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
Agenda ALAT Training Manual Chapter 29-Birds Chapter 30-Amphibians Chapter 31-Fish
Birds
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
Taxonomy: Aves Order: Galliformes Poultry Turkey Chicken (Gallus gallus domesticus) Robust immunity Eggs used in Abproduction flu vaccines Quail
Taxonomy: Aves Order: Columbiformes Pigeon White Carneau (Columba livia) Dove Atherosclerosis Learning & memory
Taxonomy: Aves Order: Passeriformes Common name: passerines Sparrow Crow Canary Finch Zebrafinches (Taeniopygia guttata)» Learning, memory, language development models (songs)
Taxonomy: Aves Order: Psittaciformes Common name: psittacines Parrots Parakeets/budgies Cockatoos
Physical Characteristics Adapted for flight Forelimbs wings Fused & hollow bones strength, lightness Lightness = fragility injury during handling
Unique Biological & Physical Spur Features 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 ~107 o F) No sweat glands thermoregulation via panting
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
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
Sexing Sexual dimorphism Size Feather conformation Color pattern Other indicators Color change (male) Cloacal change Featherless breast (female)
Sexing Surgical inspection/pme Internal gonads Female Left side only functional Male No accessory sex organs or true penis
Sexing Female Male
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
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
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
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
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?? Surgical removal of distal tip of wing Wing clip 1 st 10 primary feathers; one wing! Beak mgmt cuttle bone
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
Housing Guide (poultry): Air temperature: 16-27 o C (61-81 o F) 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
Diet Water Suspended automatic watering system Feed is species dependent Grit Commercial formulated pellets Passeriformes: seeds (canary grass) + vegetables, fruits, cooked egg yolks Psittacines: seeds (sunflower, canary grass) + peanuts, monkey biscuits Necessary, remains in gizzard aids in grinding of seeds during digestion Includes oyster shells and granite (autoclave/sterilize) Cuttlebone (passerines & psittacines) beak maintenance + Calcium
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
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
Amphibians
Amphibians Vertebrates Scaleless, smooth-skinned, ectothermic Need aquatic or very moist environments Most begin life as gill-breathing larvae (tadpole) metamorphosis
Frogs Common Species in Research Bullfrog (Rana catesbeiana) Physiology and acoustics Motion sickness in space travel They can vomit!!! Leopard frog (Rana pipiens) Hormone studies
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
Besides frogs! Tiger Salamander (Ambystoma tigrinum) Ophthalmology Limb regeneration Red Spotted Newt (Notophthalmus viridescens) Limb and lens regeneration Axolotl (Ambystoma mexicanum) Limb regeneration
Anatomy
Skin Absorb O 2 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
Digestion & Excretion Adults: Carnivores w/short GIT + welldeveloped tongue Xenopus no tongue, use front limbs to feed Cloaca (like birds!)
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
Tympanic Membrane
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
Development Metamorphosis Neoteny Retain larval characteristics throughout life
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!
Frog Restraint
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)
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
Housing Temperature Tropical species 21 29 o C (70 85 o F) Temperate 18 22 o C (65 72 o F) High humidity: 80% need moisture Lighting Full wavelength spectrum Reduce airflow to prevent habitat & animal desiccation
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
Caging Terrestrial Moist, misty environment Semi-aquatic Sloping floor Hiding places Bark PVC piping Pads Dark-colored caging
Water Quality Monitor & adjust: Water hardness Dissolved CO 2 level O 2 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
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
Euthanasia Varies per species Acceptable Injected barbiturates, dissociative agents and anesthetics as specified, topical buffered tricaine methanesulfonate (MS-222) or benzocaine hydrochloride Acceptable w/conditions Inhaled anesthetics as specified, CO2, penetrating captive bolt or firearm, manually applied blunt force trauma to the head, rapid freezing
Fish
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
Taxonomy Zebrafish Danio rerio Model for genetic conditions in human development and metabolism e.g. scoliosis Salmon & trout Onchorhychus Catfish Ictalurus
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
Anatomy & Physiology Most fish have laterally compressed, elongated muscular bodies Ectotherms Fins for locomotion Gills O 2 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
Anatomy: Skin Mucus layer Protects against infection Waterproof skin Scales 2 sets of paired fins and 1 or 2 dorsal fins, anal & tail fin
Anatomy & Physiology
Anatomy & Physiology
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
Sexing
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 C. female & male fishes d. The reinvention of Al Simmons aka Spawn
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 24-33 o C; will feed on own 4-6d post-fertilization
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
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
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
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
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
Water Quality ph Varies with species; Ideal ph 6 8 Water and room temperature Adjustments should be gradual, no more than 1 o C per hour Specific gravity, conductivity, salinity Salts and minerals in water, removed by reverse osmosis
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
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)
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
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)
Questions? Happy Holidays & Happy New Year!!!