LABORATORY ANIMAL SCIENCE
(CORE MODULE)
INHOUDSOPGAVE
Introduction ................................................................................................................................. 4
EDA – Experimental Design Assistant ................................................................................................ 6
History and ethics............................................................................................................................ 8
Legislation .................................................................................................................................. 14
Part 1 .............................................................................................................................................14
Animal welfare law 1986............................................................................................................. 15
Origin (art 3 KB2013) .................................................................................................................. 16
Animal experiment ..................................................................................................................... 19
Animal husbandry ...................................................................................................................... 25
Personnel and their responsabilities ........................................................................................... 26
At the end of the experiment ....................................................................................................... 29
Part 2 .............................................................................................................................................32
Ethics committee ....................................................................................................................... 32
Animal welfare body (dierenwelzijnscel)...................................................................................... 38
National committee ................................................................................................................... 40
Animal care health and management ........................................................................................... 41
Number and biological principles of frequently used species ............................................................41
Mouse ....................................................................................................................................... 44
Rat ............................................................................................................................................ 49
Guinea pig ................................................................................................................................. 51
Rabbit ....................................................................................................................................... 52
Gerbil ........................................................................................................................................ 54
Hamster .................................................................................................................................... 54
Nutrition of laboratory animals ........................................................................................................55
Animal facility ................................................................................................................................61
Physical facilities ....................................................................................................................... 61
Environmental parameters ......................................................................................................... 68
Daily care, cage, transport .......................................................................................................... 70
Health monitoring and safety ..........................................................................................................71
Health monitoring ...................................................................................................................... 71
Hazards – risks........................................................................................................................... 73
Humane killing ............................................................................................................................ 76
Minimally invasive procedures ..................................................................................................... 83
Identification – marking of animals ..................................................................................................83
1
, Administration of substances ..........................................................................................................87
Collection of body fluids .................................................................................................................90
Animal welfare ............................................................................................................................ 93
Lecture ..........................................................................................................................................93
E-learning .................................................................................................................................... 104
Project design ........................................................................................................................... 114
Part 1: good experimental design – sample size calculation – study designs .................................... 114
Basics for good experimental design ......................................................................................... 115
Statistical methods to support experimental design .................................................................. 124
Experimental designs ............................................................................................................... 128
Arrive ....................................................................................................................................... 135
Part 2: alternatives – severity assessment – humane endpoints....................................................... 135
Alternatives to animal-based research ...................................................................................... 135
Severity assessment ................................................................................................................ 139
Humane endpoints .................................................................................................................. 144
Genetic modification lab animals .................................................................................................. 147
Anesthesia – analgesia .............................................................................................................. 158
E-learning .................................................................................................................................... 158
Pre-anesthetic preparations ..................................................................................................... 158
What is anesthesia .............................................................................................................. 158
Why anesthesia in lab animals? ............................................................................................ 159
Training animals .................................................................................................................. 161
Pre-anesthetic considerations.............................................................................................. 161
Preparing for anesthesia ...................................................................................................... 162
Anesthesia of animals in poor health .................................................................................... 165
General principles anesthesia .................................................................................................. 166
Introduction ........................................................................................................................ 166
Planning for anesthesia ........................................................................................................ 167
Injectable agents ................................................................................................................. 169
Inhalational agents .............................................................................................................. 172
Assessing anesthetic depth .................................................................................................. 173
Supporting & monitoring body functions ............................................................................... 174
Anesthetic equipment .......................................................................................................... 176
Post-op considerations ........................................................................................................ 178
Choice anesthetic .................................................................................................................... 180
Selecting an inhalational anesthetic ..................................................................................... 180
Selecting an injectable anesthetic ........................................................................................ 184
Choosing anesthetic regimen ............................................................................................... 187
Balanced anesthesia ........................................................................................................... 192
Monitoring ............................................................................................................................... 196
Why monitoring of anesthetized animals? ............................................................................. 196
Depth of anesthesia ............................................................................................................. 198
Body temperature ................................................................................................................ 200
Respiratory function ............................................................................................................ 202
2
, Cardiovascular function ....................................................................................................... 207
Fluid balance ....................................................................................................................... 210
Animal posture and comfort ................................................................................................. 211
Anesthetic equipment .......................................................................................................... 212
How should we monitor animals? ......................................................................................... 212
Lecture ........................................................................................................................................ 217
Surgical techniques: wound healing & suturing ........................................................................... 234
Wound healing ............................................................................................................................. 234
Suturing ....................................................................................................................................... 236
Practicals.................................................................................................................................. 240
Rub-scrub & gloving...................................................................................................................... 240
Suturing ....................................................................................................................................... 241
3
,INTRODUCTION
• We don’t have to know extra legislation documents, everything to know is in the
hand-outs
• Specifications for education and training in laboratory animal science
o Member states shall ensure that
each breeder, supplier, and user has
sufficient on-site staff
o The staff shall be adequately
educated and trained before they
perform any of the named functions
A–D
o Member States shall publish minimum requirements with regard to
education and training and the requirements for obtaining, maintaining,
and demonstrating the requisite competence for the named functions
o Specific function quires certain set of theory and skills for the person that
is executing this function
• EU modular education and training framework
o You need to know what is allowed, what you need to do à you need to
master core modules
o Function-specific modules (function B) à you don’t need to know about
other functions that you are not doing
o Task-specific modules
4
,• Personnel all need to acquire the same basic knowledge = Core modules 1 – 6
• Function specific modules are required to perform the tasks specific to function
A - D. They are prerequisites for fulfilling any of the functions A – D
o We need to know appropriate procedures for animal species
o Don’t be able to perform, but know pros & cons and make decision
o Only in function A (carrying out procedure): skill training to be able to
perform it
• FELASA Certified
o We will get 3 certificates after passing this course
o Europe works with functions, Belgium with categories
o Extra functions for other animals
• Teaching method
o Theory (recorded)
§ 40% of exam (you need at least 20% to pass the course)
§ 20 short open answer questions
> 10 from Van Ginneken & 10 from Van Dam
5
, > Van Ginneken: theory (e.g. definition lab animal) & applied
question (e.g. is this a lab animal)
o Practicals
§ 10% of exam on skills
§ 100% participation mandatory
§ Preparation
§ Practical 1: explanation suturing/ gloving + practice
suturing/gloving
§ Practical 2: assessment rub/scrub & gloving + practice suturing
§ Practical 3: assessment suturing
§ Assignment: presentation
o Assignment
§ 50% of exam (minimally 10/20 to pass the course)
§ File for an experiment with lab animals = 2/3 of grade
§ Defend it for ethical committee + HBA = 1/3 of grade
§ Put link of EDA in thread
EDA – EXPERIMENTAL DESIGN ASSISTANT
• Improves your experimental set-up
o Recommendations for statistical analysis
o Power calculation
o Randomization
o Blinding
• Different templates available: https://eda.nc3rs.org.uk/
• Template 1 – effect of drug A on plasma glucose levels
o Experiment
o Practical steps: different animals, treatment groups, surgical
interventions, measurements
o Analysis
o Hypothesis
§ Drug A has an effect on plasma glucose levels in diabetic mice
o Experimental design: practical steps
§ Two treatment groups
> Group 1 = control group = saline injection (vehicle)
> Group 2 = test group = drug A injection 10 mg/kg
> Experimental unit = animal
> Syringes are prepared by a third part
§ Measurement
> Plasma glucose levels are measured 1 hour after injection
> Outcome = plasma glucose concentration
6
, > Carried out blind
§ 1 independent variable
> Drug A
> Two categories: Vehicle or Drug
> Unpaired t-test
o Analysis
§ Carried out blind
§ Sample size
> A difference of 100 mg/dL between two groups = biologically
significant
> Previous study: SD = 50 mg/mL
> Significance level 0.05%
> 85% power
> Effect size: (m1 – m2) = 100
> Variability (SD) = 50
> Significance level = 0.05
> Power = 0.85
> Calculator returns N = 6 based on previous information
> 6 animals per group are needed to test the hypothesis with
sufficient power
> Randomization is done in EDA spreadsheet
o Start from scratch with new diagram or build with an already existing
template
o Start from scratch
§ Drag shape to board
> Features at bottom right of shape
> Red box around shape: something is missing
> Question mark says what is missing
7
, § If you have 2 mice groups of which one gets drug and one is control
à start with 1 pooled group
> If it’s a logical step, a green box occurs
> Connect with arrows in a logical way
> Then 2 arrows to 2 new shapes: 1 drug & 1 control
> You can connect 2 groups to 1 measurement (1 shape), e.g.
plasma glucosa level
> Then outcome measure
> Then analysis
§ Goal: end EDA without errors, there can be some warnings but
preferably not
§ Drag treatment group into animal group so drag saline into
pharmalogical intervention of control and drug into
pharmacological intervention of drugged group
HISTORY AND ETHICS
• Laboratory animals
o Prehistorical times
§ Aristotle (384-322 BC)
> Scala naturae
> From low, simple to high, nearly perfect
> Introduction to the thinking of higher and lower beings
§ But – Plutarchus (46-120)
> Pythagoras (570-500 BC)
> Involve animals in ethical considerations ‘on the cleverness
of animals’
> Vegetarism
§ Galenus (129-216)
> Vivisection and dissection (while still alive, just restrained)
> Taboo on using human bodies (but Roman Games)
> Uses monkeys and pigs as proxy for the human body
> His knowlegde prevailed until 17th century
o Middle ages
§ Augustinus Hippo (354-430)
> Natural order added with Angels and God
- Increase the distance
- Foresake animal instincts
> Man to God’s image
> Animals serve man
> Ladder with God at top, then man, then animals
8
, § No real progress
> Disease = curse
> Cure = good faith
o Renaissance (14-17th century)
§ Revival and critical view on the Ancient times
§ Vesalius (1514-1564)
> Corrected Aristotle and Galenus – used human bodies
§ Van Leeuwen
> Invention of the microscope
§ A different view on the position of man
§ The birth of a more scientific approach
o Age of Enlightenment (17-18th century)
§ Descartes (1596-1650)
> The theory of innate knowledge; all humans were born with
knowledge through the higher power of God
- Animals not born with this innate knowledge
- Combated by philosopher John Locke (1632–1704),
an empiricist: all knowledge is acquired through
experience
> Automata
> ‘Je pense donc je suis’
> Importance of ‘reasoning
§ Halen
§ Industrialization
> Less contact with animals
> More time
§ Hogarth (1751)
> Four stages of Cruelty
§ Kant (1724-1804)
> Deontology
§ Bentham (1748-1832) and Mill
> Utilism
• Kant – deontology
o 1742-1804
o Actions that we must take (= duties) based on reasoning and not
considering the outcome or consequences
o Categorical imperative
§ Universal (for all and under all circumstances)
§ Treat human(ity) as a goal, never as a means
o Only (hu)man
9
, § The ‘reasoning’ (thinking, knowledge) is the key that decides what
has dignity and what does not
> If no dignity or intrinsic value … it may be used (the
categorical imperative only applies to those having dignity)
> Humans have absolute worth (absolute dignity) because
they are rational; animals only have relative worth
(instrumental value)
> We have to take care about animals, not because animal is
important but because it says something about man, about
our dignity
o Tom Regan (see later)
• Bentham & Mill
o 1748-1932
o Sentient beings
§ ‘The question is not Can they reason, nor Can they talk, but Can
they suffer …’
o Greatest happiness principle – utilism (utilitarisme)
§ Reasoning is less important than happiness
§ Considers the consequences of actions (>< Kant)
o Calculus
§ Everybody to count for one, nobody to count for more than one
o Agrees to the use of animals as long as the suffering is exceeded by the
benefit for man
o This is the predominant view that we have now (harm vs benefit)
• Laboratory animal
o Modern times …
§ Charles Darwin (1809-1882)
> Origin of Species > Scala naturae
> Start of zoocentrism
§ Claude Bernard (1813-1878)
> Assistant of Magendie
> Foundation of animal experiments (should be properly
controlled) – first test in animals (humanist)
§ Anesthesia (1846)
o UK: Cruelty against animals act (1867)
§ Cruel treatment of Cattle Act (°1822)
§ Cruelty against Against Animals Act (°1835)
> A reaction against Magendie (France) because of
showcased vivisection à people could buy ticket to come
see it
10
(CORE MODULE)
INHOUDSOPGAVE
Introduction ................................................................................................................................. 4
EDA – Experimental Design Assistant ................................................................................................ 6
History and ethics............................................................................................................................ 8
Legislation .................................................................................................................................. 14
Part 1 .............................................................................................................................................14
Animal welfare law 1986............................................................................................................. 15
Origin (art 3 KB2013) .................................................................................................................. 16
Animal experiment ..................................................................................................................... 19
Animal husbandry ...................................................................................................................... 25
Personnel and their responsabilities ........................................................................................... 26
At the end of the experiment ....................................................................................................... 29
Part 2 .............................................................................................................................................32
Ethics committee ....................................................................................................................... 32
Animal welfare body (dierenwelzijnscel)...................................................................................... 38
National committee ................................................................................................................... 40
Animal care health and management ........................................................................................... 41
Number and biological principles of frequently used species ............................................................41
Mouse ....................................................................................................................................... 44
Rat ............................................................................................................................................ 49
Guinea pig ................................................................................................................................. 51
Rabbit ....................................................................................................................................... 52
Gerbil ........................................................................................................................................ 54
Hamster .................................................................................................................................... 54
Nutrition of laboratory animals ........................................................................................................55
Animal facility ................................................................................................................................61
Physical facilities ....................................................................................................................... 61
Environmental parameters ......................................................................................................... 68
Daily care, cage, transport .......................................................................................................... 70
Health monitoring and safety ..........................................................................................................71
Health monitoring ...................................................................................................................... 71
Hazards – risks........................................................................................................................... 73
Humane killing ............................................................................................................................ 76
Minimally invasive procedures ..................................................................................................... 83
Identification – marking of animals ..................................................................................................83
1
, Administration of substances ..........................................................................................................87
Collection of body fluids .................................................................................................................90
Animal welfare ............................................................................................................................ 93
Lecture ..........................................................................................................................................93
E-learning .................................................................................................................................... 104
Project design ........................................................................................................................... 114
Part 1: good experimental design – sample size calculation – study designs .................................... 114
Basics for good experimental design ......................................................................................... 115
Statistical methods to support experimental design .................................................................. 124
Experimental designs ............................................................................................................... 128
Arrive ....................................................................................................................................... 135
Part 2: alternatives – severity assessment – humane endpoints....................................................... 135
Alternatives to animal-based research ...................................................................................... 135
Severity assessment ................................................................................................................ 139
Humane endpoints .................................................................................................................. 144
Genetic modification lab animals .................................................................................................. 147
Anesthesia – analgesia .............................................................................................................. 158
E-learning .................................................................................................................................... 158
Pre-anesthetic preparations ..................................................................................................... 158
What is anesthesia .............................................................................................................. 158
Why anesthesia in lab animals? ............................................................................................ 159
Training animals .................................................................................................................. 161
Pre-anesthetic considerations.............................................................................................. 161
Preparing for anesthesia ...................................................................................................... 162
Anesthesia of animals in poor health .................................................................................... 165
General principles anesthesia .................................................................................................. 166
Introduction ........................................................................................................................ 166
Planning for anesthesia ........................................................................................................ 167
Injectable agents ................................................................................................................. 169
Inhalational agents .............................................................................................................. 172
Assessing anesthetic depth .................................................................................................. 173
Supporting & monitoring body functions ............................................................................... 174
Anesthetic equipment .......................................................................................................... 176
Post-op considerations ........................................................................................................ 178
Choice anesthetic .................................................................................................................... 180
Selecting an inhalational anesthetic ..................................................................................... 180
Selecting an injectable anesthetic ........................................................................................ 184
Choosing anesthetic regimen ............................................................................................... 187
Balanced anesthesia ........................................................................................................... 192
Monitoring ............................................................................................................................... 196
Why monitoring of anesthetized animals? ............................................................................. 196
Depth of anesthesia ............................................................................................................. 198
Body temperature ................................................................................................................ 200
Respiratory function ............................................................................................................ 202
2
, Cardiovascular function ....................................................................................................... 207
Fluid balance ....................................................................................................................... 210
Animal posture and comfort ................................................................................................. 211
Anesthetic equipment .......................................................................................................... 212
How should we monitor animals? ......................................................................................... 212
Lecture ........................................................................................................................................ 217
Surgical techniques: wound healing & suturing ........................................................................... 234
Wound healing ............................................................................................................................. 234
Suturing ....................................................................................................................................... 236
Practicals.................................................................................................................................. 240
Rub-scrub & gloving...................................................................................................................... 240
Suturing ....................................................................................................................................... 241
3
,INTRODUCTION
• We don’t have to know extra legislation documents, everything to know is in the
hand-outs
• Specifications for education and training in laboratory animal science
o Member states shall ensure that
each breeder, supplier, and user has
sufficient on-site staff
o The staff shall be adequately
educated and trained before they
perform any of the named functions
A–D
o Member States shall publish minimum requirements with regard to
education and training and the requirements for obtaining, maintaining,
and demonstrating the requisite competence for the named functions
o Specific function quires certain set of theory and skills for the person that
is executing this function
• EU modular education and training framework
o You need to know what is allowed, what you need to do à you need to
master core modules
o Function-specific modules (function B) à you don’t need to know about
other functions that you are not doing
o Task-specific modules
4
,• Personnel all need to acquire the same basic knowledge = Core modules 1 – 6
• Function specific modules are required to perform the tasks specific to function
A - D. They are prerequisites for fulfilling any of the functions A – D
o We need to know appropriate procedures for animal species
o Don’t be able to perform, but know pros & cons and make decision
o Only in function A (carrying out procedure): skill training to be able to
perform it
• FELASA Certified
o We will get 3 certificates after passing this course
o Europe works with functions, Belgium with categories
o Extra functions for other animals
• Teaching method
o Theory (recorded)
§ 40% of exam (you need at least 20% to pass the course)
§ 20 short open answer questions
> 10 from Van Ginneken & 10 from Van Dam
5
, > Van Ginneken: theory (e.g. definition lab animal) & applied
question (e.g. is this a lab animal)
o Practicals
§ 10% of exam on skills
§ 100% participation mandatory
§ Preparation
§ Practical 1: explanation suturing/ gloving + practice
suturing/gloving
§ Practical 2: assessment rub/scrub & gloving + practice suturing
§ Practical 3: assessment suturing
§ Assignment: presentation
o Assignment
§ 50% of exam (minimally 10/20 to pass the course)
§ File for an experiment with lab animals = 2/3 of grade
§ Defend it for ethical committee + HBA = 1/3 of grade
§ Put link of EDA in thread
EDA – EXPERIMENTAL DESIGN ASSISTANT
• Improves your experimental set-up
o Recommendations for statistical analysis
o Power calculation
o Randomization
o Blinding
• Different templates available: https://eda.nc3rs.org.uk/
• Template 1 – effect of drug A on plasma glucose levels
o Experiment
o Practical steps: different animals, treatment groups, surgical
interventions, measurements
o Analysis
o Hypothesis
§ Drug A has an effect on plasma glucose levels in diabetic mice
o Experimental design: practical steps
§ Two treatment groups
> Group 1 = control group = saline injection (vehicle)
> Group 2 = test group = drug A injection 10 mg/kg
> Experimental unit = animal
> Syringes are prepared by a third part
§ Measurement
> Plasma glucose levels are measured 1 hour after injection
> Outcome = plasma glucose concentration
6
, > Carried out blind
§ 1 independent variable
> Drug A
> Two categories: Vehicle or Drug
> Unpaired t-test
o Analysis
§ Carried out blind
§ Sample size
> A difference of 100 mg/dL between two groups = biologically
significant
> Previous study: SD = 50 mg/mL
> Significance level 0.05%
> 85% power
> Effect size: (m1 – m2) = 100
> Variability (SD) = 50
> Significance level = 0.05
> Power = 0.85
> Calculator returns N = 6 based on previous information
> 6 animals per group are needed to test the hypothesis with
sufficient power
> Randomization is done in EDA spreadsheet
o Start from scratch with new diagram or build with an already existing
template
o Start from scratch
§ Drag shape to board
> Features at bottom right of shape
> Red box around shape: something is missing
> Question mark says what is missing
7
, § If you have 2 mice groups of which one gets drug and one is control
à start with 1 pooled group
> If it’s a logical step, a green box occurs
> Connect with arrows in a logical way
> Then 2 arrows to 2 new shapes: 1 drug & 1 control
> You can connect 2 groups to 1 measurement (1 shape), e.g.
plasma glucosa level
> Then outcome measure
> Then analysis
§ Goal: end EDA without errors, there can be some warnings but
preferably not
§ Drag treatment group into animal group so drag saline into
pharmalogical intervention of control and drug into
pharmacological intervention of drugged group
HISTORY AND ETHICS
• Laboratory animals
o Prehistorical times
§ Aristotle (384-322 BC)
> Scala naturae
> From low, simple to high, nearly perfect
> Introduction to the thinking of higher and lower beings
§ But – Plutarchus (46-120)
> Pythagoras (570-500 BC)
> Involve animals in ethical considerations ‘on the cleverness
of animals’
> Vegetarism
§ Galenus (129-216)
> Vivisection and dissection (while still alive, just restrained)
> Taboo on using human bodies (but Roman Games)
> Uses monkeys and pigs as proxy for the human body
> His knowlegde prevailed until 17th century
o Middle ages
§ Augustinus Hippo (354-430)
> Natural order added with Angels and God
- Increase the distance
- Foresake animal instincts
> Man to God’s image
> Animals serve man
> Ladder with God at top, then man, then animals
8
, § No real progress
> Disease = curse
> Cure = good faith
o Renaissance (14-17th century)
§ Revival and critical view on the Ancient times
§ Vesalius (1514-1564)
> Corrected Aristotle and Galenus – used human bodies
§ Van Leeuwen
> Invention of the microscope
§ A different view on the position of man
§ The birth of a more scientific approach
o Age of Enlightenment (17-18th century)
§ Descartes (1596-1650)
> The theory of innate knowledge; all humans were born with
knowledge through the higher power of God
- Animals not born with this innate knowledge
- Combated by philosopher John Locke (1632–1704),
an empiricist: all knowledge is acquired through
experience
> Automata
> ‘Je pense donc je suis’
> Importance of ‘reasoning
§ Halen
§ Industrialization
> Less contact with animals
> More time
§ Hogarth (1751)
> Four stages of Cruelty
§ Kant (1724-1804)
> Deontology
§ Bentham (1748-1832) and Mill
> Utilism
• Kant – deontology
o 1742-1804
o Actions that we must take (= duties) based on reasoning and not
considering the outcome or consequences
o Categorical imperative
§ Universal (for all and under all circumstances)
§ Treat human(ity) as a goal, never as a means
o Only (hu)man
9
, § The ‘reasoning’ (thinking, knowledge) is the key that decides what
has dignity and what does not
> If no dignity or intrinsic value … it may be used (the
categorical imperative only applies to those having dignity)
> Humans have absolute worth (absolute dignity) because
they are rational; animals only have relative worth
(instrumental value)
> We have to take care about animals, not because animal is
important but because it says something about man, about
our dignity
o Tom Regan (see later)
• Bentham & Mill
o 1748-1932
o Sentient beings
§ ‘The question is not Can they reason, nor Can they talk, but Can
they suffer …’
o Greatest happiness principle – utilism (utilitarisme)
§ Reasoning is less important than happiness
§ Considers the consequences of actions (>< Kant)
o Calculus
§ Everybody to count for one, nobody to count for more than one
o Agrees to the use of animals as long as the suffering is exceeded by the
benefit for man
o This is the predominant view that we have now (harm vs benefit)
• Laboratory animal
o Modern times …
§ Charles Darwin (1809-1882)
> Origin of Species > Scala naturae
> Start of zoocentrism
§ Claude Bernard (1813-1878)
> Assistant of Magendie
> Foundation of animal experiments (should be properly
controlled) – first test in animals (humanist)
§ Anesthesia (1846)
o UK: Cruelty against animals act (1867)
§ Cruel treatment of Cattle Act (°1822)
§ Cruelty against Against Animals Act (°1835)
> A reaction against Magendie (France) because of
showcased vivisection à people could buy ticket to come
see it
10
