Training presentation 2014
Theoretical
Training for personnel involved
with the use of animals in
research, teaching and testing
The Responsibility for the Well - being of Animals Used in
Research
Good science requires good animal care.
Animals that are in poor condition, discomfort
or pain are poor research subjects.
Animal well-being supports the integrity
of the research.
Guide for the Care and Use of
Laboratory Animals
From National Research Council.
Provides information that
will enhance animal wellbeing, the quality of
biomedical research, and
the advancement of
biologic knowledge that is
relevant to humans or
animals.
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The AUB Faculty of Medicine has established
in 2003 an “Institutional Animal Care and
Use Committee” IACUC.
The aim of the IACUC is to make sure
that laboratory animals used in
research, teaching, testing and training
at AUB are treated in accordance with
the “US Government Principles for the
Utilization and Care of Vertebrate
Animals used in Testing, Research and
Training”.
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The IACUC is responsible for:
(1) Overseeing the use and care of animals at
the American University of Beirut Faculty of
Medicine (AUB-FM) and for
(2) Reviewing of research and teaching activities
involving animals conducted on AUB-FM premises
by faculty, students and staff.
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Abdo Jurjus, Ph.D., Professor, Anatomy, Cell Biology and Physiology,
Chairman.
Ali Bazarbachi, M.D., Ph.D., Professor and Associate Dean for Basic
Research- Faculty of Medicine (ex-officio).
Laura Dosh, Officer (ex-officio).
Fouad Farhat, D.V.M, Veterinarian.
Asad Zeidan, Ph.D., Professor, Anatomy, Cell Biology and Physiology.
George Nemer, Ph.D., Associate Professor, Department of Biochemistry and
Genetics.
Hiba El-Hajj, Ph.D., Assistant professor, Department of Internal Medicine.
Hala Ghali-Muhtasib, Professor, Department of Biology
Robert Habib, Ph.D., Associate Professor, Department of Internal Medicine.
Raya Saab, M.D., Assistant Professor, Department of Pediatrics and
Adolescent Medicine.
Said Saghieh, M.D., Assistant Professor of Clinical Surgery.
Siham Radi, Community Representative; Non-Scientific Concerns; Not
Affiliated with AUB.
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The IACUC must review all activities involving the care
and use of animals in research or teaching. (The USDA
Animal Welfare Act requires that the IACUC perform the
same duties for all activities involving animals covered
by the Act, regardless of funding source.)
All new proposals for animal use and significant changes
to existing proposals for animal use must be reviewed
and approved by the IACUC before animal use can
begin.
No animals may be used for experimental procedures,
including field studies, pilot studies, breeding, or
euthanasia for sample collection without receiving prior
IACUC approval.
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Review and approve, require modification in, or withhold
approval to, proposals for research and teaching that
require the use of animals.
Review and approve, require modification in, or withhold
approval to, changes regarding the use of animals in
ongoing, previously approved, activities.
Suspend any activity that is not in compliance with the
policies and guidelines that govern the use and care of
animals at AUB-FM.
Inspect the institutional animal care facilities at least once
every 6 months.
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Review the Animal Care Program for the humane
care and use of animals at AUB-FM at least once
every 6 months.
Submit reports to the Dean or designated
institutional official on the review and inspection
processes at least once every six months.
Make written recommendations relating to the
Animal Care Program, animal care facilities and
related personnel or programs.
Review concerns involving the care and use of
animals at the AUB-FM.
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The IACUC must have oversight of all areas where
animal procedures are performed and may determine
the appropriate interval for inspection of procedural
spaces:
a. This includes spaces in research laboratories where
routine procedures are performed
b. This includes any non-surgical procedures, such as
weighing, blood collection, euthanasia.
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The IACUC must inspect surgical records, drug records,
and experimental records as appropriate to monitor for
protocol adherence.
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There are two valid methods of animal proposal
review by the IACUC
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i. Full committee review by a convened quorum
of the IACUC. This can be an in person meeting,
or a real time meeting by teleconference, video
conference, etc.
ii. Designated review by one or more members of
the committee only after all voting members
have been provided the opportunity to review the
proposal and call for full committee review.
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After review of animal proposals, the
IACUC may take one of the following
actions
i. Approve as written
ii. Require modifications to protocol to secure
approval
iii. Disapprove the proposal.
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The Animal Use Form
If the scientist plans to use animals as part of the
research, he or she must explain in an
Animal Use Form
WHY animals are needed to accomplish the
aims,
What procedures will be performed on the
animals and
How the animals will be housed and cared for
throughout the project.
Description of the pain category for the
research animals (Pain category C, D or F).
CATEGORIES OF INVASIVENESS IN ANIMAL EXPERIMENTS
“PAIN CATEGORIES”
Pain Category
C
Type of Experiments / Procedures
Experiments / Procedures
causing little discomfort or stress
D
Experiments / Procedures
causing moderate to severe distress or pain
using anesthesia and / or painkiller
E
Experiments / Procedures
causing prolonged or severe clinical distress or pain
without the use of anesthesia and / or painkiller
Pain Category C
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Category C procedures include:
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Experiments causing minor stress of short duration
Level C procedures should not cause significant changes in the animal’s
appearance,
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Category C procedures include:
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Injection of material in amounts that will not cause adverse reactions
Acute non-survival studies in which the animals are completely
anaesthetized and
do not regain consciousness
Approved methods of euthanasia following rapid unconsciousness
Short periods of food and/or water deprivation equivalent to periods of
abstinence in
nature
Cannulation or catheterization of blood vessels or body cavities under
anesthesia
Minor surgical procedures under anesthesia- biopsies, laparoscopy
Short periods of skillful restraint beyond that for simple observation or
examination
Blood sampling
Pain Category D
Category D procedures include:
Experiments causing moderate to severe distress or pain
using anesthesia and / or painkiller
Level D procedures should not cause prolonged or severe
clinical distress
Category D procedures include:
 Major surgical procedures conducted under anesthesia with
subsequent recovery
 Prolonged (several hours or more) periods of physical
restraint
 Induction of behavioral stress - like maternal deprivation,
aggression, predator-prey interactions
 Procedures causing severe, persistent or irreversible
disruption of sensorimotor organization
Pain Category E
Category E procedures include:
Experiments causing prolonged or severe clinical distress and / or pain without
the use anesthesia and / or painkiller
Procedures inflicting severe pain near, at, or above the pain tolerance
threshold of not anesthetized, conscious animals
Not confined to surgical procedures, but may include exposure to noxious
agents or those having unknown effects
Exposure to drugs or chemicals at levels that may impair physiological
systems and cause death, severe pain, or extreme distress
New biomedical experiments having a high degree of invasiveness
Behavioral studies having unknown degree of distress
Muscle relaxant or paralytic drug use without anesthetics
Burn or trauma infliction on not anesthetized animals
Toxicity testing and experimentally-induced infectious disease studies that
have death as the endpoint
Pain definition
The American Academy of Pain Medicine
defines pain as:
“An unpleasant sensation (that can range
from mild, localized discomfort to agony)
and emotional response to that sensation”.
Distress definition
(Merriam – Webster)
Synonyms: distress, suffering, misery, agony
meaning the state of being in great trouble.
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Distress implies an external and usually
temporary cause of great physical or mental
strain and stress
Suffering involves conscious endurance of pain
or distress
Misery stresses the unhappiness, poverty
Agony means intense feelings of suffering; acute
mental or physical pain;
Assessment of pain or distress may be based on
many different criteria
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Decreased activity
Abnormal postures, hunched back, muscle flaccidity or rigidity
Poor grooming
Decreased food or water consumption
Decreased fecal or urine output
Weight loss (generally 20-25% of baseline), failure to grow, or
loss of body condition (cachexia)
Dehydration
Decrease or increase in body temperature
Decrease or increase in pulse or respiratory rate
Physical response when touched
(withdrawal, lameness, abnormal aggression, vocalizing,
abdominal splinting, increase in pulse or respiration)
Teeth grinding
Self-aggression
Inflammation
Photophobia
Vomiting or diarrhea
Objective criteria of organ failure demonstrated by:
hematological or blood chemistry values, imaging, biopsy, or
gross dysfunction
Ethical Considerations
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An important ethical principle of animal use in
biomedical research is that alternatives to live
animals should be used whenever possible.
Documentation of a search for alternatives and an
explanation for why these alternatives were not
found to be suitable or how alternatives were
incorporated into the experimental design is a
mandatory requirement.
Exploring alternatives to animal use may be
accomplished by using the three Rs; Replacement,
Reduction, and Refinement
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The Three Rs
- Replacement
- Reduction
- Refinement
Replacement alternatives
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Replacing “higher” animals with “lower”
animals. Microorganisms, plants, eggs, reptiles,
amphibians, and invertebrates may be used in some
studies to replace warm-blooded animals. Alternatively,
live animals may be replaced with non-animal models
such as:
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Dummies for an introduction to dissection for teaching the
structure of the animal
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Mechanical or computer models
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Audiovisual aids
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In vitro modeling
Reduction alternatives
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Minimizing the number of animals needed to perform an
experiment or teach a concept, using replacement
whenever possible. Methods to achieve this may include:
Performing pilot studies to determine some of the
potential problems in an experiment before numerous
animals are used
Gathering a maximum amount of information from each
animal, perhaps gathering data for more than one
experiment concurrently
Consulting with a statistician to use only the numbers of
animals required to achieve significance
Minimizing variables such as disease, stress, diet,
genetics, etc., that may affect experimental results
Performing appropriate literature searches and consulting
with colleagues to ensure that experiments are not
duplicated
Using the appropriate species of animal so that useful
data are collected
Refinement alternatives
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Refining experimental protocols to minimize pain or distress
whenever possible.
Examples of refinement may include:
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Using proper handling techniques and receiving adequate training
prior to performing a procedure
Ensuring that:
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– Procedures to be performed on the animal are reasonable for
that species
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– Drug doses are correct and that the drugs used are not expired
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Identifying pain and distress and making plans for preventing or
relieving it using appropriate analgesics and anesthetics for
potentially painful procedures
Refinement alternatives
Performing:
 – Surgeries and procedures aseptically to prevent
infection
 – Appropriate post-surgical care, including
thermoregulation and fluid balance
 – Only one single major survival surgery procedure on
any one animal, whenever possible
Setting the earliest possible endpoint for an experiment.
Explanation: If the necessary information can be gathered
before the animal experiences prolonged suffering from
the experimental procedure, this should be defined as the
endpoint and the animal subsequently euthanized.
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Research Animals
Rats and mice are
good research models
Rats and mice
 Are Handy, easy to care for and handle, and
are inexpensive.
 Are small in size which means they can be
housed in large numbers.
 Have short generation time and high
reproductive potential which contribute to their
usefulness in genetics research and to the
economy of their production.
 Are amenable to germfree and
pathogen-free production techniques
The rat's age in human years
Rat's age in months
Rat's age in years
1.5 months (puberty)
6 months (social maturity)
12 months
18 months
24 months
30 months
36 months
42 months
45 months
48 months
0.125 years
0.5 years
1 year
1.5 years
2 years
2.5 years
3 years
3.5 years
3.75 years
4 years
Rat's age in human years
12.5 years (puberty)
18 years (social maturity)
30 years
45 years
60 years
75 years
90 years
105 years
113 years
120 years
SPECIAL BIOLOGY OF RODENTS
Highly adaptable animals
Nocturnal (active at night)
Social animals, like to live in groups
Aggression among adult male mice
Sensitive to ultra sound
Omnivorous, generally feeding ad libitum
Animal Models
MOUSE and RAT PHYSIOLOGY,
NORMAL VALUES
Respiration rate:
Heart rate:
Body temperature:
Adult weight:
Feed intake:
Water intake:
Urine output:
Breeding age:
Estrous cycle:
Weight at birth:
Gestation period:
Weaning age:
Blood pressure:
Blood volume:
Hematocrit:
Mice
100 – 200 / min
300 – 800 / min
36, 5 – 38, 0 °C
20 – 40 g
3 – 6 g / day
4 – 7 ml / day
1 – 3 ml /day
8 – 10 weeks
4 days
0, 5 – 1, 5 g
18 – 21 days
21 days p.p.
147 / 106 mm Hg
2.4 ml (30g)
39 – 49 %
Rats
70 – 110 / min
300 – 500 / min
37, 5 – 38, 5 °C
250 – 300 g
12 – 20 g
15 – 35 ml / day
10 – 15 ml / day
12 – 16 week
4 – 5 days
≈5g
21 – 23 days
21 days p.p.
145 / 105 mm Hg
18 ml (250 g)
36 – 48 %
RODENT HUSBANDRY
Suitable cages for mice( 4 mice in a 200 cm², 20 mice in a
810 cm²)
special approval for use of wire mesh cages
Bedding: shavings, (nesting material, environmental
enrichment)
Optimal temperature: 20 – 24 °C
Optimal humidity: 50 – 60 %
Air changes: 15 /hour
Light cycle (12h / 12 h)
Handling, examination, and restraint
techniques
Secure animal by holding it by the base of the tail.
Remove animal from the cage
Place animal on arm or table.
Clinical examination: Eye discharge (porphyrin),
Nose discharge
Hair coat, parasites
Body orifices – discharge,
diarrhea
Restraint techniques: Restrainer, Fixation grips
Loose Restrain
for IP and SC Injection
Secure Restrain
for IP and SC Injection
ANIMAL IDENTIFICATION
Short term (days) :
- permanent marker
Medium term (weeks) : - hair colors
Long term (life) :
- ear punch
- toe amputation
(newborns only!)
- ear tags
- tattoo on hairless skin
- micro chip transponder
Performing a Clinical Exam
1. Observation
a.
Observations without disturbing the animal:
Activity level (hypoactivity, hyperactivity, restlessness, lack
of inquisitiveness)
Attitude (arousal, depression, awareness of surroundings)
Spontaneous Behavior (vocalization, self-trauma, isolation
from cage mates).
b.
Observations after disturbing the animal:
Provoked Behavior (vocalization, hiding, aggressiveness,
minimal response).
1. Observation
of Animal Behavior and Appearance
c. Other observations:
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Body condition
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Food and fluid intake
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Fur and skin (greasy or dull fur; cyanotic, pale, or congested mucous
membranes or skin lesions;
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Eyes (porphyrin
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Posture (hunched back, tucked abdomen, head tucked down)
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staining)
Locomotion (way of walking, ataxia, lameness, action of each limb,
position of tail when ambulating, tremor, convulsion, circling, paralysis,
head tilt)
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Vital signs (respiratory distress - e.g. open mouth breathing, pronounced
chest movement)
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Other clinical parameters that are relevant to your study (presence
and status of tumors, infection, or surgical wounds )
2. Quantifiable characteristics
of clinical parameters
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can be tracked over time
compared to a starting baseline or to normal, untreated
control animals
Quantifiable characteristics of clinical parameters are:
Body weight
Hydration status
Body temperature
Behavior
Surface lesions
Blood parameters
Specific Examination Procedures depending on
research procedures
1. Body Weight
Body weight changes are a sensitive indicator of rodent health
A baseline weight measurement allows monitoring the impact of the
experiment on the animal.
Reduction in body weight may reflect starvation
and / or dehydration
Failure of young animals to gain weight is equivalent to a loss of body
weight.
Most rodents used in research are still growing.
Therefore, body weight changes should be interpreted in terms of both :
actual loss of weight and lack of expected growth
2. Hydration Status
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Observe the animals’ behavior.
Assess the animals’ appearance: Skin turgor, hair coat,
eye clarity,
3. Body Temperature_________________________
 Due to their large ratio of body surface area to mass and
high metabolic rate, rodents lose body warmth faster than
do larger animals.
 A mouse can lose 1 degree of body temperature per 5
minutes.
 Body temperature measurements are also used for
monitoring of humane endpoints.
How often the animals should be monitored ?
depends on:
The severity of the animals' condition,
The expected rate of change in the animals'
status, and
The impact of the procedure on the animals.
The animals should be monitored at least once a day.
But some situations require hourly observation.
Methods for minimizing pain and distress in rodents
Alleviation of Pain ad Distress
When animals are found to be in pain or
distress,
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The veterinarian and the investigator
should be contacted .
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Decide on a treatment
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Treatment may include the administration
of analgesics, antibiotics, warmth, fluid
therapy, nutritional supplements, etc.
Methods
for minimizing pain and distress in rodents
Nutritional Support
Special Diets
are:
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Commercial rodent surgical recovery diets; Surgical
Transgel® (Charles River Laboratories).
Peanut butter or jelly – mixtures
to stimulate appetite and to increase food intake in cases of
a)
b)
c)
Reduced food (and water) intake 1-2 days post surgery
For animals that are experiencing pain and distress
Any study in which morbidity and reduced food intake occurs
Methods
for minimizing pain and distress in rodents
Treatment of Imbalance of Fluids and Electrol
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Lactated Ringers Solution,
0.9% Saline,
Glucose-saline
Inject prior to a study and continue once daily
Therapeutic fluids should be warmed prior to injection
Analgesic treatments may be combined with daily fluid
administrations
Inject mouse s.c.: 3 ml/25 g
rat s.c.:
15 ml/ 250 g
Methods for minimizing pain and distress in rodents
Temperature support
There are many practical ways to provide temperature support to rodents, either
individually or in cages.
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Warming pads:
- Chemical warming pads (often too hot)
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- Circulating water warming pads
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Heat
- Electrical heating pads (use discouraged)
lamps (use discouraged)
Keep animals warm until their activity has returned to normal
If recovering animals are warmed within a cage, offer an area
device.
for escape from the heating
Caution! Warming devices should provide gentle heat only (maximum of 40°C).
Methods for minimizing pain and distress in rodents
Alleviation of Pain ad Distress
Pain Treatment
Commonly used analgesics are:
Ketoprofen
Tramal
Carprofen (lasts 24 hours)
Flunixine
Tylenol derivatives
Conduct a pilot study to determine whether the analgesic may affect the study
or not,
Reassess the animal for pain as the analgesic effect wanes. Perform a clinical
exam for signs of pain to determine if another dose is needed.
Refer to the veterinarian for treatment
recommendations.
Documentation of health monitoring for rodents Documentation of Post-Procedure Care
Record System
1. Cage identification system
2. Health record ( documenting the
clinical observations and physical exam
findings)
Rodent health monitoring sheet
PI:
IACUC protocol:
Experimental Procedure:
Date of Experimental Procedure
Date
Animal/Group
ID
Body
Condition
Posture
Mobility
Responsiveness
Haircoat
Breathing
Eyes and
Nose
Injection/Incision
site
Comments
Immediate Euthanasia Criteria:
 Lethargy or non-responsiveness
 Ataxia or circling
 Labored breathing
 Uncontrolled bleeding or discharge from injection/incision site
 Emaciation or significant loss of body condition (greater than 20% loss of normal body weight)
 Significant pain or pain that does not respond to appropriate treatment
Document all observations on the experimental health monitoring sheet. Alert the Attending Veterinarian of any ill or injured animals.
Initials
Humane Endpoint
A Humane Endpoint can be defined
as the point at which
an experimental animal's pain and/or distress is
terminated, minimized or reduced,
by taking action such as euthanize the animal
humanely, terminating a painful procedure, or
giving treatment
to relieve pain and/or distress.
Unless otherwise approved by the IACUC, animals
should be treated or euthanized before they
become moribund or die due to tumor load.
Endpoint in Cancer Research
In cancer research animals should be
euthanized before the tumor mass becomes
excessive, ulcerates, or impairs the animal's
bodily functions or behavior
The criteria for endpoints in tumor
development should be established in the
animal protocol (scoring system):
Tumor mass or burden
Body condition, e.g. cachexia
Impairment of body functions, e.g. way of walking
Ulceration
E. Summary
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Good science requires good animal care. Animals that are in poor
condition, discomfort or pain are poor research subjects. Animal
well-being supports the integrity of the research.
In studies where animal morbidity is an expected outcome of the
procedure humane experimental endpoints should be established
that do not conflict with the scientific objectives.
The strategies for assessing animal well-being and pain or distress
are guidelines that can assist you in developing animal assessment
methods that are appropriate for your experimental procedures.
Alleviation of pain and distress in animals is not achieved solely by
the use of analgesics. Experimental procedures offer many
opportunities for enhancing the animals' well-being by the refinement
of procedures to reduce the severity of injury or stress and by the
provision of supportive care.
Using a system to assess animal well-being will help document
the improvements in technical procedures and the benefits from
supportive care.
The Research Support Team
welcomes you
Chairman: Dr. Abdo Jurjus
Veterinarian: Dr. Fouad Farhat
ACF Manager: Ms. Laura Dosh
Transgenic Unit Manager: Ms. Yolla Bou Moughlabay
Animal attendants:
Mr.
Mr.
Mr.
Mr.
Driver:
Hussein Yunis,
Pierre Boulos,
Omar Haydar,
Abed Al Ghani Jamal.
Jean Ghanem
THANK YOU.