber for intestinal, urinary and reproductive
contents/material to empty – Female = lay eggs; male = evaginates to pass sperm
• Waste – Thick-white pasty urine – Green-brown feces
Laboratory Animal Care and Use Program and Facilities
Laboratory Animal Care and Use Program and Facilities
Sexing
• Sexual dimorphism – Size – Feather conformation – Color pattern
• Other indicators – Color change (male) – Cloacal change – Featherless breast (female)
Laboratory Animal Care and Use Program and Facilities
Sexing
• Surgical inspection/PME – Internal gonads
• Female – Left side only functional
• Male – No accessory sex organs or true penis
Laboratory Animal Care and Use Program and Facilities
Sexing Female Male
Laboratory Animal Care and Use Program and Facilities
Handling
• Birds are easily alarmed or frightened injury (fly into wall, trampling), overheating
• Calm, cool, correct handling imperative – Move slowly and deliberatly – Handle, change cages, manipulate in dim
lighting calming state – Attempt capture once, if fail, give extended
break before attempting again
Laboratory Animal Care and Use Program and Facilities
Handling Galliformes • Chickens
– Can carry upside down by legs – Restrain wings & legs with hands, tuck
under arm for support (pic) – Add’n aids: tape legs together, drape
cloth over head/eyes calm
• Turkeys – DO NOT carry by legs – Stronger, more aggressive than
chickens – One hand encircles humerus of both
wings over back, restrain head with fingers around neck, hold legs with other hand
Laboratory Animal Care and Use Program and Facilities
Handling Columbiformes & Passerines
• Small, swift difficulty in safe capturing
• Nets, small cage + hands • Can restrain large
pigeon/dove like chicken (pic)
• Parrots/parakeets: – Restrain head to avoid bites
(+/- gloves); cover head – Stockinette: wings restrained,
head & tail exposed
Laboratory Animal Care and Use Program and Facilities
Identification
• Leg bands – Color-coated and/or
alphanumeric – Radiofrequency ID chip
(larger birds) – Size & place considered
along with growth of bird – Checked as part of bird health
exam • Wing tags • Dyes
Laboratory Animal Care and Use Program and Facilities
Behavior
• Easily excited – Respond to sudden noise & movements with
vocalizations calm, quiet handling • Groups can have strict hierarchy
fighting, food possession – Nail trims – Restrict flight
• Pinioning?? • Wing clip 1st 10 primary feathers; one wing!
– Beak mgmt cuttle bone
Surgical removal of distal tip of wing
Laboratory Animal Care and Use Program and Facilities
Housing • AWA does not address birds • Physical Environment
– Prevent exposure to wild birds/rodents – Perches – variable size – Adequate space for freely flapping wings
• Psychological well being – Solid panels between cages if conflicts
• Environmental – Humidity 45 – 70% – Lighting
• Wide spectrum or incandescent should be used – Temperature
• Ventilation important, susceptible to heat stress
Laboratory Animal Care and Use Program and Facilities
Housing • Guide (poultry):
– Air temperature: 16-27oC (61-81oF) – Relative humidity: 30-70%
• Cage litter: – Species dependent – Cover floor with sand, gravel or shavings – Place low plexiglass frame around perimeter to contain
litter
• Design – Flexibility accommodate other species, group sizes – Avoid mixing species, variable age groups – Away from swine/dogs (noise) & shield from visual
disturbances – Have external heat source/brooder for very young birds
Laboratory Animal Care and Use Program and Facilities
Diet • Water
– Suspended automatic watering system
• Feed is species dependent – Commercial formulated pellets – Passeriformes: seeds (canary grass) + vegetables,
fruits, cooked egg yolks – Psittacines: seeds (sunflower, canary grass) + peanuts,
monkey biscuits
• Grit – Necessary, remains in gizzard aids in grinding of
seeds during digestion – Includes oyster shells and granite (autoclave/sterilize)
• Cuttlebone (passerines & psittacines) beak maintenance + Calcium
Laboratory Animal Care and Use Program and Facilities
Husbandry
• Feed & water containers – Several locations at varying heights
reduced aggression – Scatter feed on floor + dish to adapt bird
to bowl feeding • Environmental enrichment
– Music – Colored objects/toys – Nesting material
Laboratory Animal Care and Use Program and Facilities
Euthanasia Method Acceptable Acceptable w/conditions
Avian IV barbiturates Inhaled anesthetics, CO2, CO, N2, Ar, cervical dislocation (small birds and poultry), decapitation (small birds)
Poultry Injected barbiturates and anesthetic overdose
CO2, CO, N2, Ar, cervical dislocation (as anatomically appropriate), decapitation, manual blunt force trauma, electrocution, gunshot, captive bolt
Laboratory Animal Care and Use Program and Facilities
Amphibians
Laboratory Animal Care and Use Program and Facilities
Amphibians
• Vertebrates – Scaleless, smooth-skinned, ectothermic – Need aquatic or very moist
environments – Most begin life as gill-breathing larvae
(tadpole) metamorphosis
Laboratory Animal Care and Use Program and Facilities
Common Species in Research
• Frogs – Bullfrog (Rana catesbeiana)
• Physiology and acoustics • Motion sickness in space
travel – They can vomit!!!
– Leopard frog (Rana pipiens)
• Hormone studies
Laboratory Animal Care and Use Program and Facilities
Common Species in Research
• Frogs – African-clawed frog
(Xenopus) • Entirely Aquatic • Originally used in
pregnancy assays • Developmental studies year round reproduction with hormone therapy
• Genetics & molecular biology oocytes
X. laevis X. tropicalis
Laboratory Animal Care and Use Program and Facilities
Besides frogs!
• Tiger Salamander (Ambystoma tigrinum) – Ophthalmology – Limb regeneration
• Red Spotted Newt (Notophthalmus viridescens) – Limb and lens regeneration
• Axolotl (Ambystoma mexicanum) – Limb regeneration
Laboratory Animal Care and Use Program and Facilities
Anatomy
Laboratory Animal Care and Use Program and Facilities
Skin • Absorb O2 through moist skin breathing!
– Have lungs, no diaphragm • Larve breath via gills
– Chemicals can permeate skin too • Mucous Glands
– Slimy protective layer • Granular Glands
– Head & shoulders – Compounds & chemicals
• Defense compounds (neurotoxins, hallucinogenics) • Magainins (inhibit growth of pathogens) • Pheromones
Laboratory Animal Care and Use Program and Facilities
Digestion & Excretion
• Adults: – Carnivores w/short GIT + well-
developed tongue • Xenopus no tongue, use front limbs to
feed – Cloaca (like birds!)
Laboratory Animal Care and Use Program and Facilities
Sexing
• Xenopus: – F > M (female is pear-shaped) – Male has roughened black
nuptial pads & large thumbs
• General: – Tympanic membrane 2x larger in
males (bullfrogs) – Species specific mating
behaviors
Laboratory Animal Care and Use Program and Facilities
Tympanic Membrane
Laboratory Animal Care and Use Program and Facilities
Mating
• Induce ovulation in female & stimulate sexual activity in male with hormone injections
• Egg collection: – Collect before adults eat them – Can collect unfertilized eggs
• Manual expression • Surgery
Laboratory Animal Care and Use Program and Facilities
Development
• Metamorphosis
• Neoteny – Retain larval characteristics throughout
life
Laboratory Animal Care and Use Program and Facilities
Handling & Restraint
• Only handle when necessary • Handle w/moist, powder-free gloves
– Nets or jars – Eye protection: cane toads toxic skin
secretions from parotid gland • Restraint:
– Salamander: grasp head and forelimbs w/one hand, hindlimbs w/other (never hold by tail!)
– Axolotl: never use net!
Laboratory Animal Care and Use Program and Facilities
Frog Restraint
Laboratory Animal Care and Use Program and Facilities
Identification
• Cage card may be sufficient • Individual ID:
– Photographs of skin patterns – Tattoo (fades as skin sheds) – Toe-clipping (discouraged; IACUC approval) – Freezing (branding-method) – PIT = passive integrated transponders
(microchips)
Laboratory Animal Care and Use Program and Facilities
Behavior • Cannibalism
– Adults may eat eggs, tadpoles, growing adults due to overcrowding, poor diet
• Sensitivity – Environmental temperature (ectothermic) – Overhandling, too much enrichment – Skin water quality key!
• Pain, distress, illness – Lethargic, poor appetite – Bacterial infection: ‘red leg’ – Parasitic infection: thick, pitted, sloughing of
skin
Laboratory Animal Care and Use Program and Facilities
Housing
• Temperature – Tropical species – 21‐29oC (70‐85oF) – Temperate – 18‐22oC (65‐72oF)
• High humidity: 80% need moisture • Lighting
– Full wavelength spectrum • Reduce airflow to prevent habitat &
animal desiccation
Laboratory Animal Care and Use Program and Facilities
Housing: Aquatic Systems System Features
Static • No recirculation of tank water • Water change by draining & refilling • Clean frequently due to waste accumulation • Replace old water with same temperature new
water
Recirculating • Filters remove waste & debris • Less frequent cleaning, but must replace filters
regularly
Flow-through • Constants stream of water in and out of tank • Fresh water is always available, but must make
sure chlorine is reliably removed from this incoming water
Laboratory Animal Care and Use Program and Facilities
Caging
• Terrestrial – Moist, misty environment
• Semi-aquatic – Sloping floor
• Hiding places – Bark – PVC piping – Pads – Dark-colored caging
Laboratory Animal Care and Use Program and Facilities
Water Quality
• Monitor & adjust: – Water hardness – Dissolved CO2 level – O2 level
• Bubbling filtered air through water: – Maintains correct concentration of dissolved
gases • Keep light if new hatchlings present
• No detergents or disinfectants for cleaning – Remnants can be absorbed – Heat sterilization & scrubbing recommended
Laboratory Animal Care and Use Program and Facilities
Nutrition
• Adults: Carnivores – Commercial frozen gel/pellet (Xenopus) >
crickets, mealworms, brine (Salamander exception)
– Axolotls: beef muscle/organs + vitamins – Daily‐twice weekly (species & life-stage
dependent) – Larval salamanders: Carnivores
• Larval amphibians – Tadpoles: Herbivores – Spinach, yeast, lettuce
Laboratory Animal Care and Use Program and Facilities
Euthanasia
• Varies per species
Acceptable Acceptable w/conditions
Injected barbiturates, dissociative agents and anesthetics as specified, topical buffered tricaine methanesulfonate (MS-222) or benzocaine hydrochloride
Inhaled anesthetics as specified, CO2, penetrating captive bolt or firearm, manually applied blunt force trauma to the head, rapid freezing
Laboratory Animal Care and Use Program and Facilities
Fish
Laboratory Animal Care and Use Program and Facilities
Uses of Fish in Research
• Models: – Endocrinology – Bacterial diseases – Genetics
• Conditions in human development – Toxicity studies – Health & husbandry of fish themselves
• Importance of fish in aquaculture • Fish as a source of food
Laboratory Animal Care and Use Program and Facilities
Taxonomy
• Zebrafish – Danio rerio – Model for genetic conditions in human
development and metabolism • e.g. scoliosis
• Salmon & trout – Onchorhychus
• Catfish – Ictalurus
Laboratory Animal Care and Use Program and Facilities
General Considerations
• Fish are typical vertebrates in many respects (500 million years old) – One can apply basic concepts you
already know regarding mammalian biology
• Possess the same/similar organ systems as in other animals – Have some unique anatomical and
physical characteristics
Laboratory Animal Care and Use Program and Facilities
Anatomy & Physiology
• Most fish have laterally compressed, elongated muscular bodies – Ectotherms – Fins for locomotion – Gills O2 exchange
• Operculum: flexible bony plate that covers/protects gills
– Lateral line: sense organ along each side of fish detects movements/vibrations
– Swim bladder: maintain buoyancy
Laboratory Animal Care and Use Program and Facilities
Anatomy: Skin
• Mucus layer – Protects against infection – Waterproof skin
• Scales • 2 sets of paired fins and 1 or 2 dorsal fins,
anal & tail fin
Laboratory Animal Care and Use Program and Facilities
Anatomy & Physiology
Laboratory Animal Care and Use Program and Facilities
Anatomy & Physiology
Laboratory Animal Care and Use Program and Facilities
Sexing & Reproduction
• Most lay eggs, some bear living young – Over 97% fish lay eggs & fertilization takes
place outside the body – Fertilization may occur internally or externally
• Sperm released over eggs after female has laid them
– Variable parental care: abandon or nest • Live-bearing
– Female > male; Male more brightly colored • Egg-layers
– Female has swollen abdomen when ready to spawn
Laboratory Animal Care and Use Program and Facilities
Sexing
Laboratory Animal Care and Use Program and Facilities
Question?!?
1. What is spawning? a. The metamorphosis of a fish from egg
to hatchling to adult b. Nest building behavior in fish c. The release of egg & sperm from
female & male fishes d. The reinvention of Al Simmons aka
Spawn
C.
Laboratory Animal Care and Use Program and Facilities
Zebrafish Breeding
• Either: – 10 gallon ‘breeding tank’: lots of males &
females together – Small tank with 1 or 2m:2f
• Breeding tanks have perforation to keep adults from eating eggs
• 1/week: place fish in breeding tank late in day collect eggs next morning & return adults to home tanks
• Keep embryos at 24-33oC; will feed on own 4-6d post-fertilization
Laboratory Animal Care and Use Program and Facilities
Procurement • Usually shipped in plastic bags/foam
containers – Bags must have an air pocket
• Minimize shipping time as much as possible
• Temperature adjust transport water to tank – Can also add tank water to shipping water
• Should unpack immediately upon arrival • Ideally, all incoming fish should be
quarantined for 30d – Usually only embryos go to primary system
Laboratory Animal Care and Use Program and Facilities
Handling, Restraint, Identification
• Handling and Restraint – Fish NEVER acclimate to being handled
• Can anesthetize if needed • Wear wet, non‐powdered gloves (+/- full
arm length)
• Identification Methods – Records can be kept at tank level or per
animal – SQ dye, tags, fin clipping, SQ or
abdominal transponders
Laboratory Animal Care and Use Program and Facilities
Pain, Distress, Illness
• Signs – Listlessness – Change in feeding – Erratic movements/flashing – Isolation; staying near bottom/top
of tank – Gulping air at surface – Loss of buoyancy – Clamped fins, operculum flaring – Lesions/altered skin/scales
Laboratory Animal Care and Use Program and Facilities
Facilities
• Waterproof surfaces • 1 gallon = 3.78kg (8.33lb) • Tanks should have dedicated
equipment – Disinfected and rinsed between uses
• Drainage (normal usage and spills)
• Electrical – Overhead supply or high on wall – Emergency power
Laboratory Animal Care and Use Program and Facilities
Facilities: Holding Systems • Closed
– “Static” = fishbowl
• Flow‐through – Fresh water enters & passes through system
• Recirculating – Like flow‐through, but water is reused after being filtered
Laboratory Animal Care and Use Program and Facilities
Water Quality
• pH – Varies with species; Ideal pH 6‐8
• Water and room temperature – Adjustments should be gradual, no
more than 1oC per hour • Specific gravity, conductivity, salinity
– Salts and minerals in water, removed by reverse osmosis
Laboratory Animal Care and Use Program and Facilities
Water Quality
• Hardness – Calcium and magnesium in the water
• Dissolved oxygen – Fish need to breath; decr as temperature incr
• Nitrogenous waste – Ammonia, nitrite, and nitrate – Removed by biofilter
• Lighting – Most fish require a low light level for growth &
development, also limits algae growth
Laboratory Animal Care and Use Program and Facilities
Husbandry
• Cleaning tanks: – NO disinfectants or detergents;
residue health problems – NO leftover feed, feces, salt
deposits – Autoclave: kills algae & sanitizes
• DON’T autoclave biofilter kill good bacteria
• Room cleaning – Disinfect between studies – Use disinfectant according to SOP
(e.g. bleach)
Laboratory Animal Care and Use Program and Facilities
Nutrition
• Quantity – Typical amount eaten in 3‐5 minutes – Excess = source of nitrogen waste!
• Natural ingredient lab diet (commercial)
• Live food – Artemia, rotifers, Paramecium
• Schedule – Zebrafish 2x a day
Laboratory Animal Care and Use Program and Facilities
Euthanasia Method Acceptable Acceptable w/conditions
Finfish Immersion in buffered benzocaine or benzocaine hydrochloride, isoflurane, sevoflurane, quinaldine sulfate, buffered tricaine methanesulfonate (MS-222), 2-phenoxyethanol, injected pentobarbital, rapid chilling (appropriate zebrafish/research setting)
Eugenol, isoeugenol, clove oil, CO2-saturated water (aquarium-fish facilities/fisheries), decapitation/cervical transection/manually applied blunt force trauma followed by pithing, rapid chilling followed by adjunctive method (aquarium-fish facilities), maceration (research setting)
Laboratory Animal Care and Use Program and Facilities
Questions?
Happy Holidays & Happy New Year !!!