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 !!!