Examenvragen

Question 1

Definition of laboratory animal

Answer 1

Every living vertebrate animal that is used or intended for laboratory experiments:
• Including: free living and/or reproducing larval forms
• Excluding: other foetal or embryonic forms
Changed:
• Including: some invertebrates, like cephalopoda
• Including: mammalian embryonic forms from the last third of term (pregnant mouse foetuses are also seen as lab animals)

Question 2

Definition of animal experiment

Answer 2

Every use of a living animal for experimental and other scientific purposes that can inflect pain, suffering, discomfort or permanent injury.

• Including: every treatment that has the purpose/consequence of the birth of an animal in such a condition.
• Exception of the least painful, in modern practice accepted methods to kill or mark an animal
• Excluding: non-experimental treatments in agriculture and veterinary practice

!!! (an animal should not be used more than once in a experiment that inflicts serious pain and suffering -> different degrees of pain: mild, moderate, severe)

Question 3

For what purpose can laboratory animals be used?

Answer 3

1. Production and control of sera, vaccines and diagnostics
2. Toxicological and pharmacological research
3. Diagnosis of diseases
4. Education
5. Answer scientific questions
   !!! NOT for cosmetics!!

Question 4

Source and identification of laboratory animals

Answer 4

Source:
1. No stray (= straat, zwerf), lost or abandoned animals
2. Mice, rats, guinea pigs, hamsters, rabbit, primates
o Purpose-bred by licensed breeding companies
o Exemption (=vrijstelling) (if you want to use an animal that is not purpose-bred): if difficult to find, strong motivation
3. Farm animals
o Pig, sheep
4. Threated species living in the wild: special demand needed
Identification:
• Dogs, cats and primates need to be marked individually and permanent immediately after weaning
• Register must be kept of all animals coming in and leaving the lab

Question 5

Explain the three R’s of Russel and Burch + give 2 examples

Answer 5

Reduction
= Reduce number of animals to the minimum
1. Choosing well the experimental design
2. Standardizing the laboratory animal population and the experimental procedures
3. Sharing animals, pilot studies

Replacement
= Replacing the animal experiments with another method or an invertebrate if possible (non-sensitive)
Ex: 
1. Organs of dead animal/human
2. in vitro research of cell/tissue
3. organ cultures

Refinement

1. Less discomfort, minimum of pain/stress
2. Gathering knowledge about biological needs of the animal
3. applying the analgesia
4. Environmental enrichment (cage)
5. well-trained staff

=> First replace, if not then reduce and refine

Question 6

What do you need before starting animal experiments?

Answer 6

1. Laboratory licence
   It’s a governmental laboratory licence given by the Federal Public Service, department Animal Welfare
   In the application form:
   • Overview and map of the rooms (description and function)
   • Overview of the kind of experiments that will be performed
   • List of the species and their source (van waar ze komen)
   • Overview of the staff, responsible for the projects and working with the animals
   o Laboratory director (RESPONSIBLE manager)
   o Expert (protection of the health and well-being)
   o Project leader (set up, logbook)
   o Biotechnicus (conduct the experiments , deze voeren de exp uit)
   o Animal caretakers
2. Permission from the Ethical Committee
   The Ethical committee is obligatory in labs and institutes since 2001 (tasks and composition p 22)
   Ethical committee: has a professional secrecy for all the members
    tasks:
   - evaluation of the experiments
   - setting up the ethical criteria concerning an experiment
   - advising labo’s and the government
   - reporting to the government
    composition: lab director, project leader, bio-technician, veterinarian or expert, independent members (governmental inspector is no longer a member)
   In the application form:
   • Staff responsible for the project
   • Purpose and description of the project
   • Number and species of the animals
   • Degree and duration of pain, suffering and injury
   • Anaesthesia and analgesia that will be used
   • Alternative methods
   • Human endpoints and euthanasia

In the new legislation:
• Competences! At least 7 members are present and at least all the competences are present to improve the project
• No conflict of interest allowed ( eigen project -> niet zelf bëoordelen)
• Non-technical summary to inform the public
• Post experimental evaluation
o Evaluation of the number of animals
o Evaluation of the pain and suffering
o What was the gain of the project?

3. Education (FELASA and BCLAS)
   On every level (since 2004 obligatory):
   - project leaders (80h - FELASA C)
   - researchers and lab technicians (40h – FELASA B)
   - animal caretakers (25h – FELASA A)
   You need continuous education and every year you need to follow a training about treating animals
4. Animal welfare body, Dieren welzijnscel (begrip) NEW!
   Set up by every user (include animal caretaker, scientist, veterinarian and expert)
   • Advice on animal welfare (care, use, accommodation)
   • Review internal operation processes (you have to do what you promised to the EC)
   • Improve animal welfare and transparency (ultimate goal: better follow-up of human endpoints, improve protocols, rules/laws followed?)
   • Genetically altered animals (p28) (every animal that has a likely harmful phenotype can suffer due to genetic alternations)
   • Reporting all animals used in the creation of the new line (p 31 of 32)
5. Other paperwork
   • Import license: if importing animals from non-EU member states (Q: do you need a licence to import animals? -> yes)
   • Bio-security dossier
   • Radio-activity form (when you use it)

Question 7

Zootechnique (Begrip)

Answer 7

All knowledge about the animals to keep them in good health: knowledge of housing, taking care, feeding and breeding.

Question 8

Bruce effect (Begrip)

Answer 8

When another is introduced into a cage 24h after a successful mating, there won’t be an implantation -> spontaneous abortion. It’s a priming effect, directed by pheromones from the urine

Question 9

Orbitapunction (Begrip)

Answer 9

Behind the eyeball of the mouse and rat, there is a net of venous blood vessels (Plexus Opthalmicus). If you take blood from this: orbitapunction

Question 10

Whitten effect (Begrip)

Answer 10

When a male is introduced by the females -> oestrus synchronisation (also priming effect)

Question 11

Lee-Boot effect (Begrip)

Answer 11

When females are kept in group, there is a tendency to anoestrus. (priming effect)

Question 12

Harderian gland (Begrip)

Answer 12

i.e. tear gland, found in animals with a nictitating membrane, 3th eyelid (knipvlies). Found in rat and mouse.
• It produces liquid with a red-brown colour that contains porphyrins
• Can’t see it because they wash it away, unless something is wrong

Question 13

Coprophagy/Coecotrophy(Begrip)

Answer 13

Rabbit and rodents: soft faeces pass at night from the caecum to the colon, overlaid with mucus (rich in vitamin B and K). They are taken up (eaten) from the anus directly. During the day they become hard droppings.

Question 14

Altricial (Begrip)

Answer 14

They stay in the nest and are dependent of the mother, they are incapable of moving around after being born. Mostly they are blind, nude,… (e.g. mouse, rat, hamster,.. )( precocial)

Question 15

Precocial (Begrip)

Answer 15

In a few hours they can feed themselves (solid feed), they are capable of moving around. They go away from the nest because they can live without their mother. They are relatively mature when they are born (e.g. guinea pig)( atricial)

Question 16

Diastema (Begrip)

Answer 16

Gap between the teeth of rodents because of the structure

Question 17

Hip glands (Begrip)

Answer 17

i.e. a scent gland. Especially in male they are well developed,they use it to determine their territorial. (e.g. hamster)

Question 18

Xenopus Laevis

Answer 18

• Frog live in the water and breath trough their lungs. They come to the surface regularly to breath trough their nostril
• They are used for research in organogenesis and genetics because they are very big and deposited in the water.

Embryo’s

1. Big and available in large number
2. Robust and suited for micro implantations
3. Accessible in all stages of development (maps!)
4. Transgenic embryo’s can be made in a simple and fast way

Housing and feeding
= Chlorine and cupper free water and transparent cages, with flow to prevent water pollution.
1. Avoid direct sunlight
2. pH 7.5-8.5, T=18-20°C
3. 3x per week feeding (lean meat, pellet food)
4. 1.5 h after feeding, the water is skimmed (= thin layer on the water)

Breeding: Amplexus (Begrip) Type of mating behaviour in Xenopus Laevis.
= The male grasps the loin of the female with his forelegs, female sets off her eggs and they are inseminated with the sperm.

Handle: around the hind legs + palm of hand

Marking:

1. autotransplant
2. picture
3. chip

!!! Species specific behaviour: get to know everything
• Use the right animal model
• Femals are twice as big as males

Question 19

Housing of the zebrafish (danio rerio): tropical fresh water fish

Answer 19

• Optimal measures: water pH 6.9-7.5, T= 28.5°C, ammonia, clorine and copper as low as possible
• Water changes: 10-20% a day
• Feed: small amounts: 2-3 times per day (flakes, pellets), remove surplus of food 1h after feeding
• Male is leaner than female and more yellow, female is more silver
• Larves and embryos are transparent: easy to follow development

Question 20

Nude mouse

Answer 20

(zie cursus, later)

Question 21

Germfree, gnotobionten, SPF, conventional animals

Answer 21

zie cursus

Question 22

What are the sources of infections and how can you prevent this in an animal facility?

Answer 22

By performing prophylaxis measures the infection pressure can be decreased. It also helps to protect from pathogens and prevent spreading of disease. These measures can be medical (e.g. vaccination of the staff and large animals and preventive antibiotic administration) or there is sanitary prophylaxis, which can be divided by the source of infection:
1. Animals: use only animals that are free from pathogens (know the microbiological status of your animal!), put new animals in quarantine, do a clinical investigation upon arrival.
2. Personnel and researchers: there is a strict protocol (e.g. not taking care of animals you have home RESPONIBILITY) and rules to enter into barriers (48h rule), personal hygiene, shower, clothing (sterile, mask, gloves, hair cap, overshoes), wash hands and disinfect when entering or leaving the animal facility, after using toilet. Don’t wear any jewellery or watches.
3. Biological materials (e.g. cells, sera, tumours,…): should be tested before using, with PCR or MAP/RAP-test: inoculation of virus-antibody free material and do after 4 weeks a serum sampling and identification.
4. Insects/vermin: design of the building, disinfection of rooms, air lock, flycatcher,…
5. Other material: should be sterilized and controlled after delivering. First clean, rinse and dry to remove biological material and use then physical methods (heat-radiation-filtration) or chemical method (liquid-gasses). E.g. feed need to be irradiated and autoclaved
• Health monitoring (eventueel)
• Disease management (eventueel)

Question 23

Sanitary prophylaxis, explain and give 2 examples

Answer 23

Prophylaxis: infection pression can be decreased, protect from MO and prevents the spreading of disease. These measures can be medical (vaccine + AB) or sanitary which can be divided by the source of infection:
•	Animals
•	Staff
•	Biological material
•	Insects and vermin
•	Other material

Question 24

Disinfection, sterilization methods

Answer 24

Physical:

1. heat (flame, dry heat, humid heat ~> autoclaves)
2. radiation (UV, gamma)
3. filtration.

Chemical:

1. liquids (alcohols, halogens, acids,..)
2. gasses (ethylene oxide, formalin gas, hydrogen peroxide)

E.g. food = irradiation, autoclaving
E.g. bedding material = autoclaving, irradiation (different order!)
E.g. water: autoclaving, filter (0,2), chlorination, UV

Question 25

Why do animals become ill and what to do?

Answer 25

• Combination of harmful environment factors that exceeds the capacity to adapt
  • Food (shortage of vitamins)
  • Housing conditions (too hot, too humid)
  • Experimental conditions (incompetent application of techniques, administration of some products)
  • Infections
  • Genetic factors (some strains are more sensitive)
• Adaption mechanism itself is decreased and equilibrium is disrupted
  • Irradiation
  • Administration or immunosuppressants
  • Nude-, scid-, and beige- mutations
• Adaptation mechanism is exceeded by abnormal metabolism
  • Diabetes or tumours
  Diagnosis: tracing and defining the problem
• case history and syndrome: general information (species, age, nature of experiment,..), determined problems, changes in environment
• clinical investigation: appetite, fur, behaviour
  -additional behaviour: biopsies, fur- and skin monsters, blood sampling, sampling from throat, nose,..
• post-mortem investigation: bring the animal to the lab as soon as possible because the autolysis and decay starts immediately after death. Put the organism in 4% formaldehyde.

Question 26

Causes of infections

Answer 26

• Viruses
• Bacteria and mycoplasma
• Protozoa
• Fungus and yeast
• Endo-/ectoparasites

Question 27

Pinworm (begrip)

Answer 27

Endoparasite: lives in the intestines -> eggs in faeces -> spreading and indigestion of faeces -> infection keeps going
• no symptoms, hard faeces, losing weight, …

Question 28

Why is there health monitoring, how do you implement this in practice and why is this important? (p38 e.v.)

Answer 28

Health monitoring is important:

1. since knowing the microbiological status can give you an idea of disease and mortality
2. but it can also interfere with the experimental results
3. and there is a risk for the staff for zoonosis.

So barriers should be checked but also the animal.
You can do this by using direct methods
• Isolation/grow the agent
• PCR
• Visualization of the agent by histology
Or by using indirect methods
• Demonstration of antibodies
• Seroconversion after 3-4 weeks
It consists of: (pre-) necropsy, necropsy, bacteriology, parasitology,..

You can screen animals:
• From the colony: young animals have more parasites and it’s easier to detect recent viral antibodies. In older animals it’s better to detect the history of the colony (chronic infections).
• Sentinels: animals that are brought into the colony to test. Mostly Swiss or DBA mice, it must be able to produce Ab’s ant it’s housed in open cages (airborn pathogens can enter) where it receives dirty bedding material of the other cages (a lot of infections are passed by faeces).

The animal facility is tested 4x a year, 3 standard and 1 additional screening.
!!! Tests are not completely sound -> false-positive and false-neg are possible

Question 29

Zoonose (begrip)

Answer 29

MO that is carried by a lab animal and that can cause a disease in humans. E.g. LCM: disease management = depopulation, disinfection and repopulation or herpes.

Question 30

Anthropocentric (begrip)

Answer 30

= humanopocentric. Animals were excluded from ethical consideration: only human beings (persons) had intrinsic value because they are considered as subjects. Animals were objects, things, ‘instruments’. This position gave legitimation to any kind of animal use: killing for food/fur, sport hunting, use for research and educational purposes.

Question 31

Zoocentric (begrip)

Answer 31

The opposite of humanopocentric. Animals have a moral standing. Animals also have a intrinsic value/wordt so they are also considered as members of the royal community = ‘moral objects’.

Question 32

Animal welfare body

Answer 32

= traditional animal protection movement; animal welfare consists of five different schools:

• Functioning school: creating the best conditions for optimal functioning
• Behaviour school: fulfilling natural behavorial needs
• Feelingsschool: non-suffering, being happy, fun, pleasure
• Integrity school: respecting physical and mental integrity
• Robustness school

Question 33

Anaesthesia (begrip)

Answer 33

pathological sleep: reversible, predictable, verifiable

• Repress consciousness and pain perception temporarily

Question 34

How can you know if the anaesthesia is deep enough?

Answer 34

• Respiratory frequency, depth
• Heart frequency
• Reaction to pain stimuli
• Testing reflexes (turn-round, eyelid, pupil,..)

Question 35

Different types of anaesthesia

Answer 35

1. Local anesthesia
2. Sedation
3. Premedication
4. Neurolept anesthetics (REAL anaesthetics)
5. Hypnosis
6. Dissociative anesthesia
7. Alpha-adrenergic agonists
8. Muscle relaxation
9. Injection anaesthesia
10. Inhalation anaesthesia

Question 36

Local anaesthesia

Answer 36

= Effect limited to a part of the body. Not really anaesthesia since there is no loss of consciousness

Bv.: Lidocaine or procaine
I/ Used for puncture, biopsies, removing skin tumours

Sorts:
1. Superficial administration
o Added on the surface where it’s needed
o Droplet, cream, gel
o Surface must take up the anaesthetic (e.g. mucous membrane)
2. Intra- and subdermal infiltration
o Injected in tissue to anesthetise the nerve fibres (limited area: small depot, larger area: diamond)
o Often adrenaline is added to cause vasoconstriction: decrease the blood flow (increase of effectiveness and decrease of blood flow)
3. Regional/conduction anaesthesia
o Perineural injection: complete innervation area of the nerve is anesthetised
4. Spinal block: complex of nerves is anesthetised. Epidural injection + this can cause stress because the animal is completely paralysed (motor nerves are switched off) -> give sedation.

Advantages:

• Influence on normal physiological processes outside the anesthetised area is minimal
• Catheter: anaesthetics can be given for a long time to maintain the anaesthesia

Question 37

Sedation

Answer 37

= Tranquilizing effect without considerable pain killing, to calm down nervous animals
• Benzodiazepines (hypnotic, myorelaxing, anxiolytic)
o Binds to GABA receptor -> Cl- influc -> hyperpolarisation -> decrease sensitiveness
o Often combined with ketamine to decrease it’s convulsive effects
• Used for diagnose, taking samples, as preparation for general anaesthetics (premedication).
• Can be induced orally, intramuscular, intravenous
• Metabolised in liver and eliminated through kidneys
• Cardiovascular and respiratory effects are rather weak: only little vasodilatation and hypotension

Question 38

Premedication

Answer 38

= pre-treatment with farmaca to prevent unwanted effects of anaesthesia (e.g. bronchoconstriction, saliva secreation,…)
1. Induce general anaesthesia smoothly
2. Atropine
3. Decreases tear production: use synthetic tears, eye cream (to prevent eye from drying out)
!!! Rabbits don’t always react on it: atropine-esterase inactivates it

Question 39

Neurolept anaesthetics (REAL anaesthetics)

Answer 39

= basal general anaesthesia is induced by combination of neurolepticum and morphine like analgeticum to allow minor surgery.
• Often a minor tranquilizer like diazepam, midazolam is added to reduce muscle tension

Question 40

Hypnosis

Answer 40

= state of deep sleep when it’s deep enough, the pain perception is reduced

Bv.: Barbiturates, urethane
~> Different types with a different duration of effectiveness
o Short acting thiopental: larger animals
o Medium term acting pentobarbital: small animals (rodents)

!!! In physiological experiments: combination of alphachloralose and urethane often used so that the physiological parameters are little influenced (terminal experiments)

Question 41

Dissociative anaesthesia

Answer 41

= state where junction between cortical and subcortical cerebral activities is lost
~> the animal no longer feels connected and aware of its environment.

Mechanism:

1. Painkilling
2. Causes muscle tension
3. It is hallucinogenic
4. Increases saliva secretion

Bv.: Ketamin is mostly used
!!! Drug for human -> specific legislation -> drug is stored locked

Question 42

Alpha-adrenergic agonists

Answer 42

= mild to moderate analgetic
• causes moderate muscle contraction
• used for premedication/sedation
• metabolised in liver + excretion by kidneys

Mechanisme:
1. have specific affinity for alpha-2-receptors
o by stimulating the peripheral vascular receptors, an intravenous injection causes severe vasoconstriction -> hypertension
o rapidly followed by a hypotension because of centrally increased vagustonus and bradycardia
2. sedative effect by inhibiting release of NA at reticular system
3. myorelaxation and analgesic, mostly by inhibiting the nociceptive afferent-vessels
4. stimulating the contraction of the sphincters and relax the smooth muscles in the digestive system
5. causes hyperglycaemia and a decrease of insulin in the plasma
6. because of the direct influence on the centre for thermoregulation, an important hypothermia develops
=> cause several side effects on blood pressure, heart rate,… (check this before using them in your experiment.)

Question 43

Muscle relaxation

Answer 43

=>stimulation of the striated muscles is blocked at the crossing nerve-muscles
!!! animal is completely motionless, but is fully conscious
-> use this only in combination with anaesthetic and analgetic never by itself
!!! ventilate because also the respiratory muscles are paralysed (animal can’t breathe)

Bv.: curare, succinylchorine,..

Question 44

Inhalation anaesthesia

Answer 44

= substances that evaporate well at room temperature and can be administered through the respiratory system

Types:
1. chloroform: older, but forbidden because of health hazards
2. ether: cheap, irritating, coughing, salivary bronchial excretion, flammable and explosive
o causes stress to the animal: effects the eyes -> rubbing the face (EC says NO)
3. halothane: no longer sold, hepatotoxic
4. !!! NO (carrier gas) and isoflurane are normally used

Remarks:
• necessary concentration depends on species and anaesthetic depth
o MAC= minimum alveolar concentration= concentration where 50% of the animals no longer react to standard stimuli
• limited metabolism (inert, unchanged in the body)
• Need expensive equipment to see the depth of the anaesthesia (disadvantage)
• Gaseous anaesthetics are given in combination with a breathing gas (30% O2) into respiratory tract -> taken up in bloodstream via alveola until gaseous tension blood=lungs (application by a cap or a mask)
• Endotracheal tube (p2 deel2)

Question 45

Injection anaesthesia

Answer 45

• intravenous, intramuscular, intraperitoneal

* overdose!

Question 46

Urethane is a hypnotic compound with a big advantage and disadvantage. Explain

Answer 46

Disadvantage:

1. psychological side effects. The combination of urethane and alfachloralose is frequently used in psychological experiments. Often, psychological parameters are being influenced.
2. Urethane is also carcinogenic so only use it in terminal experiments

Advantage: It stays effective for a long time

Question 47

Explain open, half-open and half-closed gas anaesthesia system

Answer 47

1. Open system
   The animal breathes in free air mixed with liquid anaesthesia gas, released from a cotton humidified with anaesthetics. E.g. mouse in a jar with cotton close to the nose or mixed with oxygen.
2. Half-open system
   Air or oxygen is directed via a liquid anaesthetic or a gas anaesthetic to a cap or a trachea tube. A breathing balloon and a valve can be added to the system to remove exhaled air.
3. Half-closed system
   Between the balloon and the valve there is a CO2 absorber -> the valve can be closed more, so less anaesthetic is blown into the open air.
4. Fully closed system
   Now there are two valves: during breathing in one is open and the other is closed: exhale and inspire valve. The CO2 absorber filters the exhaled air, so that it can be inspired again.

Question 48

Why is it important to know how animals in pain behave? + treatment

Answer 48

Because you can see if your animal is in pain and you can do something about it. KNOW YOUR ANIMAL!

1. Less activity
2. Tremor, dirty eyes (no cleaning behaviour)
3. Very aggressive when they are normally very quiet
4. Scream when picked up
5. Less taking up of food and water
6. ANS: increased hart frequency, blood pressure and respiration
7. Twitching: involuntary muscle movements (begrip) (usually when animal is resting)
8. Back-arching: stretching hind and forelegs, raining the abdomen from the floor and arching the back (begrip)
9. Falling (loss of balance)
10. Writhing: contraction of lateral muscles (begrip)

Treatment (p13-14) with analgesia (=pain killers) (kan ook vraag op zich zijn)
1. Central acting analgetics
• Opiate analgetic = narcotic analgetic: morhine and related substances -> work on the brain
• Side effects: cardiovascular and respiratory effects, gastrointestinal, immune system
2. Peripheral acting analgetics (oral, subcutaneous, intramuscular and intravenous)
• NSAID (= non-steroidal anti inflammatory drug)
• Act on peripheral nerves and receptors
• Usually used for post-operative pain killing
• Possible side effects: e.g. renal damage

Question 49

Post-operative care

Answer 49

1. Housing and general care
   • In quite environment, individual housing
   • No respiratory obstruction (no bedding material), no sharp edges in the cage
   • Feed and water: administered only when the animal is fully awake
2. Temperature regulation and humidity control
   • Keep the animal warm until it has enough muscle activity to keep himself warm
3. Pre- and post-anaesthetic sedation, analgesia
   • Carefully check the animal to make sure it has no discomfort: behaviour, water and feed consumption

Question 50

Human endpoints: how are they determined and what is the importance

Answer 50

There is often a conflict between how far one can go in the framework of scientific work and what is unacceptable.
• Euthanasia is too early: necessary to repeat the experiment (data loss), delaying scientific work, extra use of lab animals
• Euthanasia is too late: loss of data (animal is dead, tissue cannot be used anymore; we are not allowed to wait until the animal is dying)
• If you have to take a human endpoint = euthanasia

It’s determined by Standard Operating Procedures (to determine if the scientific value does no longer meet the Animal Welfare)
1. Describing clinical signs and criteria
o If they don’t eat or drink anymore
o Looses body weight ( >15% short time or >20% larger time)
o Respiratory, circulating problems
o Behaviour and movements of the animal are abnormal
o Tumour causes serious symptoms or the tumour mass is growing too big (10% of the normal body weight in mouse and rat)
2. Determining observation schemes for a rapid identification of the human endpoints
o Score set up
3. Approinting staff to evaluate, keeping data and inform the researcher and the veterinarian for euthanasia

Question 51

Human endpoint (begrip)

Answer 51

The animal must be euthanised if it has more pain than necessary for the research and if this can’t be avoided or decreased by some method. Therefore, an expanded knowledge about the animal is needed together with a good observation of the animal. With the help of evaluation criteria and scores a decision can be made about implementing euthanasia or not.

Question 52

Euthanasia methods

Answer 52

Euthanasia = deliberate ending of life through means of a gentle and painless death
(at the end of an experiment, for the benefit of the research in organs, if the scientific importance is outweighed by the inconvenience of the animal)
Techniques:
- Pharmacological-methods
1. Overdose of an anaesthetic
 E.g. injection of barbiturates (intracardial or intravenous)
 bloodcongestion
2. Use of CO2
 In saturated close container
 Oedema in lungs
3. Overdose of potassium intravenously
 Cardiac arrest, blood in organs and metabolic acidosis
 Animal must be anesthetised (too painful)
- Mechanical psychical methods
o Small animals
 Decapitation (onthoofden :o)
 Cervical dislocation
o Larger animals
 bullet

Question 53

Explain possible hazards

Answer 53

Physical
1. Trauma
o Sharp objects (needles, scalpels -> special container, never recap)
o Machines and material (sharp edges -> protective clothing and safety rules)
o Maintenance (keep places clean -> wet floor!)
o Light (poor visibility: dangerous)
o Ergonomic hazards (heavy object, always doing same movement)
o Bite and scratch wounds
 Training needed
 Secondary infection (chance of infection is determined by site and size, contamination)
 Dog>cat>rodent, but usually rodents
2. Fire and burning (combustible material, gasses, electrical apparatus, liquid nitrogen)
3. Noise (animals, machines -> protection)
4. Apparatus under pressure, gasses and vapours (e.g. autoclave, gas bottle -> protection)
5. Electricity (shock) and electrocution
6. Radiation: UV (!eyes and skin) and ionisation (!DNA) + lasers

Chemical
1. Disinfectants, anaesthetic gasses, chemicals to preserve tissue…
2. Corrosiveness, toxicity, risk of explosion,… (predictable, sometimes less predictable)
Protocol related
= Chemicals of unknow risk and infectious agents, 4 classes of biological risk:
• No disease in healthy human = class 1
• Moderate risk, after oral intake, mucus exposure -> disease = class 2
• Respiratory transmission: serious and lethal = class 3
• High risk, lethal disease -> no therapy = class 4
 Different facilities needed
Risk depends of the characteristics and complexity of the experiment

Allergy (zie verder)

Zoonosis (zie verder)

Question 54

What are the measures for using radioactive compounds?

Answer 54

• Internal contamination by breathing or oral intake of radioactive substances (e.g. radioactive dust on hand or face)
• External contamination: the radioactive source sends out ionisation within the space in which you are present
o Work in a hood (kap) and use drip-trays (mors bakjes)
o Prevent aerosol and dust: bedding material
o Animals are only allowed to transport below a certain limiting value
o Wear gloves
o Keep distance to the source
o Use a protection screen, wear proactive gear (beschermende uitrusting)
o Follow proper training
• Animals that hare treated with radioactive substances are housed in appropriate rooms
o Approval of Radio Protection Service (RPS)
o At the end of the experiment: the cages are checked by the responsible of RPS
o Garbage (bedding,..) must be removed as radioactive waste
o Cadavers of radioactive animals must be kept in a special freezer -> nature and dose of administered tracer

Question 55

Prevalence of allergens

Answer 55

Everyone having contact with animals is liable to get an allergy, but some are more sensitive (lifestyle and environmental factors, previous diseases, genetic predisposition (atopy))
 Degree of exposure is not important for the development of an allergy
Most important allergens: urine of rats (protein), urine and saliva an fur particles of guinea pig.
Through the air: surgical masks aren’t always efficient, better use real mask
Purpose of the preventive measures
• Reduction in frequency of sensitisation
• Reduction in symptoms
Mostly nasal symptoms, skin rash and watery/itchy eyes
Develops over 1 or 2 years
Consists of
1. Screening programs
• Intracutaneous test
• Antibody measurement in the blood (IgE)
• Specific IgE-antibodies
• Contact allergy test
• Lung function test
 Yearly screening and evaluation of the allergic employee
2. Design of the animal facility
• Adjustment of ventilation and filtration systems: increase in ventilation and humidity and separate ventilation for housing and treatment rooms
• Cage cleaners to empty the cages
• IVC units and filtertop cages
3. Work organisation
• Decrease of cage density
• Choice of bedding material (absorbing instead of wood)
• Other tasks: choose other species, less procedures baring risks
• Cleaning cages: vacuum instead of wiping dust
• Clothing: lab coats in the animal facility
4. Personal protective equipment (PPE)
• Skin contact: gloves, lab coat
• Through the air: surgical mask is not always efficient, better to use real dust masks

Question 56

Zoonosis (begrip

Answer 56

MO carried by laboratory animals that can cause disease in humans and many vary from subclinical to lethal.
• Higher possibility in conventional animal or animals from the wild
• Risks limited: using isolation measures and personal protection
• E.g. herpes B, harta virus, cow-pox disease, … (rodents can indirectly spread a number of human diseases, like plague)

Question 57

Factors that are important in housing and consequences: Ventilation

Answer 57

• Supplied air is filtered (no dust and insects)
• Correct temperature and humidity
• 15-20 changes of air per hour
• No blind corners (air everywhere) -no draught (tocht)
• Exhaust of air (remove of e.g. ammonia ‘luchtuitlaat’) -ideally close to the floor

Question 58

Factors that are important in housing and consequences: Temperature

Answer 58

• Checked every day
• Homeothermic: animals like to be in a typical T
• Changes in T or compensating factors are potentially capable of interfering with the experimental data
• Animals try to adapt to T changes by changing
o Behaviour: restricted by cage size/density/cage material…
o Metabolism: depends on body condition, health and age
• Optimal environmental T varies with the species
o Rodents: 20-22°C (actually hot, but this is not possible to work in + aggression among males)
o Rabbit: 18-20°C
• Difference between micro and micro climate
• Consequences
o T too high: low activity, less breeding, death (rabbits)
o T too low: disrupt the oestrus cycle
• Anesthetized animals must be monitored well. Especially small lab animals can die after a successful operation because of hypothermia -> use heat pad

Question 59

Factors that are important in housing and consequences: Relative humidity

Answer 59

HIGH
• Growth of bacteria and fungi
• Encourages ammonia building up in cages
• More difficult to loose heat when air is humid: animals are unable to sweat
• React to heat stress by increasing respiratory rate
• Difficult thermoregulation
LOW
• Ringtail (rat, if you see this -> Relative humidity is too low or dehydration (lack of drinking))
• Dehydration in nude mice and rats
• Death in pups

Question 60

Factors that are important in housing and consequences: gasses

Answer 60

CO2 gas
•	Originates from respiration
•	Causes no problems
•	<1500 ppm
NH3 gas
•	Originates from urine
•	Irritating for respiration and eyes
•	<20 ppm/<10 ppm for rabbits
Solution: filtertop cages and changing the cages regularly

Question 61

Factors that are important in housing and consequences: Noise

Answer 61

• Animals have a better auditory system than human, hear ultrasonic sounds (> computer).
• Sudden loud, unexpected and unfamiliar sounds: more disruptive than constant loud noise
o Avoid background and unexpected noise by putting on the radio
• Mechanisms by which sound can affect the mammalian body: complex, dose-dependent, relating to the intensity, loudness and frequency of the sound.
• Consequences:
o Cannibalism
o Disturbance oestrus cycle, less fertility
o Less growth (mice)
o Hypertension (rats)
o Changed blood chemistry
• E.g. Silentone fire alarm

Question 62

Factors that are important in housing and consequences: LIght

Answer 62

• Crepuscular/nocturnal in their habits: eyes are adapted to dim light conditions and very restricted to colour vision.
• Light induced retina damage is not uncommon in albino animals
o Effect increases when recovery periods in darkness are too short
o Area should be foreseen with < 25-40 lux
• But room lighting level of around 125-130 lux is adequate for all routine procedures (caretakers have to see) and special areas have a level of +- 500 lux.
• Very bright light (operating lamp) can cause irreversible damage within 1h -> cover eyes during the procedure (maatregel tegen licht tijdens de operatie)
• Difference in light intensity at the top/base of the racks (rek). So don’t put cages on top of the racks.
• When changes are introduces in the day-night rhythm: problems with breeding
• Light-dark cycle can be reversed when doing behaviour studies (data gathered in active phase of the animal), so that the caretaker doesn’t have to do the nightshift

Question 63

Factors that are important in housing and consequences: Smell/olfactory system

Answer 63

• Mediating social encounters, competitive aggression among males, influence physiology trough priming effects (e.g. Whitten effect in mice)
• Cage cleaning:
o necessary
o scent-marking patterns are too frequently disturbed. Smells make the animal feel home so put some old bedding material in a new, clean cage -> refinement.
• Inbreeding/transgenesis might have an effect on the olfactory system:
o Influence on behaviour (aggression)
o Affect responses in behaviour experiments
• Some animals should not be housed in the same room
o Rats are the natural predators of mice, they’ll show fear responses when they encounter anaesthetised rats
o If housed in the same room: mice might become aware of rat by olfaction, even if they don’t see them
• Change clothing and wash hands after handling predator species (rat and cat) or their bedding to avoid causing fear reactions in mice.

Question 64

Factors that are important in housing and consequences

Answer 64

1. Ventilation
2. Temperature
3. Relative humidity (optimal: 50-55%)
4. Gasses
5. Noise (This and the following parameters are not directly linked to the cages)
6. Light
7. Smell/Olfactory system

Question 65

What is environmental enrichment/cage enrichment

Answer 65

= everything that improves the display of natural, species-specific behaviour and that decreases or stops abnormal/pathological behaviour.
Goal:
• Enhancing quality of life and research (less stress)
• Form of refinement (>3R’s)
Cons:
• Higher cost and workload
• Hygiene problems
• Toys can sometimes provoke fighting
Different forms:
• Social enrichment: contact with cage mates and humans
o Q: can animals be housed together: Yes -> social enrichment (apart cage but with transparent wall, but no if prey and predator
• Dietary enrichment: offering hay/straw, way of presenting feed, animals have to search for it (foraging).
• Sensory enrichment: auditive (radio), visual, olfactory (dirty bedding in new cage)
• Physical enrichment: dimensions in the cage, design of the cage, toys
Practice: carton, carton toilet paper (autoclaved) and kitchen paper, shredded paper (nesting), commercial enrichment (mouse house, happy mats), bottles, carton boxes…
Effects:
• Less fighting by aggressive males
• Less aggression in certrain inbred mouse strains
• Calm the animal and provide a better living environment (pigs, primates)
• Les barbering in mice (begrip) -> afknabbelen van de huid door de dominante muis
o If 5 male in a cage, usually 4 have spots without fur -> fighting
• Also important: space, workload, hygiene, cost, possibility to observe the animal

Question 66

Barbering (begrip)

Answer 66

afknabbelen van de huid door de dominante muis

Question 67

Barrier systems + pro’s and con’s

Answer 67

= keep MO out and keep the animals as healthy as possible
Preventive hygiene measures -> applicable to different levels (complete/practical facility, 1 or several rooms, 1 rack or group of cages or 1 single cage)
Purpose:
• Protection of the animal, personnel and environment
• The higher the risk, the tighter the barrier
1. Barrier unit
= 5 essential characteristics
• Complete germ-tight unit (a room or a group of rooms) , from which the clean zone can be disinfected
• Lock for transfer of material or animals
• Ventilation: sterile air goes in and out
• Lock for staff
• Possible to observe and control the clean area from outside (so that you don’t have to go in)
2. Isolator
PCV (flexible film) or Trexler isolator
• Pros: cheap, simple to handle, peracetic acid as disinfectant.
• Cons: perforation of cage for the permanent gloves
• 5 essential characteristics
o Closed sterile room -> from which the content and the inside can be sterilized
o Observe content from outside (one or more transparent walls)
o Lock for material and animals
o Ventilation: sterile air supply and exhaust of dirty air
o Possibility to work and handle animals from outside
(differences with barrier unit: gloves instead of personnel lock, no direct contact with the annimals)
3. ventilated cabinets
• Pros: Sealed and airtight (luchtdicht); used under pos/neg pressure; controlled environment (T, RH) and light cycle
• Cons: animals must be handled in laminar flow
4. Filtertop cages
• Easy removable and airtight filter (polyester)
• Pros: effective hygiene barrier -> housing of immunodeficient animals, low cost, prevention of cross-contamination
• Cons: handling in laminar flow, high T, humidity,…
5. Individually ventilated cages (IVC)
• Superior micro-environment (air changes 70 times/h)
• Less cage changes necessary (less bedding and autoclaving)
• Protection of the staff and the environment
• Can be placed in elder animal houses
• Cons: expensive
 Used to create a barrier within a conventional animal facility

Question 68

A2-facility

Answer 68

= area where animals get infected by MO or viral vectors
Here it is very important what comes out: staff, air, garbage, lab animals, biological or experimental products:
 Protect the environment
The facility stands alone and has its own washing area, lab, autoclave, can be disinfected/ sterilized, animals are not able to leave,.. -> minimum risk of spreading MO in environment

Question 69

SPF facility

Answer 69

= specified pathogen free facility, no MO ( A2)
Here it is very important what goes in: staff, air, animals, material, feed, water, products,…
General build up:
• 1-corridor barrier (clean and dirty material pass through same corridor ->more infection risk)
• 2-corridor barriers, with single rooms
o Pros: protection against environmental influences, supplies, 1 or several rooms can be extracted, separation of clean and dirty material
o Cons: need extra space, corridor= lot of space, less space for animal rooms
• 2-corridor barrier with double rooms (2 rooms)
• Three corridor barrier
• All: Autoclave and hatch (luik) to bring in material, feed, cages and bedding
• All: Metabolic cages: measure feed intake, water intake, excretion of urine, excretion of faeces.
• All: washing- and animal rooms ( similar to others, but extra features, like extra filters for air that comes in and out, door between the dirty and clean area of the washing room,..)

Question 70

What is species specific behaviour?

Answer 70

• Genetically determined: actual form can change during the course of life through experience
• Adaptation of behaviour during evolution
o Result of elimination of individuals that were less adapted (survival of the fittest)
• Product of selection
• Behaviour that is of great importance for survival (forage behaviour, nesting, social behaviour) is anchored in their inheritance and is past on to the offspring
• This behaviour is being displayed even if the environment is unfit to do and the effect (the actual function) stays away.
• Such behaviours are the attitudes and movements that are
o Genetically defined
o Stereotypic and species-specific
o Not or very difficult to change by experiments
• Aim: keep the balance, homeostasis

Question 71

What is an ethogram and how can it help in determining pain?

Answer 71

In an ethogram the behaviour repertoire is layed down.
• The frequency, duration and succession of behavioural patterns
• ‘How will the mouse react when you put it in a cage’, ‘How they interact with other mice’…
• In general it contains information about species-specific behaviour.
During pain there will be changes in the standard behavioural. That’s how one can see that the animals are in pain and need analgesia. ETHOGRAM: when this deviate from standard -> problems

Question 72

What is the effect of stress on the behaviour?

Answer 72

Stress occurs when the predictability or possibility to affect a situation decreases, the animal feels like it losses grip on the environment. It’s not the pain itself, but the fact that they can’t predict it.
• Acute (a sudden change) - chronic stress (long unpredictability of changes in environment)
Whether an animal can cope with its environment in a certain situation
• depends on the means that are available
• depends on the way how the animal thinks about the stimuli: threat for its homeostasis or not? –> the way the different physiological and neuro-endocrine systems are activated
• Depends on the individual
Acute stress: ACTH release (cortisol, cortisone release from cortex of adrenal glands), adrenaline and NA (bloodpressure and rate increases, stimulates the liver for more glucose, increases the flow in brain and muscles, less flow in organs)
Chronic stress: more acid in stomach (and more alkaline enzymes of the pancreas), damage and shrink of the hippocampus, an enhanced memory

2 extreme reactions:
• Active coping strategy: active attempts (pogingen) to change the situation
o Defending the territory
o Flight when a dominant animal is around
o Fight/flight
• Passive coping strategy: animals do nothing to change their situation
o Little or no defending of the territory
o Quietly sitting in a corner
The strategy that is chosen depends on the genotype and experience of the individual. Environmental enrichment can increase the controllability and consequently decrease stress. E.g. the animal can build a nest, can hide itself from an aggressor,..

Question 73

Measuring wellbeing

Answer 73

1. Based on biological parameters

2. Based on behaviour (dit moet nog bij vorige vraag ‘effect van stress op behaviour’)

Question 74

Measuring wellbeing

Based on biological parameters

Answer 74

• Productivity decreases: if something is going wrong, this is noticed in the reproduction very quickly (less breeding)
• Physical health: animals get sick quicker
• Psychological criteria (stress hormones, immunological status)
• Aggression, cannibalism

Question 75

Measuring wellbeing Based on behaviour

Answer 75

1. Test of preference: let the animal choose. From a central cage the animal can choose between 4 systems of housing. Photo sensors in the corridors measure where the animal stays most and for how long.
2. Abnormal behaviour
   • behaviour in acute stress situation = conflict behaviour.
   o Ambivalent behaviour (2 types at the same time: e.g. scared and aggressive)
   o Redirected behaviour towards animals, humans, others or completely different behaviour.
   • Stereotype behaviour: This kind of behaviour is provoked when the stress stays and the animal can’t get rid of it. E.g. tail biting in pig, pacing up and down like polar bears
   • Cognitive functioning: animals have difficulties to learn, solve problems when they are in a situation they are not comfortable in.
   • Latency time eating: animal that is not at ease -> it will take long before it will start to eat.
   • Also physiologic changes can occur: less breeding and big amount of stress hormones.

Question 76

Explain gavage/gastric tube + in practice

Answer 76

A gastric tube/stomach canula is sometimes used for enteral administration (sonde voeding), so to administer substances trough the mouth. That’s how you put the substances directly in the animals stomach. The gastric tube is a curved tube with a blunt end.
In larger animals it’s sometimes necessary to keep the mouth open with a special equipment or to bring in a tube through the nose.
In practice: Restrain the animal well. Let the tube slide along the palate, this makes the animal swallow. Prevent the tube to get into the trachea -> inject substances gently -> animal coughs when wrong.

Question 77

Pre-operative treatment during surgery

Answer 77

= to prevent wound-infection
• Asepsis of the operative site
o Remove hairs: clipping, shaving, depilatory cream (hypothermia!)
o Positioning and securing the animal (fixation)
o Antisepsis of the skin: never completely sterile e.g. isobetadine
o Risk of hypothermia associated with shaving over a large surface (pattern = spiral) and excessive disinfectant use
o Draping of the operative site: create and maintain a sterile field around the operative site. The drapes are impervious to water and bacteria, lint free (puisvrij) and fire resistant. (reusable or disposable)
• Instrument sterilisation
o Moist het (steam) sterilization (=autoclaving) -> not for all instruments
o Dry sterilisation (quick, e.g. instruments if 10 mice)
o Chemical sterilization: ethylene oxide, liquids (alcohols, Dettol)
o Radiation (gamma)
• Asepsis of the surgeons and helper
o Surgical scrub, hairnet and face mask, gloves
• Anaesthesia
• Fixation
• Placing of the catheter
• Eye protection (oil, artificial tears, eyes are open!)
• Fasting (take away food) to avoid aspiration pneumonia (rat/mice)

Question 78

Per-operative treatment during surgery

Answer 78

• Wound incision
• Procedure: The actual surgery, correct surgical techniques (limit duration of wound exposition, limit dead space between tissue, minimize tissue trauma/blood or liquid accumulation, absence of foreign material)
• Wound closure
• Homeostasis
o Fluid: surgical irrigation, secretion pumps: to avoid blood loss
o T: heating pad
• Monitor depth of anaesthesia
• Surgical homeostasis = preventing from blood loss
o Important to control blood loss, improve visualisation of the surgical field
o Methods: pressure, electrocautery, ligature or vascular clamps or clips,…

Question 79

Post-operative treatment during surgery

Answer 79

• Wound care: cover wounds (spray, bandages), drain wound
• In animals avoid licking, scratching or biting (with jackets, collars)
• Antibiotics to prevent infection
• Analgesia
• Recovery
• Hypothermia: possible because of disinfectant and shaving -> keep them warm
• Hypovolemia: animal looses a lot of blood -> saline

Question 80

Prevent blood loss during surgery (3 R’s) (Zie ook surgical homeostasis)

Answer 80

Animals have a much smaller blood volume so it’s important to limit every blood loss. The amount of blood one can withdraw at a time is limited and described precisely. The animal should be hydrated with saline to prevent blood loss. Other ways to develop a surgical haemostasis:
• Pressure
• Electrocautery
• Ligature, vascular clamps or clips
• Surgical repair of important vessels
• Haemostatic agents (cellulose, collagen)
REFINEMENT => good knowledge of the anatomy of the blood vessels

Question 81

Explain an inbred strain

Answer 81

• Inbred= breeding closely related individuals resulting in an increase in homozygosity of the pups
• Degree of inbreeding = expressed as inbred coefficient F (= fraction of the original heterozygous genes that become homozygous)
o The more generations of inbreeding, the more F increases
o Increase of F per generation is determined by the relation between parents (degree of consanguinity)
• Beginning in the 20th century because cancer research demands genetically uniform animals. Tumour tissue maintained by successive transplantation.
o Now: > 400 mouse and rat inbred strains
• Inbred: after at least 20 successive generations of brother x sister breeding or mating of offspring x youngest parent, F = 98.4 % so there is still some variation, but quite genetically uniform => isogenic strain
• Inbred strains: growth, fertility and vitality can decrease.
o Most properties are set by different genes (polygenic)
o In natural populations: combination of genes that function best and that survive the natural selection, are the most common => balanced system
• Random mating: chance is small of a descendant becoming homozygous for a recessive allele.
• Mating between related parents: increase chance
• By inbreeding
o Animal is fixed into an arbitrary combination of genes
o Not necessarily the most beneficial -> inbred depression
o Animals function is worse compared to the original population
o Effects of unfavourable alleles are more pronounced in homozygous conditions.
• First 4-8 generations
o Lot of drop out because of unfavourable gene combinations (lot of dying)
• > 10 generations: most unfavourable alleles are dropped out and a new balanced system develops
• Genetic defects compared to human diseases
o E.g. obesity, muscular dystrophy
o A lot of strains are available with a specific disease

Question 82

Co-isogenic strain

Answer 82

• Arise from spontaneous mutations in the inbred strain that differentiates in 1 gene of the original strain
• Sub strains can be maintained next to original inbred strain e.g. when mutant represents a genetic model for a human disease or involves a gene of general interest
• Pros:
o Research in 1 particular gene
o Control group is available

Question 83

F1 hybrid = Filial 1

Answer 83

• Resulting from a cross between two inbred strains
• Features that make them useful:
o Genetically and phenotypical uniform
o Possess hybrid vigour (sterke kruising): more resistant to disease, survive better under stress, live longer, have larger litters => Heterosis: pups are doing better, have more advantages than the parents
o Useful as hosts for tissue transplants (e.g. tumour) from either parental strain
o For many studies they are more viable than parental strains
• In some cases the F1 hybrid react more uniform than the strains they originate from (e.g. reacting more uniform to pentobarbital)
• F1 hybrids can show more variation under certain circumstances -> due to improved ability of F1 to adapt (can choose between behaviour of both parents) -> perform pilot experiment (=Tyron effect)
• F1 hybrids generally:
o More uniform response: morphological parameters
o More variable response: behavioural characteristics

Question 84

Congenic strain

Answer 84

• Too see what the effect is of the background
• Produced by introducing certain genetic qualities into an inbred strain by backcrossing repeatedly
• Most used method to bring in recessive characteristics => cross-intercross-backcross method
o Recessive characteristics mm of strain D(donor) is imported in strain A(acceptor)
o After initial cross, a cycle of intercrossing-backcrossing is repeated at least 10 times (the animals selected for the backcross must carry the gene of interest mm)
• As result of crossing-over during meiosis and selection of proper (with mm) backcross animals, donor gene is introduced in the genome of strain A.
o Practically identical stream, expect for the gene that was selected and some genes of donor strain D to the left and right of the mutant gene that will be reduced with successive generations of backcrossing
• By using DNA-markers: speed congenic method (5-7 back-crossings)
• Double congenic lines are produced to study interactions between genes.

Question 85

Outbred strains

Answer 85

• If a random-bred-population is kept as a closed colony during at leas 4 generations and the increase of the inbred coefficient (F) does not exceed 1% per generation, we get an outbred strain.
 At least 25 breeding pairs needed
• Phenotypic variation is greater in outbred strain than in inbred strains
 Cons: more animals to get reliable data
• During long term experiments, in particular if consistent genetic variation is essential, use of an outbred stock is inadequate due to gradual change in the gene pool.
• Genetic variation can be standardized through a system of reciprocal hybridising of the inbred strains
• For long lasting experiments is where a certain variation of the population is wanted, same genetic variance can be made over and over again
• Result is a hybrid or mosaic population

Question 86

Chimeric mouse (Webinar: figuur!)

Answer 86

This is the kind of mice you get by performing the transgenesis method of gene targeting.
• First the inner cell mass of the embryonic stem cells is isolated and cultured.
• Transfect them with the targeting construct: specifically made to inactivate (KO) a gene or to overexpress a gene (KI).
• Selection of cells with the genetic manipulation, so we get ESC with the targeted gene
• Screening for HR (resistentie gen?) and expand correctly the targeted clones.
• Then there are 2 methods to introduce them in a mouse
o Blastocyst injection
o Morula aggregation
So we get a chimeric blastocyst that consist of non-manipulated cells from the host mother and manipulated cells that have been re-introduced
• Implantation in a carrier mother
• Grow out into a chimeric mouse, you can recognize it by the fur colour (black and white)
• Breed the chimeric mouse with wild type mouse until you have a germline mouse with the targeted gene in all of its cells.

Question 87

Different kind of feed + dis(advantages)

Answer 87

1. Natural ingredients
   = natural product type diet
   Advantage: relatively inexpensive and readily available from large suppliers
   • Closed formula: guarantees a minimal % of fat, ash (as), protein and carbohydrates, lists ingredients without stating exact quantities
   o Disadvantage: differ from batch to batch
   o Prove very adequate for maintenance, growth and reproduction
   • Open formula: gives the amount of each component and guaranteed analysis (qualitative) of the range of each major component.
   o More repeatable, though still somewhat variable in actual nutritive value
   • In situations where even slight variations in diet are considered critical:
   o Purchase a sufficient volume of 1 single batch for the total experiment (storage)
2. Defined diets
   • Semi-synthetic feed or semi-purified feed: more refined and restricted sets of ingredients
   o Quantity and quality of nutrients are exactly reproducible
   o E.g. casein and soybean as source of protein, sugar and starch as source of carbohydrates, vegetable oil and lard as source of fat
   o Often used in studies of specific nutrient deficiencies and excesses
   • Synthetic feed or purified (chemically defined) feed: entirely from pure chemicals (individual AA, essential fatty acids)
   o Max control over quality of ingredients
   o For studies in which a strict control over nutrients is essential

3. Pros and cons
Closed, Open, Semi-synthetic, synthetic
* Cost
\++	+	-	--
* Ease of formulation
-	-	+	++
* Variation between deliveries/brands
-	+	++	++
* Difference formulation- analysis
--	-	+	++
* Contamination risk
-	-	+	+
* Bio-availability
-	-	+	+
* Taste
\++	++	-	--

Question 88

Natural feeders and contamination risk

Answer 88

1. Plants
   • They have their own ‘means of defence’ (against predators) -> possibly toxic
   • Antinutritional compounds (ANF):
   o Reduce efficacy of digestive enzymes
   o Harmful effect on growth
   o Bad utilisation of feed components
   o Discomfort
   • Phytoestrogens (e.g. isoflavones in soy)
   o Similar structure as oestrogens
   o Can affect several parameters in which oestrogen is involved (e.g. reproduction)
2. External induced toxins
   • Herbicides, heavy metals
3. Botanic contamination
   • Weed (e.g. nightshade, Datura: when they are not filtered out during the preparation of food: toxic)
   • Fungus infection in the field
   • Problems with mould: some of them also have phytoestrogenic action
   => Quality control is very important!

Question 89

Which factors affect the nutrient requirement?

Answer 89

1. Genetic differences among species, gender or individuals
   • Certain species or genotypes lack certain enzymes
   • Mouse strains may differ in requirement for riboflavin, pantothenic acids and other nutrients
2. Stage of life
   • Animals needs more metabolizable energy during: growth, pregnancy and lactation
3. Environmental factors
   • T decrease, below lower threshold of thermoneutral zone:
   o Increase in nutrient requirements
   o Increase in feed uptake
   • T increase, disturbance, stress
   o Decrease in feed uptake
   o Sometimes feed with higher nutrient content necessary
   • Housing type can affect nutrient needs
   o Stainless steel cages with solid floors: lower need for zinc
   o If animals ingest bedding material: possible source of nutrients and toxins
4. Microbiological status
   • MO in intestinal tract
   o Intestinal flora produces nutrients (e.g. Vit K)
   o If faeces cannot be consumed, these nutrients are lost
   o Feed must be supplemented
   • Germfree animals
   o Lost their microbial systems
   o Influences dietary requirements
   o Adjustments in feed necessary (when you give them normal food: fur around their eyes will disappear)
5. Research conditions
   = Experiment itself can cause changes in feed uptake
   • Surgical procedure
   • Test compounds that alter appetite or that are not tasty (gavage)
   • Stress/pain
   • Experimental protocol requires feed restriction
6. Nutrient interactions
   • Amount of feed that is taken up: mostly determined by energy density of feed
   o Animals will take up less from feed with a high energy content (because their energy need is already satisfied)
   o The rest of feed needs to be adjusted in nutrients to compensate for decreased intake
   • Some nutrients compete with each other for active resorption through gut wall, due to use of same transport mechanism
   o If diets have unusual composition, potential effects on other nutrients must be considered.

Question 90

How to give a substance with a quite unpleasant flavour?

Answer 90

The animals will eat than less from their feed (mixed with the unpleasant) than from their usual feed.
• Problem when body weight is an important factor
• When the animal eats extremely less than expected, also think of a physiological effect of the substance
Solutions:
• Try to mask the unpleasant taste and smell (add sugar)
• Forced-feeding (gavage, trechter): effect of the substance
• Pow-fed feeding: the control group will also eat less

Question 91

Alternatives and Replacement (1 of the 3 R’)

Answer 91

= Replace the animal by another technique (= non-sentient alternative)
1. In vitro research: cell- and tissue culture -> create an environment that mimics normal physiological situation

• E.g. cells expressing a specific human receptor (receptor-binding studies for screening medication)
• E.g. test possible cell damage
o Before needed a lot of animals: now only a few
• E.g. Draize test: screening for skin irritation (e.g. make-up), but forbidden -> NOW: examine cells under the microscope, RNA screen (only in living cells), human
• Advantages: very few animals needed (for cells, tissue), homogenous and therefore a reliable substrate to experiment on
• Disadvantages: no info on bio kinetics, bio transformation/metabolism

2. Use of vertebrates at early stages of development => embryo’s

• Very young: nerves are not developed yet -> no pain perception
• Embryos of chicken, rats, frog and fish are already used
• EC recommendation: give anaesthetics and analgetics to foetal forms
• E.g. CAM-test: incubate the CAM after day 10 with a test substance, rinse CAM, evaluate coagulation, haemorrhage,… -> testing for irritating substances or carcinogenic compounds
3. Use of lower organisms
• Pyrogenic test in rabbit (unwanted side effect of medication): intravenously inoculated with product, monitor body T for evidence of febrile (koortsige) responses
• Alternative = LAL test (Limulus Amoebocyte Lysate test)
o Blood from the horseshoe crab, Limulus: after blood is taken, the crab is released unharmed back into the sea. Amoebocytes are incubated with small amounts of test substance -> formation of blood clots when bacterial toxins are present
o Blood from 1 crab can substitute several live rabbits, but test is not perfect yet -> false results
4. Audio-visual means
• E.g. video films for experiments that are performed each year again by students
• Computer programs and simulations
• Mostly for education

5. Organs of dead animals/humans
   • Slaughterhouses: e.g. pig organs, cow eyes or chicken eyes
   • Eyes used for Draize eye irritation test
   • Learning suturing (naaien, hechten) techniques
6. Other: human volunteers, artificial animals: PVC rat (learn to inject),…

Question 92

Alternatives and Reduction (1 of the 3 R’s)

Answer 92

= Decrease the number of animals to a minimum
• Ethical consideration: Before the study, make an ethical judgement to prevent using unnecessary animals, unnecessary pain. The research proposal will be judged by the EC.
• Experimental design (statistic): make a good estimate of how many animals are needed per group to get reliable results (with statistical program). Not too many, not too little.
• Pilot studies with a limited number of animals can indicate if more studies, other methods are appropriate
o e.g. minipig used for caries research -> feed sugar, candy,.. -> no effects on teeth because not under critical pH to develop caries -> choose other animal models)
• Sharing animals: if one needs one part, another researcher can use another part -> make use of the same animal
• Cryo-preservation: freezing cell or embryos in liquid nitrogen for preserving purposes
o No need to keep breeding animals just to keep the strain
• Computer models: find substances that are suitable as medicine
o Change substances and simulate -> choose best for cell culture test -> choose best for testing in animal model
• …

Question 93

Alternatives and Refinement (1 of the 3 R’s)

Answer 93

= providing less discomfort, minimum of pain and stress
• Housing, taking care, health and normal behaviour
o Prevent diseases and death
o Attention for the animals’ needs (cage enrichment) -> increase well-being
• Pain control, anaesthesia and euthanasia
o Determining human endpoints
o Appropriate staff (trained)
• Well trained staff
o Training and education for all
o Handle in correct way
o Avoid wrong use of experimental techniques -> discomfort
• Environmental enrichment
o Cage enrichment -> Less stress, increasing species-specific behaviour
• Non-invasive-methods
o CT-scan/ CAT-scan: painless, but can cause tumours due to high radiation dosage
o PET: bad anatomical detail -> use in combination with CT; tracers (FDG)
o MRI: anatomy, morphology and function; e.g. fat distribution and oedema; following diseases

Question 94

Alternatives and Responsibility

Answer 94

= ‘The 4th R’
• Make sure you do all you can for the animal wellbeing, preventing infections, reducing pain, no excessive use, using well experimental techniques,..

Question 95

Op een bepaald ogenblik daalt de kweek van muizen (kleine nesten, spontane abortus), het blijkt een probleem te zijn over de hele dieren faciliteit te zijn. Wat doe je?

Answer 95

• Autopsie, biopsie van een muis (inwendig onderzoek)
• Sentinel inoculeren met bloed van muizen -> kijken naar effect
• Omgeving inspecteren: T, humiditeit, licht, geur, geluid (teveel ultrasounds?: elektronische toestellen, computers?), contact met buitenwereld
• Bloedstalen, faeces stalen onderzoeken

Question 96

Er sterven in een kamer vele (transgene > niet-transgene, geen significant verschil) dieren. Hoe achterhaal je de oorzaak?

Answer 96

• Omgeving inspecteren (T, ventilatie, humiditeit, contact met buitenwereld,..)
• Bloed nemen en test met Ab’s doen -> screenen of infecties, MO, virussen
• Sentinel binnenbrengen + bloed -> Ab’s?
• Experiment van de transgene bekijken en proberen de oorzaak te achterhalen
• Andere labo’s contacteren die met dezelfde stam werken, om te vragen of zij ook problemen hebben

Question 97

Mogen biologisch gevaarlijke middelen ook in gewone labo’s?

Answer 97

• Nee: er zijn sowieso extra maatregelen nodig zoals HEPA filters, bescherming voor personeel en dier, afvalverwerking, ..

Question 98

Hoe contact met proefdieren hebben bij een stalen isolator?

Answer 98

• Je neemt een kooi met aan meerdere kanten een glazen plaat, zo zien ze mensen en eventueel andere dieren
• In contact komen via opening met daarin grote handschoenen die daar permanent vast aan hangen. Dieren strelen, observeren,..

Question 99

Er is een probleem met de kweek. Wat zou er aan de hand kunnen zijn? Hoe kan je dit achterhalen?

Answer 99

Het zou kunnen dat de dieren zich in chronische stress verkeren. Dit kan door gewijzigde omgevingsfactoren en het onvermogen van de dieren om zich hieraan te adapteren. Ten eerste kan je nagaan welke factoren er gewijzigd zijn (temperatuur, vochtigheid, geluid, geur, …) en vervolgens kan je het gedrag van de dieren observeren en vergelijken met het normale soort-specifieke gedrag m.b.v. een ethogram. Het zou ook kunnen dat het dier ziek is of pijn lijdt. Daarvoor neem je best bloed monsters of biopsies om een klinisch onderzoek uit te voeren.

Question 100

Wat doe je als een infectie bij een dier is vastgesteld?

Answer 100

Afhankelijk van verschillende factoren:
• Diersoort: verschil tussen grote en kleine dieren, de stam van het individu
• Type van de ziekte: examinatie van de pathogenesis en de zoönose
• Beschikbaarheid van therapie
• Effect van het experimen: duur, inteferentie, …
• Beleid van het instituut omtrent microbiologische kwaliteit
Mogelijke opties:
• Acute infecties die snel verspreiden: quarantaine en kweekstop voor 5 weken
• Acute infecties met lage verspreiding: isolatie van enkele kweekparen, gebruiken van negatieve kweekparen om nakomelingen te testen.
• Beginnende infecties met lage transmissie: testen en selectieve methode
• Persistente en resistente infecties: depopulatie, desinfectie en repopulatie
• Transplacentaal verspreidende infectie: testen voor kweken
• Zoönose: depopulaite, desinfectie en repopulatie

Question 101

Fossa perinealis (begrip)

Answer 101

De zone van de geslachtorganen bij de cavia, hier liggen veel klieren

Question 102

Nude mouse (begrip) hoort overal en nergens

Answer 102

A mouse without fur, they have no thymus, so they don’t make T-lymphocytes. Therefore, they are extremely susceptible to infections. They are used for e.g. immunity experiments.

Question 103

Wat zegt de wet over herkomst van dieren en hun identificatie? H1

Answer 103

Het is vaak heel interessant om kleine dieren individueel te kunnen identificeren. Voor grotere dieren zoals katten, honden, primaten,… is het gedefinieerd door de wet dat ze permanent moeten kunnen geidentificeerd worden vanaf dat ze geboren zijn. Boederijdieren, zoals varkens en schapen hebben een Sanitel nummer wat hun traceerbaar maakt in de vleesketen. Identificatie kan gedaan worden door oor, poot en vleugen ring tattoos.
Het dier moet goed vastgehouden, vastgezet zijn tijdens het inbrengen van de tube. Vermijden dat de tube in de trachea komt (zien of het dier hoest als je een beetje toedient)