Laboratory Animal Science (2023-2024)

Praktisch
1 Severity assessment
2 History
3 Legal
4 Safety
- Hazards
- Allergens
- Zoonosis
5 Zootechnique
6 Housing
7 Hygiene
- Pathology and microbiological standardisation
- Disease management
8 Anaesthesia
9 Humane endpoints
- Overview table (background info)
10 Behaviour and wellbeing
11 Experimental techniques
12 Surgical techniques (guest)
13 Genetics (guest)
14 Ethics (guest)
15 Alternatives
- Replace
- Reduce
- Refine
16 Feeding

Praktisch

keuzevak
3 stp
eerste (of tweede) semester
e-learning modules
opdrachten
- verplicht practicum bijwonen
- korte paper schrijven (ECD report)
  - alleen of in groep
examen
- meerdere momenten doorheen jaar
- schriftelijk
- open vragen

1 Severity assessment

classifications
- no harm
  - less than introduction of needle
  - excl. killing for organs/tissues
- non-recovery or terminal
  - after procedure under anaesthesia
- mild
  - short term mild pain
  - mild wellbeing impairment
- moderate
  - short term moderate pain, or long term mild pain
  - moderate wellbeing impairment
- severe
  - short term severe pain, or long term moderate pain
  - severe wellbeing impairment
- post-mortem (only at KUL)
  - euthanized for tissue collection
factors
- procedure
  - surgery
  - effect
  - analgesia / anaesthesia
  - new procedure
  - restraint
  - environmental: housing, food
  - stress
- animal-specific
  - species
  - strain, phenotype
  - gender, age
- frequency, intensity, duration
- refinement
  - analgesia / anaesthesia
  - environmental: housing, food
non-factors
- affect wellbeing but not severity
- anything not part of planned procedure
  - flooding
  - fighting
  - unrelated sickness
  - trauma
types
- actual severity
  - reported
- prospective (expected) severity
assessment
- define study procedure w.r.t. animals
- define animal experience
- define mitigation measures + humane endpoints
- decide on severity
table
- columns
  - What does this study involve doing to the animals?
  - What will the animals experience? Adverse effects? What might make it worse?
  - How can suffering be reduced to a minimum?
  - Endpoints
- rows
  - actions on animals / step in procedure

2 History

background info

3 Legal

ethology: study of natural animal behaviour
1986-11-24 EU directive
- housing should meet the animal’s health and well-being and should not limit their physiological and ethological needs
1991-10-18 caretaking and housing per species
2010-04-06 KB: lab animal housing
- meet animals needs
- daily control
- regular control
  - large animals: by veterinarian
  - small animals: expert
2010-09-xx EU directive on protection of lab animals
2013-05-29 KB van EU directive
2014-07-xx BE: federal -> regional
definition: lab animal
- living vertebrate in lab
  - incl. larva
  - incl. embryo in last trimester
- some invertebrae (e.g., cephalopoda)
definition: animal experiment
- can inflict pain, suffering, ...
  - animal should only be used once when serious pain and suffering
- incl. birth
- excl. humane methods
- excl. non-experimental treatments in agriculture or veterinary
purpose
animal ID
- dogs, cats, primates: permanent, individual marker
- keep register
- statistics
  - per species
  - per experiment type
source
- no strays
- purpose-bred by licensed companies
- farm animals
- threatened species: special demands
- exemptions: strong motivation required
responsible use
- choose species with lowest neurophysiologic degree
- 3R's (Russel and Burch)
  - reduction
    - experimental design
    - standardize animal population and procedures
  - replacement
    - alternative methods
  - refinement
    - avoid pain, suffering, injury
      - use anaesthesia, analgesics
      - never subject to severe pain
    - biological needs
    - environmental enrichment
    - analgesics
what do you need to start animal experiments
- lab license
  - van Vlaamse overheid
  - rooms
  - experiment types
  - species + sources
  - staff
    - lab director
    - expert
      - large animals: vet
      - small animals: FELASA C training (PI) under vet supervision
    - project lead
      - keeps log
    - biotechnicians
      - conduct experiments
    - animal caretakers
- permission from ethical committee
  - mandatory since 2001
  - composition
    - lab director
    - project lead
    - biotech
    - vet / expert
    - independent members
    - (government inspector as non-member guest)
  - competencies (ethics, alternatives, wellbeing, ...)
  - at least 7 members
  - no conflict of interest
- education
  - courses
    - Federation of EU Lab Animal Science Associations (FELASA)
    - Belgian Council for Lab Animal Science (BCLAS)
    - continuous education
  - caretakers: course FELASA A (25h)
  - biotech: course FELASA B (40h)
  - project lead: course FELASA C (80h)
- Animal Welfare Body (AWB)
  - set up by lab or breeder ("user")
  - who
    - biotech
    - vet / expert
    - caretakers
  - why
    - improve welfare
    - improve transparency
  - what
    - advise on welfare
    - review internal processes
misc
- project types
  - GS1: wildtype
  - GS2: genetically modified with harmless phenotype
  - GS3: genetically modified with harmful phenotype
    - perform Animal Welfare Assessment (AWA)
      - describe strain and modification
      - info about possible harm
        
        pain?
        
        disability?
        
        short lifespan?
        
        tumors?
        
        neurological issues?
      - check for abnormal behaviour and breeding
      - assessment
        
        severity
        
        harmfullness (i.e., GS2 or GS3)
      - mitigations
        
        refinement
        
        humane endpoints
  - creation of new genetic line
    - report
  - experimental
- retrospective assessment
  - animal count
  - pain assessment
  - project gain
- non-technical summary
- only if relevant
  - import license (from outside EU)
  - bio-security dossier
  - radio-activity use

4 Safety

standard operating procedures (SOP)
- prevent accidents and disease
bio security
- class 1
  - lockable rooms
  - windows don't open
  - animals cannot escape
  - sink in room
  - window in door
  - disinfectable floors, walls
  - washing area
  - lab coats per room
    - coat hangers
  - no eating, drinking, smoking
- class 2
  - biohazard sign
  - sink with elbow tap
  - laminar flow class II
  - autoclave
  - airtight rooms
- class 3
  - airlock entrance
  - negative pressure
  - hepa filters at exhaust
  - disinfect rooms with gas
  - autoclave before exit
  - collection for urine, faeces
- class 4

Hazards

Physical

trauma
- sharp objects
  - needles
- machines
- maintenance
- light
  - low light -> more risk
- ergonomics
- bite and scratch wounds
  - dogs > cats > rodents
  - cats: deep wounds
  - risks
    - direct trauma
    - infection
      - dogs, cats: Capnocytophaga canimorsus
      - mice
        
        Streptobacillus moniliformis
        
        Spirillium minus
        
        "rat bite fever"
      - check tetanus vaccination
      - check by doctor
      - vet checks animal for diseases
  - primates
    - lab coat, gloves, mask, ...
fire
- combustible material
  - wood
  - paper
  - gasses and liquids (alcohol, ether, ...)
  - liquid nitrogen
noise
- animals
- machines
- solution: ear protection if >85 dB
electricity
pressure
- autoclaves
- gas bottles
- heating -> face protection
radiation
- lasers
- UV-C (255 nm)
  - bio peneration: 1mm
  - acute: affects eyes, reversible, 24-48h
  - long term: lens turbidnes, skin tumours
- ionising
  - types
    - alpha
      - stopped by piece of paper
    - beta
      - stopped by plastic
    - gamma (photons)
      - stopped by thick wall of lead
  - contamination
    - internal: breathing or oral intake
    - external
      - wear hood, gloves, protective gear
      - keep distance
      - prevent dust (bedding!)
  - radio-active waste disposal
    - cadavers: special freezer

Chemical

predictable
- combustability
- reactivity
- corrosiveness
- explosiveness
unpredictable
- toxicity
use personal protection
- gloves, mask, goggles
icons
- flammable
- toxic
- corrosive
risk analysis
examples
- DEN
  - liver cancer
- tamofixen
  - trigger in many genetically modified animals

chemicals with unknown risks
infectious agents
- class 1: no disease
- class 2: moderate risk
- class 3: respiratory transmission, serieus disease
- class 4: no therapy, lethal
transgene animals
- increased risk of human pathogens

Allergens

prevalence: 15-30% of staff
- 10% asthma
factors
- lifestyle, environment
- previous diseases
- genetic predisposition (atopy)
  - 30% of population
  - cats, dogs, pollen
  - hay fever, eczema, asthma
- not: degree of exposure
pathogenesis
- sensitisation: 6-36 months
- effect
  - usually immediately
  - 2-8h possible
sources
- urine
- saliva
- fur particles
symptoms
- nasal
- eyes
- skin rash
- asthma (10%)
  - coughing
  - wheezy
  - shortage of breath
  - can become chronic
- anafylaxis
  - typically from bite
  - minutes - hours
  - mild - lethal
prevention
- reduce sensitisation
- reduce symptoms
- yearly screening programs
  - tests
    - lung
    - intracutaneous
    - antibodies (IgE)
    - contact allergy test
- design of facility
  - ventilation, filtration
  - IVC and filtertop cages
  - clean cages: vacuum > wiping
  - decrease cage density
  - bedding: no wood shavings
- work organization
  - surgery < animal handling < cleaning
  - choice of species
- personal protective equipment (PPE)
  - gloves, lab coat, dust masks, goggles
  - Versaflo 3M

Zoonosis

exam: define + give 1 example
MO in lab animals
- can cause disease in humans
- subclinical - lethal
risks
- wild, conventional animals > SPF
prevention
- isloation measures (cage type)
- PPE
examples
- rats
  - reservoir for plague via fleas
- Leptospirosis = Weil's disease = canicola fever
  - bacterium Leptospira
  - fever, headache, chills, muscle ache, vomit, jaundice, ...
  - spreads via urine
- Hanta virus
  - kidney damage
- Lymphocytic Choriomeningitis Virus (LCMV)
  - adults: ~mild flu
  - pregnant women: neurological disorders in foetus
- salmonellosis
  - diarroea
  - serious in children and weak people
- Streptobacillus moniliformis
  - rat bite fever = Haverhill fever
  - contaminated feed or water
- cow pox disease
  - virus
  - laesions
  - swollen lymph nodes
  - muscular pain
  - fever
  - endemic in rodents
- trichophyty
- herpes B
  - in primates: little effect
  - in humans: can be lethal
  - transmission: blood, urine, saliva
    - contact
      - scrub with brush/disinfectant for 15min
      - blood sample
      - antiviral cure (if needed)
  - incubation: 2-30 days
  - symptoms
    - herpes vesicles
    - headache
    - tired
    - fever
    - conjunctivitis
    - neurological problems
  - PPE: lab coat, mask, goggles

5 Zootechnique

FAQ
- background info
  - taxonomy
  - heart rate, etc.
  - mouse pup development stages

Rodents

teeth
- I 1/1, C 0/0, P 0/0, M 3/3
- keep growing
nocturnal
social hierarchy
newborns
- precocial = newborns are self-sufficient
  - guinea pigs
- altricial = newborns are helpless
  - mice
  - rats
  - hamsters
diet
- omnivore
  - mice
  - rats
- herbivores
  - hamsters
  - guinea pigs
coprophagy
- ingestion of faeces

Mice and rats

Mus musculus
Rattus norvegicus
most used animals (80%)
- easy to breed
- small
- cheap
research
- cancer
- drugs
- toxicology
strains
- dwarf mouse (C3H/HeJ)
  - growth hormone deficiency
- obese mouse
- nude mouse
  - no thymus -> no competent T-lymfocytes
  - sensitive to infections and cold
- Zuckerrat
  - obesity research
Harderian gland
- = tear gland behind eye
- in animals with nictitating membrane (3rd eyelid)
- secretes red-brown porphyrines (cf. heme)
oestrus cycle (4-5 days)
- pre-oestrus: 0.5d
- oestrus: 0.5d
- met-oestrus: 1d
- di-oestrus: 2-3d
gender
- anogenital distance
  - short: female
  - long/double: male
breeding
- monogamous, trio, harem
- Lee-Boot effect
  - no male in group
  - suppressed oestrus cycle
- Whitten effect
  - male introduced
  - oestrus sync in females
  - less clear in rats
- Bruce effect
  - successful mating
  - 2nd male introduced within 24h
  - no implantation
  - not in rats
- copulation plug (CP)
  - proof of mating (24h)
  - no proof of fertilization
anatomy
- plexus ophthalmicus
  - orbita-puncture
- lungs
  - left: 1 lobe
  - right: 4 lobes
- stomach
  - pars non-glandularis
  - pars glandularis
- rat: no gallbladder
- uterus
  - duplex
handling
- pick up by tail
- scruff

Hamsters = Mesocricetus auratus

rarely used
research
- breeding
- hypothermia
sensitive to antibiotics
hip glands (esp. in males)
breeding
- monogamous or harem (difficult)
- no fostering
gender
- anogenital distance
  - short: female
  - long/double: male
handling
- cupping
- scruff
anatomy
- big pouches
- lobed liver
- lungs
  - 1 left lobe
  - 4 right lobes

Guinea pigs = Cavia porcellus

strain: Dunkin-Hartley
related to
- Chinchilla
- capibara
purpose
- control of serums and vaccins
  - easy to sensitize
- immunology research
- feeding research
no clear day/night rhythm
very sensitive to antibiotics
gender
- push on abdomen to reveal sex
breeding
- monogamous or harem (1:8-10)
- before 7 months, else symphysis pubis
handling
- support abdomen

Rabbits = Oryctolagus cuniculus

research
- toxicity tests
- pyrogenity tests
- antisera
- atherosclerosis
strains
- Dutch (< 2 kg)
- New-Zealand White (2-5 kg)
teeth
- 2(I 2/1, C 0/0, PM 3/2, M 3/3)
- keep growing
glands to mark territory
- chin
- anus
breeding
- no retrieval
- no need for colostrum (milk)
  - passive immunity through placenta
- little feeding
gender
- pull skin flap under tail
coecotrophy
- type of coprophagy
- night: soft, mucus-covered faeces produced by caecum
  - rich in vit B and K, low fiber
  - re-ingested
- day: hard droppings
handling
- lock hind legs
  - powerful kicks can break its bones/back
anatomy
- marginal ear vein
- big caecum

African clawed frogs = Xenopus laevis

live in water
lungs
organogenesis research
pros
- very big eggs, deposited in water
- robust embryos
- simple transgenic embryos
housing
- copper, chlorine free water
- transparent cages
- water flow
- pH: 7.5 - 8.5
- T: 18-20C
- no direct sunlight
  - 12/12 day/night cycle
feed
- 3/week
- lean meat or pellets
gender
- female: 2x size, black thumb cushions
handling
- grasp around hind legs
marking
- autotransplant skin (abdomen/light -> back/dark)
- picture
- chip

Zebrafish = Danio rerio

tropical fresh water fish
pro
- small (adult 4-5cm)
- rapid development
- transparent embroys, larva
water
- pH: 6.9 - 7.5
- T: 28.5C
- quality: no metals, ...
gender
- male: leaner, yellowish belly
- female: silver-like
feed
- 2-3/day
- flakes, pellets

Pigs = Sus scrofa

pro
- similar to humans
  - skin
  - skeleton
  - GI tract
  - pancreas
  - kidneys
  - cardiovascular
social groups
- form at young age
floor
- grid
- concrete
- bedding
avoid boredom -> enrichment
breeding
- heat lamps for piglets
- fostering OK
handling
- piglets: in arms
- adults: herd in corner with screen
transport
- approach calmly
- sedate stress sensitive animals
- keep cool
marking: ear label

6 Housing

Cages and accessories

cage material
- wood
- plastic
  - light
  - easy to clean
  - types
    - polycarbonate (Macrolon)
      - transparent
      - autoclave = steam sterilizer
    - polypropylene
      - white
        
        better for breeding
    - polystyrene
      - ~transparent
      - disposable
      - washable at 80
- stainless steel
  - heavy
  - easy to clean
  - expensive
  - acid cleaning
cage floor
- closed
  - bedding
    - purpose
      - absorp faeces, urine
      - comfort
      - insulation
      - enrichment
    - requirements
      - dust free
      - no resins (no pine wood)
      - no contaminants / micro organisms
    - types
      - wood shavings
      - corn cob
- mesh
  - pro
    - excrement falls through
    - no risk of drowning
  - con
    - possible paw injury
    - no bedding
water supply
- acidified
- chlorated
- irradiated with UV
- automatic supply system
  - pro
    - low effort
    - fresh water
  - con
    - intake control
    - clogging
    - infections
- types
  - bottle
  - bowl
    - large animals
    - small immobile animals
    - requires fixation + cleaning
feedhoppers / feedracks

Environmental enrichment

encourage natural, species-specific behaviour
- and discourage abnormal behaviour
why
- lower stress: less fighting, less barbering
cons
- cost
- hygiene
types
- social
  - group housing
- dietary
  - hay / straw
- sensory
  - auditory
  - visual
  - olfactory
- physical
  - cage size, design
  - toys

Housing systems

conventional
- rooms
  - gutter, drained, sloped floor
  - window in door
  - lab coat per room
- corridor
  - large
  - insect lamps
- washing room
  - clean side
  - dirty side
- feed and bedding storage
  - cool, dry, dark
  - near clean washing room
semi-barrier
- keeps unwanted micro-organisms out
- barrier units (single or multi-room)
  - 5 characteristics
    - germ tight
    - lock for transfer
    - ventilation with sterile air
    - lock for staff
    - observable clean zones from outside
- isolators (sealed box with two holes with gloves)
  - flexible film (PVC), Trexler
  - pro: cheap, simple, disinfectant
  - con: glove perforation
  - 5 characteristics
    - lock for materials and animals
    - observable
    - ventilation
      - sterile air in
      - dirty air out
    - handle objects from outside
- ventilated cabinets
  - airtight
  - pos/neg pressure
  - controlled env
    - temp
    - relative humidity (RH)
    - light cycle
  - handle in laminar flow
- filtertop cages
  - removable, airtight filter
  - effective
  - low cost
  - handle in laminar flow
    - protects contents from handler
    - not the other way around
- individually ventilated cages (IVC)
  - input
  - output
  - calamity filter
  - 70 air changes/h
  - less cage changes required
  - protects staff and environment
A2 facility
- focus on what comes out
SPF facility (specified pathogen-free)
- focus on what goes in
metabolic cages
- measure feed, water, excrement
transport
- stress depends on species, age
- low density
- no sick animals
- acclimatisation upon arrival

Legal

homeostasis
ventilation
- filtered air
- temperature
  - homeothermic
  - rodent: 20-22C
    - use heat pad during surgery
  - rabbit: 18-20C
- relative humidity (RH)
  - too high
  - too low
    - ringtail in rats
- cycle: 10-20/h
- no blind corners
- no draught
- exhaust (NH3, CO2) close to floor
noise
- incl. ultrasonic
light: dim
- crepuscular: active during twilight
- nocturnal
- tips
  - cover eyes during surgery
  - do not put cages on top of racks
smell
- Whitten effect in mice
  - male urine induces estrus in females
  - cf. zootechniques
- cage cleaning
- mice fear predators (rats, cats, ...)
  - wash hands
  - change clothes
  - keep in separate rooms (overkill?)

7 Hygiene

prophylaxis
- medical
  - vaccination
    - staff: tetanus, hep B
    - small animals: N/A
    - large animals: myxomatosis, Rabbit Haemorrhagic Disease Virus
  - preventive antibiotics
- sanitary
  - source
    - animals
      - microbiological status
        
        germfree
        
        gnotobionts: fully known microflora
        
        specific pathogen free (SPF)
        
        conventional: uncontrolled
      - quarantine
      - clinical investigation upon arrival
    - staff
      - no pets of same species
      - barrier protocols
      - personal hygiene
        
        shower
        
        clothing
        
        SPF: sterile, mask, gloves, hair cap, overshoes
        
        conventional: lab coat, mask, gloves, hair cap
        
        wash hands (alcogel)
        
        no jewellery in barrier
    - biological material
      - mouse antibody protection (MAP) test
        
        inoculation + test after 4 weeks
      - rat antibody protection (RAP) test
      - PCR
    - insects & vermin
      - building design
      - airlock
      - flycatcher
    - other material
      - disinfection and sterilisation
        
        methods
        
        physical
        
        heat (flame, dry, humid/steam)
        
        "STERILE 🌡️"
        
        radiation (UV or gamma)
        
        "STERILE R"
        
        filtration (0.2 um)
        
        chemical
        
        liquids
        
        phenols
        
        Dettol
        
        halogens
        
        aldehydes
        
        alcohol
        
        acids
        
        ...
        
        gasses
        
        ethylene oxide ("STERILE EO")
        
        formaline
        
        H2O2
        
        releases free oxygen radical
        
        applications
        
        feed
        
        irradiate
        
        autoclave
        
        bedding
        
        irradiate
        
        autoclave
        
        water
        
        irradiate (UV)
        
        autoclave
        
        chlorinate
        
        filtration (0.2 um)
        
        air
        
        in room
        
        formaline
        
        H2O2
        
        peracetic acid aerosol
        
        supplied air
        
        HEPA filter
        
        equipment
        
        irradiate (UV)
        
        autoclave
        
        chemical (liquids and gasses)

Pathology and microbiological standardisation

example question: "discuss 2 of the most commonly found (viruses|bacteria|parasites) in an animal facility"
disease
diagnostics
infectious causes
- pathogenicity of micro organism (MO)
  - virulent
  - obligate
  - facultative
- source
  - viruses
    - mouse hepatitis virus (MHV)
      - corona virus
      - infection: 2-3 weeks
      - transmission: direct contact, airborne
        
        very contagious
      - mostly subclinical
      - changes
        
        immune system
        
        hepatic enzymes
    - ROTA-virus
      - = epizootic diarrhoea of infant mice (EDIM)
      - subclinical in adults
      - young mice: diarrhoea, expanded abdomen
      - transmission: airborne, dust, bedding
    - ectromelia
      - poxviridae (mouse pox)
      - transmission: direct contact, mites
    - mouse adenovirus (MAD-1, MAD-2)
      - no clinical effects
      - kidney damage
      - transmission: oral
    - parvovirus
      - e.g.: Minute Virus of Mice, MPV, H-1, Kilham rat virus
      - transmission: direct contact, urine, faeces
      - usually asymptomatic
        
        else brain necrosis, hepatitis, haemorrhages
    - mouse norovirus
      - no clear symptoms
    - hantavirus
    - ...
  - bacteria
    - corynebacterium
      - gram positive
      - skin infection in immunocompromised nude mice
    - staphylococcus aureus
      - origin: humans
      - can cause abscesses
    - clostridium piliformis (Tyzzer's disease)
      - gram negative
    - pasteurella pneumotropica
      - gram negative
    - pseudomonas aeruginosa
      - gram negative
    - mycoplasma pulmonis
      - gram negative
    - helicobacter
      - enteric bacteria
      - opportunistic
      - heliobacter hepaticus
        
        liver lesions
        
        start: 1-4 months
        
        progress: 6-8 months
        
        hepatitis (incl. gal bladder)
        
        chronic IBD (incl. caecum, colon)
        
        tumors
        
        rectal prolaps
      - detection: PCR, ELISA
      - treatment: triple antibiotic
      - eradication: room sanitation + repopulation
    - salmonella enteritidis
    - streptococcus pneumoniae
    - ...
  - protozoa
    - encephalitozoon cuniculi
      - rabbits > mice
      - causes meningoencephalitis
    - pneumocystis carinii
      - protozoa? fungus?
      - subclinical
      - immunocompromised: pulmonary disease
    - entamoeba muris
    - giardia muris
    - spironucleus muris
  - fungi, yeast
    - trichophyton spp.
    - microsporum spp.
  - endoparasites
    - pinworm
  - ectoparasites
    - mites
other problems
- barbering
- fighting

Disease management

factors
- species, strain, individual
- disease
- therapy
- experiment
- institute policy
- economy
rodents
- acute + high prevalence
  - quarantine
  - stop breeding
  - example: MHV, Sendai
- acute + low prevalence
  - isolate breeding pairs, test, use negative pairs
- slow transmission
  - test and cull
  - example: Ectromelia
- persistent infections
  - depopulation, disinfection and repopulation, rederivation
  - C-section, embryo transfer
  - example: most bacteria
- transplacental transmitting agent
  - rederive and test
  - example: Parvo
- zoonosis
  - depopulation, disinfection and repopulation
  - example: LCM
microbiological quality control
- control barrier (process)
  - autoclave
  - pressure difference
- control animal (product)
  - health monitoring
    - direct
      - isolation/grow
      - visualisation by histology
      - PCR
    - indirect
      - antibodies (Ab)
        
        seroconversion: 3-4 weeks
    - techniques
      - pre-necropsy
      - necropsy
      - bacteriology
      - serology
      - parasitology
    - sampling
      - from
        
        animals from colony
        
        sentinels (brought in colony to test)
      - beware FP, FN
    - FELASA directives
      - standardization
        
        freq, samples, methods, report, ...

8 Anaesthesia

properties
- reversible
- predictable
- verifiable
important
- use healthy animals
- influence on params
- interaction with other drugs (e.g., antibiotics)

General anaesthesia

4 cornerstones
- mental block: loss of consciousness
- sensible block: no pain
- locomotory block: muscle relaxation
  - striated muscles
    - incl. diaphragma
      - requires ventilation
  - products
    - curare
    - succinylcholine
    - pancuronium
  - never use by itself
- autonomous block: repress reflexes
how
- mono-anaesthetic
  - pentobarbital
  - isoflurane
- balanced anaesthesia (combination)
  - ketamin/xylazine
phases
- premedication
  - sedation = tranquilizer
    - minor tranquilizers decrease muscle tension
    - products
      - benzodiazepines
        
        diazepam (Valium)
        
        midazolam
        
        (Xanax)
      - medetomidine
      - xylazine
      - droperidol
    - administration
      - oral
      - intramuscular (IM)
      - intravenous (IV)
    - metabolism
      - liver and kidneys
      - bind to GABA
        
        induce hyperpolarization, reducing APs (depolarization)
    - limited hypotension, vasodilation
  - prevent side-effects of anaesthesia
    - counters
      - saliva
      - bradycardia
      - bronchoconstriction
    - product: atropine
      - parasympathicolyticum = anticholinergic
        
        blocks ACh receptors in motor neurons
      - decreases tear production
        
        use synthetic tears or eyecream
      - no effect on 30% of rabbits
- induction = introduction
  - induction
    - analgesic + sedation
  - excitation
    - enhanced reflexes
    - increased muscle tension
    - pupil dilated
    - more tears and saliva
  - surgical
    - respiration decreases
      - frequency
      - depth
    - pupil narrows
    - slow reflexes (eyelid, cornea, toe)
    - decreased muscle tension
    - finally: reflexes absent
  - hypoxic
    - euthanasia due to overdose
    - breathing stops
    - heart stops
- maintenance
- recovery
depth
- factors
  - respiration
    - frequency
    - depth
  - heart rate
  - reaction to pain
  - reflexes
    - turn around
    - eyelid
    - swallow
    - pupil
    - in rodents
      - tail pinch
      - toe

Local anaesthesia

no mental block
minimal impact on params
products
- lidocaine (Xylocaine)
- procain 1-2%
types
- superficial
  - mucous membrane (eyes, trachea?)
  - examples
    - EMLA cream
    - fentanyl patches
  - max 40mg/kg
- infiltration
  - injection
    - intradermal
    - subdermal
  - combine with adrenaline for vasoconstriction
- regional = conduction
  - perineural
    - complete innervation are of nerve
  - spinal block
    - epidural between L and S outside of dura mater
    - causes stress
      - combine with sedation

Injection

neurolept_anaesthesia
- combines
  - morphine-like analgeticum
  - major tranquilizer = neurolepticum = antipsychotic
- products
  - Hypnorm = fentanyl + fluanisone
  - Thalamonal = fentanyl + droperidol
- cons
  - muscle tension
    - add minor tranquilizer = anxiolytics = anti-anxiety
      - benzodiazepines
hypnotics
- deep sleep
  - mild analgesic
- products
  - barbiturates
    - pentobarbital in small animals (medium term)
    - thiopental in large animals (short term)
    - methohexital
  - chloralhydrate
  - etomidate
  - alphachloralose
  - urethane
    - long effect
    - carcinogenic
      - only used in terminal experiments
dissociative anaesthesia
- cortical vs subcortical brain activity
- effect
  - analgesic
  - increase muscle tension
  - hallucinogenic
  - saliva +
- product
  - ketamin
    - combine with sedation (benzodiazepines/xylazine) to decrease convulsions
    - requires license
-adrenergic agonists
- mild-moderate analgesic
- moderate muscle relaxation
  - inhibit release of noradrenaline
- goal: premedication / sedation
- products
  - xylazine
  - medetomidine
- antagonists
  - yohimbin
  - atipamezole
- metabolism: liver and kidneys
- side-effects
  - IV: vasoconstriction -> hypertension -> hypotension
    - counter with parasympathicolyticum (atropine)
  - hyperglycaemia
  - low insuline
  - blood pressure
  - heart rate
  - ...
pros
- simple requirements
  - syringe
  - needle
  - scale (weight)
cons
- poor depth regulation
- long recovery
  - solution: antagonists
- overdose due to storage in fat and muscle
routes
- intravenous (IV)
  - products
    - barbiturates
  - pros
    - quick effect
    - depth regulation
- intramuscular (IM)
  - easier than IV
  - cons
    - slow
    - depth regulation
    - unpredictable effect
    - limited product choice
      - ketamin
      - Hypnorm = fentanyl + fluanisone
      - not: barbiturates (tissue necrosis)
- intraperitoneal (IP)
  - in small animals for practical reasons
  - cons
    - see IM
    - risk of hitting organs (bladder, ...)
- subcutaneous (SC)
  - slow

Inhalation

products
- chloroform (now forbidden - health hazards)
- ether
  - pros
    - cheap
    - anaesthesia + analgesia + muscle relaxation
  - cons
    - irritant
    - respiratory disease
    - flammable, explosive
    - not inert
      - induces liver enzyme activity
  - ethical committee: negative advise
- halothane
  - hepatotoxic
  - no longer sold
- enflurane
  - not irritating
  - not inflammable
- N2O, laughing gas
  - moderate effect
  - with oxygen (1:1 or 2:1)
  - often carrier for other products
- isoflurane
  - halogenated ether
  - depress BP, respiration
  - non-flammable
  - inert
    - metabolism: exhaled
  - risk
    - with adrenaline -> arrhythmias
concentration
- f(species, depth)
- MAC = minimum alveolar concentration
  - 50% of animals no longer react to standard stimuli
pro
- depth control
- inert / limited metabolisation
- fast recovery
con
- expensive equipment
- environment pollution
  - risky for pregnant women
mechanism
- apparatus
  - cylinder: O2
  - cylinder: N2O
  - valves to reduce pressure
  - evaporator for liquid anaesthesia (enflurane, isoflurane)
  - 3-way system
    - inhalable gas mixture
    - to animal
    - exhaled air
  - carbonic acid absorber
    - soda lime = NaOH (5%) + silicates (5%) + Ca(OH)2 (90%)
      - changes color when saturated
    - capture CO2
- cap or mask over nose and mouth
- min 30% oxygen
- uptake via alveola
- intubation
  - for long sessions
  - trachea tube + inflatable cuff
  - large animals: laryngoscope
consequences
- on
  - CNS
  - cardiovascular
  - respiratory
  - liver/kidney
- risks
  - hyperglycaemia
  - shock
  - no homeostasis
  - mortality
  - polluted experimental data
response to anaesthetics
- depends on
  - species
    - hamsters less sensitive to Nembutal
    - mice and cats: excitated from morphine
  - age
  - strain
  - weight
  - gender
  - ...
- breakdown ~ body surface instead of BW
conclusion
- no fixed recipe
- do literature study

Systems

open
- gas from cotton humidified with anaesthetics
- use jar or mask
- no recirculation
- no filters
- inhale: free air + gas mixture
- exhale: N/A
halfopen
- uses cap over nose, mouth
- inhale: O2 + N2O + (liquid or gas) anaesthetic
- exhale: pressure valve + balloon
- goal: reuse anaesthesia gas
halfclosed
- halfopen + cabon acid absorber
closed
- closed circuit
- re-use or exhaled air after CO2 absorption

Pain

types
- acute
- chronic
- derived
- superficial
- deep
nociceptors
- pressure
- temperature
- chemical
tissue-dependent
- brain: insensitive
- skin, eye, bone, chest, abdomen: very sensitive
principle of analogy
- painful in humans -> also in animals
- "benefit of the doubt"
animals choose painkillers if possible
signs of pain
- activity decrease
- appearance
  - hunched back
  - erected fur
  - dirty eyes (no grooming)
  - tremor
- temperament changes (agressive)
- noises
  - some ultrasonic
- food
- params
- twitching
- balance loss
- writhing: lateral muscle contraction
  - Writhing is defined as a stretch, tension to one side, extension of hind legs, or contraction of the abdomen so that the abdomen of the mice touches the floor, or turning of the trunk (twist).
- eyes
- nose
- cheeks
- ears
- whiskers
analgesia
- general anaesthetics
- specific analgetics
  - central acting
    - opiates = narcotics
      - morphine-like
        
        fentanyl
        
        methadon
        
        meperidine
      - specific antagonists in case of overdose: naloxon
      - very addictive -> need license
    - side effects
      - sedation vs excitement
      - cardiovascular
      - respiratory
      - heat regulation
      - GI, appetite
      - immune
  - peripheral acting
    - act on nerve ends
    - anti-inflammatory (NSAID)
    - post-operative
    - products
      - aspirin
      - paracetamol
      - gemirixin
      - ketoprofen
      - meloxicam
    - side effects
      - gastric ulcers
      - kidney damage
      - anticoagulation
- routes
  - oral
  - SC
  - IM
  - IV

Postoperative care

quiet
individual housing
no bedding
no sharp edges
feed and water only when fully awake
temperature
check behaviour

9 Humane endpoints

trade-off
- too early
  - repeat experiment
  - more animals
- too late
  - loss of data
standard operating procedures (SOP)
- clinical criteria
- observation schemes
- appoint evaluation staff
codes of practice
- control of welfare
- immunisation
- cancer research
important
- don't wait until dying
- when in more (incurable) discomfort than required
when
- stops eating/drinking
- loses 15% BW in 1-2 days or 20% since start
- serious respiratory or circulatory problems
- aberrant behaviour
- tumour causes clinical symptoms
- tomour too big
  - mice: 10% BW or 2 cm3
  - rats: 10% BW or 40 cm3 or 4.2cm diameter
- scoring list
decrease inconvenience
- experimental technique
- adjust care and housing
- pain control
euthanasia
- = painless death
- when
  - end of experiment
  - humane endpoint
  - organ research
- how
  - pharmacological-chemical
    - anaesthetic overdose
      - barbiturates
        
        IV
        
        intracardial
    - CO2
      - 6:4 CO2 O2 in closed container
    - K overdose
      - IV
      - cardiac arrest
      - painful -> under anaesthesia
  - mechanical-physical
    - small animals
      - decapitation (guillotine, scissors)
      - cervical dislocation
    - large animals
      - bullet
- who
  - trained staff
- impermissible
  - hypothermia
    - too slow
  - snap freezing
  - hyperthermia
    - too painful
  - drowning
    - too slow
  - strangulation
  - crush neck
  - bleeding
  - narcotic overdose
    - unpredictable
  - sedative and ketamin overdose
    - large volumes
  - air embolism
    - painful
    - unreliable
  - strychnine
  - neuromuscular blockers (KCl, curariformes)
    - respiratory arrest before unconsciousness
- double check death
- animal waste
  - specialized company (Rendac)
  - excl. radioactive or bio-contaminated waste

Overview table (background info)

1) Shall, where appropriate, be used with prior sedation. 2) Only to be used on large reptiles. 3) Only to be used in gradual fill. Not to be used for fetal and neonate rodents. 4) Only to be used for birds under 1 kg. Birds over 250 g shall be sedated. 5) Only to be used for rodents under 1 kg. Rodents over 150 g shall be sedated. 6) Only to be used for rabbits under 1 kg. Rabbits over 150 g shall be sedated. 7) Only to be used for birds under 5 kg. 8) Only to be used for rodents under 1 kg. 9) Only to be used for rabbits under 5 kg. 10) Only to be used on neonates. 11) Only to be used for birds under 250 g. 12) Only to be used if other methods are not possible. 13) Specialised equipment required. 14) Only to be used on pigs. 15) Only to be used in field conditions by experienced marksmen. 16) Only to be used in field conditions by experienced marksmen when other methods are not possible.

10 Behaviour and wellbeing

ethology: study of animal behavior especially under natural conditions
homeostasis
- disturbance -> cannot adapt? -> stress -> decreased wellbeing
species-specific behaviour
- genetics
  - evolutionary adaptation
  - still displayed in unfit environment without any effect
  - ethogram: behavioural repertoire
    - frequency
    - duration
    - succession
- learned from experience
  - Tolman experiment: animals use knowledge about territory
  - place-dependent behaviour
    - example: don't tolerate rival within specific area
    - superficial changes can feel very radical
      - ventilation
      - caretaker
      - smell
      - cage mates
  - causality and expectations
    - caretaker enters -> feed
    - unpredictable behaviour -> stress
      - Weiss experiment
        
        stress ~ predictability, influence
        
        3 rats, each in cage with light and wheel
        
        cage 1: light -> tail shock, postpone by turning wheel
        
        cage 2: no light or wheel, shocked together with cage 1
        
        cage 3: control (no shocks)
stress
- acute
  - sudden decrease in predictability or influence
  - ACTH hormone
    - adrenal cortex: more corticosterone + cortisol
  - adrenal medulla: (nor)adrenaline -> fight or flight
    - increase heart rate, BP
    - blood: brain, muscle > internal organs
    - liver: glucose + cholesterol
  - adaptive mechanism
    - no adaptation -> chronic stress
- chronic
  - stomach acid
    - prancreas: alkaline enzymes
    - hypochlorhydria = low stomach acid
  - hippocampus: damage + shrink
    - memory impaired
    - mild stress (adrenaline) enhances memory
coping
- available means
- threat to homeostasis
- individual differences
- 2 extreme reactions
  - active coping
    - fight/flight
  - passive coping
environmental enrichment
- increase controllability
  - build nest
  - hide
  - avoid light
measure wellbeing
- biological
  - reproduction
  - health
  - physiology (hormones, immunological status)
  - sound?
- behaviour
  - preference test (4 chambers)
  - abnormal behaviour
    - conflict behaviour in acute stress
      - ambivalent
      - redirected
        
        towards animals/humans/other
        
        completely different
    - stereotype behaviour
      - = ritualized conflict behaviour
      - pigs: tail biting, bar biting, pacing
  - cognitive functioning
  - eating latency
conclusion
- experiment: standardization
  - limits animal wellbeing
  - careful trade-off

11 Experimental techniques

Identification

rings
- ear
- paw
- wing
tattoos
- mostly on larger animals
- rat: tail
burnmarks
- not allowed anymore
natural marks
- unique pattern
  - e.g., cows, frogs
- take pictures (left + right)
ear notch patterns
- nr = 10 * left + right
microchip (RFID)
- in fish
- ear tag in mice

Drug administration routes

transdermal
- ointment on skin or mucosa
- works for limited substances
- inaccurate
- irritating?
enteral (GI)
- types
  - oral
    - via feed or water
    - bad taste?
    - insoluble?
    - chemically unstable in water?
    - gavage via gastric tube or stomach cannula
      - curved tube with blunt end
      - via mouth or nose (in larger animals)
      - check if not in trachea -> cough?
  - suppository
- inaccurate
parenteral (i.e., other)
- injection
  - restrain
  - use thin, sterile needle
  - types
    - subcutaneous (SC)
      - mice: neck or armpit
    - intraperitoneal (IP)
      - head down so bowels can slide up
      - inject in lower abdomen
      - aspirate to check for blood or urine
      - mice: 1 person
      - rats: 2 persons
    - intramuscular (IM)
      - mice: thigh
        
        cave: sciatic nerve (n. ischiadicus)
    - intravenous (IV)
      - rats: tail
        
        warm up -> vasodilation
        
        always aspirate
    - intradermal
      - shave first

Blood withdrawal routes

orbitapuncture
- sample from retro-orbital venous plexus in mice and rats
  - plexus opthalmicus
- procedure
  - apply anaesthesia
  - use two fingers around head to make eye pop out slightly
    - don't suffocate
  - push/rotate small capillary tube behind eye
ear vein
- in rabbits
tail vein
saphenous vein
- in leg of mice, etc.
submandibular (cheek-puch)
foot
sublingual vein
- in mice and hamsters
larger samples
- cardiac puncture
  - almost always terminal
  - inject anaesthesia overdose after puncture
- aorta puncture
  - requires surgery
  - terminal
- decapitation
  - only rodents and birds
  - guillotine
  - terminal
  - only use if no other method is possible

12 Surgical techniques (guest)

goals
- surgery training
- develop new surgical techniques
alternatives to animal experiments
- virtual endotrainer
- post-mortem humans
types
- ectomy
- transplantation
  - auto
  - allo
- implantation
definitions
- sepsis: presence of pathogenic MO in living tissue
- asepsis: absence of pathogenic MO in living tissue
- antisepsis: prevent sepsis by destroying MO
- disinfection: $10^6$ reduction of MO on non-living objects
- sterilization: complete elimination of MO
stages
- pre-operative
  - remove hairs
    - in separate area
    - cave: hypothermia
  - fixate animal
  - skin antisepsis
    - never completely sterile
    - products
      - chlorhexidine soap (or in 70% isopropylalcohol)
        
        preferred
      - isobetadine 7.5-10% in alcohol
    - pattern: circular (in -> out)
  - draping: create sterile field
    - criteria
      - impervious to water
      - impervious to bacteria
      - lint free
      - fire resistant
    - types
      - reusable
        
        gore-tex
        
        4-drape technique
      - disposable
        
        adhesive
        
        non-adhesive
  - instrument sterilization
    - autoclave
      - not for gummi, latex, PVC
      - quality control
        
        chemical indicator
        
        biological indicator
    - dry heat
      - ~30min at 180C
      - e.g., hot bead sterilizer
        
        not entire instrument
    - chemical
      - liquid (rinse afterwards)
        
        glutaraldehyde 2%
        
        alcohol 70%
        
        dettol 5%
      - ethylene oxide (EO)
        
        special equipment
        
        long aeration
      - radiation (R)
        
        gamma
  - people
    - scrub
      - nails
    - gown, hairnet, mask
    - gloves
      - size
      - no powder
      - perforated -> change
        
        double?
      - gloving technique
  - animal
    - check health status
    - fasting? (see H16 feed)
    - eye protection: artificial tears
- per-operative
  - laminar air flow, HEPA
  - minimal passage
  - correct surgical technique
  - monitor depth of anaesthesia
  - check homeostatis
    - temperature
      - heating pad with rectal probe
    - fluid balance
  - control blood loss
    - pressure
    - electrocauter
    - ligature (tie suture around vessel), vascular clamps
    - surgical vessel repair
    - hemostatic agents (cellulose, gelatin, collagen)
- post-operative
  - cover wounds
    - spray, bandage, ...
    - drain?
    - avoid lick/scratch/bite
  - antibiotics?
    - prophylactic
    - curative
rodent surgery
- large number in limited space
- instrument re-use
- smaller dimensions
surgical instruments
- criteria
  - durable
  - retain sharpness
  - no corrosion
- maintenance
  - clean after use
  - pH neutral detergent
  - rinse
- examples
  - scalpel
    - pencil grip
  - electroscalpel (mono- or bipolar)
    - pro
      - less bleeding
      - less suture
      - less time
      - sterilization
    - con
      - slower healing
      - more infection
      - muscle or nerve stimulation
  - needle holder
    - grip
    - hold needle in middle third
  - scissors
    - grip
    - types
      - sharp
      - blunt
  - forceps
    - grip
    - types
      - thumb (i.e., tweezers)
      - hinged
        
        tissue forceps
        
        Adson
        
        slightly traumatic
        
        Babcock
        
        atraumatic
        
        Doyen
        
        intestinal clamp: occlude lumen
        
        hemostatic forceps (good grip but traumatizing)
        
        Halsted-mosquito
        
        Rochester-Pean
        
        Rochester-Oschner = Kocher
  - hemostatic clamps and clips
    - bulldog clamp
    - hemoclip applier: permanent clips
  - tissue retractors
    - hand-held
    - self-retaining
  - needles
    - body shape
      - 1/2 circle
      - 3/8 circle
      - 1/4 circle
      - ...
    - radius, needle length (curved), wire diameter, chord length (straight)
    - types
      - rounded (taper)
        
        point: rounded
        
        body: rounded
      - reverse cutting
        
        point: 3 cutting edges
        
        body: 3 cutting edges
      - taper cut
        
        point: 3 cutting edges
        
        body: rounded
  - sutures
    - criteria
      - easy to handle
      - tensile strength
      - knot security
      - non-capillary
      - sterile
      - cheap
    - classification
      - size
        
        EU
        
        US pharmacopeia (USP)
        
        e.g., 11/0
      - length
      - sterilization method
      - natural vs synthetic
      - (non-)absorbable
        
        absorbable
        
        catgut: enzymes + fagocytosis
        
        polymers: hydrolysis
        
        targets: subcutis, muscle, intestine, fascia?
        
        non-absorbable
        
        ~silk (absorbed after 2 years)
        
        polyester
        
        stainless steel
        
        nylon
        
        polypropylene
        
        polyethylene
        
        targets: skin, tendon, vessels, nerves
      - mono- vs multifilament
        
        mono: poor knot security, memory, non-capillary
        
        multi: good knot security, easy handling, capillary
        
        requires coating
    - alternatives
      - staples
      - glue
        
        low tension, dry
      - mesh
    - knots
      - 3+ throws
      - by hand (one or both) or by instrument
    - patterns
      - interrupted
        
        tension adjustment along the way
        
        slower
        
        more suture material
      - continuous
        
        single point of failure

13 Genetics (guest)

Generic standardisation

goal of experiment?
- uniformity
  - transplant
  - cancer research
- variation
  - drugs
  - toxicology
causes of variability
- genetic background
  - interaction, confounding effect
- compounds
- environment
  - housing conditions
  - drug treatment
  - experimental design (e.g., overhandling)
how to standardize
- inbreeding
- IVF
- transgenesis
- cloning
genetic uniformity
- monozygote animals
  - nine-banded armadillo (Dasypus novencinctus)
    - 1 egg -> 4 monozygote pups
    - genetically identical
    - still some phenotypical variantion
      - differences during intra-uterine development
  - cloning
  - inbreeding
- inbred strains
  - increases homozygosity over generations
  - degree: inbred coefficient F
    - fraction of hetero genes that became homo
    - increases per generation
      - depends on relation (degree of cosanguinity)
        
        use brother-sister or parent-offspring
    - 98.4% after 20 generations of sibling inbreeding
      - quite uniform -> "isogenic strain"
  - challenges
    - inbred depression
      - worse performance than natural counterparts
      - decrease in growth, fertility, vitality
    - generation 4-8: lots of drop-outs
    - generation 10+: balances out
  - nomenclature
    - STRAIN/Substrain
    - approved abbreviations
  - have unique characteristics
- co-isogenic strains
  - spontaneous mutation in (isogenic) inbred strain
  - different in 1 gene -> "co-isogenic strain"
  - nomenclature
    - mouse gene: Abc
    - mouse allele - dominant: Abc ${}^{Def}$
    - mouse allele - recessive: Abc ${}^{def}$
    - STRAIN/Substrain-Gene^allele
  - not always easy to maintain line
- F1-hybrid
  - = Filial 1
  - cross two inbred strains
  - genetically and phenotypically uniform
    - more uniform in morphology
    - more variable in behaviour
      - improved ability to adapt
        
        = Tryon effect
  - "heterosis"
    - = hybrid vigor
    - more disease resistent
    - better stress coping
    - live longer
    - larger litters
  - nomenclature
    - mother always comes first
      - reciprocal cross gives different results
    - full: (ABC/De x FGH/Ij)F1
    - abbr: ABFGF1
- congenic strains
  - goal: introduce specific gene in inbred strain
  - procedure
    - cross of recipient with donor
    - repeat 10+ times
      - intercross resulting children
        
        meiosis: crossing over
      - select children with desired traits
      - backcross with original inbred strain
  - nomenclature
    - RECIPIENT.DONOR-Allele
    - use Cg for unknown/complex/F1 donor
  - use DNA markers to reduce number of repeats (5-7)
  - double congenic lines
- chromosome-substitute strains = consomic strains
  - inbred strain where chromosome is replaced by homologeous chromosome of another inbred strain
  - procedure
    - repeated backcrossing
  - nomenclature
    - HOST-Chr #^DONOR
      - superscript: non-italic, all caps
        
        cf. co-isogenic
- recombinant inbred (RI) strains
  - procedure
    - start from 2 progenitor strains (S1, S2)
      - both inbred strains
    - mating individuals from F2 generation of cross
  - nomenclature
    - S1XS2-#
    - always abbreviated
  - studies on linkage
- recombinant congenic strains (RCS)
  - series of inbred strains, which are derived from the second or third backcross generation of 2 unrelated progenitor strains
  - procedure
    - 20x inbreeding
  - nomenclature
    - prefix RCS
    - RCS S1cS2-#

Genetic variation

random breeding
- always some spontaneous inbreeding in closed population
- ad random mating
  - $\Delta F = \dfrac{1}{8F} + \dfrac{1}{8M}$
  - - $\Delta F$
    - $= \dfrac{1}{4N} + \dfrac{1}{4N}$
    - $= \dfrac{1}{2N}$
  - $M = F = 50 \implies \Delta F = 0.5\%$
  - $M = F = 10 \implies \Delta F = 2.5\%$
Poiley rotation scheme
outbred strain
- random-bred for 4 generation
- with per generation
  - min $M = F = 25$
- nomenclature
  - Company:[A-Z]{1-4}
- genes will keep varying over generations
problem
- once you have variation
- how to keep constant throughout experiment?
solution
- reciprocal hybridising of inbred strains
- = hybrid population
- = mosaic population
- start with $n$ inbred strains
- all possible combinations
  - $n$ homozygotes
  - $\dfrac{n^2 - n}{2}$ F1 hybrids

Quality control

goal
- preserve strain
- avoid genetic contamination
challenges
- contamination not always phenotypically obvious
check contamination
- skin transplant -> rejection?
- describe genetic characteristics
  - colour genes
  - immunogenetic markers
  - biochemical markers
  - zymogram
    - uses gel-electrophoresis
    - check for
      - enzyme variants = isozymes
      - other proteins
  - DNA markers
    - type I
      - polymorphism in functional genes
    - type II
      - polymorphism in DNA with unknown function
  - PCR: amplify microsatellites
cryopreservation
- freeze sperm or embryos (8 cells = morula)
- goal
  - keep strain or valuable mutant from going extinct
  - avoid genetic draft
  - easy transport
  - avoid unnecessary breeding (reduces animal count)
- how
  - draw out all water
  - freeze
    - challenge: crystals
  - store in liquid nitrogen
  - defreeze
  - sperm
    - intracytoplasmatic sperm-injection in oocyt (ICSI)
  - embryo
    - implant in receptive/pseudo-pregnant female

Transgenese

intro
- goal: insert transgene
- differentiable cell types
  - zygote
  - embryonic stem cells (ES cells)
zygote injection
- inject DNA in zygote (male pronucleus)
  - before first split
- implant in pseudo-pregnant carrier mother
  - 15-20 embryos
- 15-20% of progeny becomes "transgenic"
  - verify integration
    - PCR
    - southern blotting (DNA)
  - verify expression
    - PCR
    - northern blotting (RNA)
    - western blotting (protein)
    - enzyme linked immunosorbent assay (ELISA)
    - ...
  - phenotypical analysis
  - "founders" -> new colony
- result
  - gain of function
  - loss of function
- cost
  - mouse: 20 000 EUR
  - cow: >500 000 EUR
- alternatives
  - sperm injection (transgen ICSI)
    - sperm as carrier for the transgene
    - limited to embryo stage
  - retroviral gene transfer
    - target: 8-16 cell stage
- other animals
  - chicken
    - no zygote injection (thick membrane)
    - transfection of blastoderm cells
  - fish
    - no implantation, external development
    - zebrafish
      - transparent embryos
      - fast breeding
  - xenopus tadpole
- pro
  - fast
  - simple
  - various species
gene targeting
- homologous recombination
- extract inner cell mass (ICM) from blastocyst
  - ES cells (undifferentiated, pluripotent)
- isolate and culture
- targetting construct (skipped)
  - knock-out (KO) or knock-in (KI)
- selection
- screening for HR
- re-implant in carrier mother
  - option 1: blastocyst injection
  - option 2: morula aggregation
  - result: chimeric blastocyst
- grows into chimeric mouse
  - different fur colors (on purpose)
    - ranges from 5-95%
- breed with wild type until line is stable
- pro
  - full integration control
  - alterations: KO, KI, mutation, ...
- con
  - only mice
cloning (skipped)
- cf. Dolly
- identical copies
- via nuclear transfer
knock down (skipped)

14 Ethics (guest)

anthropocentric
- instrumentalization of animals
- = speciecism = racism
zoocentrism
- e.g. PETA
- equality: rat = dog = pig = child
KU Leuven animal testing policy
- human life > animal life
- human: highest level of consciousness
- use animals with lowest level possible
moderate viewpoint
- serious animal interests > trivial human interests
- all animal interests < basic human interests
disproportional
- using animals for fun
- ritual slaughtering
- cosmetics toxicity testing
- fur
schools of animal welfare
- functioning
  - best conditions for optimal functioning
    - growth
    - reproduction
    - fitness
    - adaptiveness
- behavioural
  - respecting animal specific characteristics
  - natural environment
  - behavioural repertoire
  - environment enrichtment
- feelings
  - non-suffering
  - happiness
- integrity
  - respect physical and mental integrity
  - no force feeding
- robustness
five freedoms
- from thirst, hunger
- from discomfort
- from pain, injury, disease
- to express natural behaviour
- from fear, distress
no-unless policy
3 R's

15 Alternatives

motives
- scientific
  - faster
  - easier to replicate
  - less variability
- ethical
- economical
  - costs time and money
1959: Russel and Burch, “The principles of Humane Experimental Technique”
- 3 R's
  - replace
  - reduce
  - refine
  - (responsibility)
BE: law of 1993
alternatives must
- be as good as system it replaces
- meet formal validation
- be completely evaluated
- accepted by users and government

Replace

options
- in vitro
  - cell and tissue cultures
  - polio vaccin test
    - 4500 monkeys/year in 60s
    - 4 monkeys/year today
  - skin irritating test
    - 1944: Draize test on rabbits
    - 2001: forbidden
    - now: commercial options (EpiDerm, Episkin)
      - more accurate than animal tests
      - measure how fast keratinocytes are killed via RNA presence
        
        4h: strong
        
        24h: moderate
        
        48h: weak
        
        else: no irritation
    - also: test on humans
  - cons
    - missing biokinetics
    - missing metabolisation, bio-transformation
- embryos
  - chickens, rats, frogs, fish
  - no nerves = no pain
  - 2010 EU directive: anaesthesia for foetal forms
  - CAM (chorioallantoic membrane) test
    - example 1
      - on day 10 in chicken eggs
      - apply test substance for 20s on membrane
      - rinse
      - evaluation
        
        hyperemia
        
        hemorrhage
        
        coagulation
    - example 2: tumor research
- lower organisms
  - invertebrates: insects, bacteria, ...
  - 2010 EU directive: includes some invertebrates
  - example: LAL (limulus amoebocyte test)
    - replaces pyrogenic tests on rabbits
    - pyrogen: substance that induces fever
    - process
      - take blood from horseshoe crab
      - extract amoebocytes
      - incubate with test substance
      - toxins -> blood clots
    - not perfect (false positives)
- audio-visual aids
  - in education
  - replace dissection
- others
  - organs of dead animals or humans
    - source: slaughterhouses
    - Draize eye test
      - on chicken or cow eyes
    - learn suturing techniques
    - test systems (perfusion, ...)
    - surgery simulation
  - human volunteers
    - typical for cosmetics
  - inner bicycle tube to learn suturing
  - artificial animals
    - Koken rat: learn to sample blood or gavage
    - PVC rat: learn to inject or cannulate
    - back of cow with intestines
    - artificial GI system

Reduce

ethics
experiment design
- calculate desired sample size (statistics)
pilot study
- first species might be unsuited
- learn new (less invasive) method
- optimize humane endpoints
sharing
cryo preservation
- instead of continuous breeding to keep strain alive
computer models
improved storage, exchang and use of data
- exchange negative, unpublished results
- databanks
telemetry
- implant transmitter
- params: BP, heart rate, temp, ECG, ...
good lab practice (GLP)
combinations

Refine

housing, care, health, behaviour
trained staff
pain control
- anaesthesia
- analgesics
- humane endpoints
environmental enrichment
non-invasive methods
- CT scan
  - tumour risk
- PET scan
  - tracer
  - can check gene expression
- bioluminiscence
  - luciferase enzyme from fireflies
  - needs O2, won't work in necrotic tissue
- MRI / MRS (spectroscopy)

16 Feeding

types
- natural
  - closed formula
    - ingredient list without quantities
    - minimum % fat, ash, carbs
    - cheapest
  - open formula
    - range for each component
    - varies from batch to batch
      - buy in bulk to ensure consistency
    - cheaper
- semi-synthetic
  - more expensive
  - worse taste
- synthetic
  - most expensive
  - little variation
  - worse taste
caveats
- soybean contains isoflavones (phytoestrogens)
  - can affect reproduction
- toxic plants
  - internal
    - tannines
    - strychnine
    - cyanide
  - external
    - herbicides
    - heavy metals
- botanic contamination
  - weeds (e.g., nightshade)
  - fungi
    - can have phytoestrogenic actions
    - Aspergillus flavus and A. parasiticus
      - produce Aflatoxine B
- antinutritional compounds (ANF)
  - e.g., trypsin inhibitors
- alfalfa
- fish
- bio-availability
  - phosphorus
    - 2/3 tied up in phytates
      - cannot be digested, except by ruminants
      - problem: too much P in manure
    - solution: add phytase enzyme to feed
excessive omega 6 + low omega 3 intake -> generational obesity
ready to use feed
- composition
- preparation
  - pelleted feed
    - pressed at high temperature
  - expanded feed
    - using steam
    - for cats, dogs, primates
    - higher cost, waste
- storage
  - 3 months (sometimes 6 or 9)
  - cool, dark, dry place
  - protect from vermin
- physical form
  - meal
  - pellet
    - can't add substances
  - gel/liquid
- sterilising
  - autoclave
    - causes loss of ingredients
      - add extra buffer to start with
  - gamma radiation
nutritional needs
- goal: max growth
- deficiencies
  - leucine (Leu, essential AA)
    - white fat: lipolysis
    - brown fat: upregulate UCP1
    - decrease fat mass
  - linolic acid
    - skin problems
    - immune problems
    - kidney malfunction
    - heart malfunction
  - vitamin D (rachitis)
    - weak bones
  - vitamin K
  - vitamin B1 (thiamine)
    - uncoordinated muscle movements
  - vitamin C (ascorbic acid)
    - muscle weakness
  - magnesium
    - weak legs
    - poor growth
  - copper
    - skin/hair proteins
- excess
  - zinc
    - alopecia
- factors
  - genetics
  - life stages
    - growth
    - pregnancy
    - lactation
  - environment
    - lower temp
      - higher needs
      - higher uptake
    - higher temp
      - lower uptake
        
        increase nutrient content
    - steel cages: less zinc
    - ingested bedding
  - microbiological status
    - conventional: intestinal flora produce vitamin K, etc.
    - germfree: requires compensation
  - research procedures
    - animals eat less if unpleasant flavour
    - solutions
      - mask taste and smell (e.g., add sugar)
      - gavage = force feed
      - pair-fed: control group also eats less
  - nutrient interactions
    - e.g., regular + experimental feed
    - active resorption competition
    - animals aim for fixed energy need
      - high energy density -> less uptake
feeding and research
- troubleshooting
  - diarrhoea
  - pathologies
  - reproduction issues
  - bad intake
  - => check feed, contact other users, exclude from experiment?
- ad libitum feeding
  - method of choice in rodents
  - has risks (aging, cancer, obesity, ...)
  - 12 "meals" per day
    - 8 during dark
  - rat: 3-6h before stomach is empty
- restricted feeding
  - during night: most natural
- cohousing
  - dominant animals eat more
- anaesthesia
  - rodents cannot vomit: no restrictions needed
  - fasting
    - can cause
      - hypoglycaemia
      - dehydration
      - hypothermia
    - worse in mice
    - solution
      - remove feed few hours before
      - remove water 1h before
  - low protein diet -> takes longer to recover from barbiturates