Animal Handling

Base Premise
“Virtually every major medical advance of the last
100 years (as well as advances in veterinary
medicine) has depended on research with
animals. Animal studies have provided the
scientific knowledge that allows health care
providers to improve the quality of life for
humans and animals by preventing and treating
diseases and disorders, and by easing pain and
suffering.”

- Foundation for Biomedical Research, “Understanding the Use of

Animals in Biomedical Research”
DASAR FALSAFAH
 Animal Act, tahun 1876
 International Code of Medical Ethics, tahun
1949
 Animal Act, tahun 1986
 Nurenberg Code
 International Guidelines for Ethical Review of
Epidemiological Studies, tahun 1991
 Helsinki Declaration, tahun 2000
 Operational Guidelines for Ethics Committees
That Review Biomedical Research, tahun
2000
 Universal Declaration for The Welfare of
Animals, tahun 2000
 International Guidelines for Biomedical
Research Involving Human Subjects, tahun
2002

 DASAR HUKUM PENELITIAN
PADA HEWAN COBA DI
INDONESIA

UU no. 23 tahun 1992 tentang

Kesehatan pasal 69 ayat 1 yang
berbunyi : ”Penelitian dan
pengembangan kesehatan
dilaksanakan untuk memilih dan
menetapkan ilmu pengetahuan dan
teknologi tepat guna yang diperlukan
dalam rangka meningkatkan derajat
kesehatan”
UU no. 36 tahun 2009 tentang
Kesehatan pasal 44 ayat 4 yang
berbunyi : ”Penelitian terhadap
hewan harus dijamin untuk
melindungi kelestarian hewan
tersebut serta mencegah dampak
buruk yang tidak langsung bagi
kesehatan manusia”
 Dari dokumen itu
tercermin hal-hal
sebagai berikut :
Hewan percobaan di dalam laboratorium
harus diperhatikan kenyamanan fisiknya,
mereka harus diperlakukan dengan baik,
diberi makanan yang memadai serta
lingkungan harus memenuhi persyaratan
Hanya hewan yang diperoleh secara legal
yang boleh digunakan sebagai hewan coba
Pemeliharaan serta penggunaannya harus
mematuhi segala peraturan perundangan
yang berlaku.
Anesthesi yang memadai harus
digunakan untuk menghilangkan rasa
sakit selama tindakan operatif. Bila
penelitian diperlukan lagi setelah lepas
anesthesia, harus digunakan cara yang
baik untuk mengurangi rasa sakit
menjadi sekecil mungkin
Relaksan otot bukan anesthesia,
sehingga untuk tindakan pembedahan
tidak boleh digunakan tanpa
anesthesia. Obat tersebut harus
digunakan bersama dengan obat yang
menimbulkan anesthesia
Bila pada penelitian tersebut hewan coba tidak
dibangunkan lagi dari anesthesia maka setelah
penelitian selesai hewan coba dibunuh secara
hewani

Perawatan pasca pengobatan terhadap hewan

coba hendaknya sedemikian rupa sehingga
mengurangi rasa tidak nyaman dan rasa sakit.

Bila hewan coba tersebut digunakan mahasiswa

untuk pendidikannya atau untuk pengembangan
ilmu, tindakan tersebut harus dilakukan dibawah
supervisi langsung atau oleh peneliti yang
berpengalaman. Peraturan untuk pemeliharaan
hewan berlaku juga seperti yang dilakukan
terhadap hewan coba untuk penelitian
What is Animal Care and
Use ?
For purposes of this session, the word
“animals” means live, vertebrate animals
used in research, testing, teaching, health
surveillance, or for related purposes.

For purposes of this session, the phrase

“care and use” means procurement,
housing, transport, husbandry, health
maintenance, experimentation, treatment and
humane euthanasia.
Tujuan

Bukti efek terapeutik

(efficacy)
Bukti keamanan (safety)
Bukti efek samping (side
effect)
Prosedur
Penentuan hewan coba
Jumlah hewan coba
Jalur pemberian dan frekuensi
pemberian
Peringkat dosis
Saat dan lama pemberian
Pengamatan dan evaluasi hasil
 Menurut FDA
Uji sitotoksis dilakukan pada empat spesies
mammalia salah satunya non rodent
Evaluasi obat baru pada spesies hewan pada
umumnya untuk :
- dose responses
- absorbsi obat
- distribusi sistemik
- metabolisme dan ekskresi
- tempat dan mekanisme kerja
- reaksi alergi
- Toksisitas akut, sub akut dan kronis
Beberapa uji dapat dilakukan in vitro (contoh otot
polos atau mikroorganisme)
Faktor-faktor yang
mempengaruhi
metabolisme obat
Variasi strain
Perbedaan jenis kelamin
Kondisi lingkungan
Diet
Umur
Cara pemberian materi
Spesies ideal
Mudah diambil darahnya dan jumlah
darah yang diambil cukup banyak
Mudah dipegang dan dikendalikan
Pemberian materi mudah dilakukan
dengan berbagai rute
Mudah dikembangbiakan dan mudah
dipelihara di laboratorium
Lama hidup relatip singkat
Fisiologi diperkirakan sesuai/identik
dengan manusia atau hewan yang
dituju
Contoh spesies yang
umum
Anjing (27%)
Tikus (22%)
Kera (21%)
Kucing (8%)
Kelinci (4%)
 ASAS ETIK PENELITIAN
(The Sundowner
Principles)

Respect for Life Asas Kejujuran
(Principle of Veracity)

 Asas Tidak Merugikan

Societal Benefit (Principle of Non
Maleficence)

Non-Maleficence  Asas Manfaat (Principle

of Beneficence)

 Asas Respek terhadap

Lingkungan (Respect of
Environment)
 SYARAT PENELITIAN
DENGAN HEWAN COBA
 Tujuan penelitian dinilai cukup
bermanfaat;
 Desain penelitian dapat menjamin
bahwa penelitian akan mencapai
tujuannya;
 Tujuan penelitian tidak dapat dicapai
dengan menggunakan subjek atau
prosedur alternatif;
 BEBERAPA PRINSIP DASAR
PENGGUNAAN HEWAN COBA
Untuk kemajuan pengetahuan biologi
dan pengembangan metode dalam
melindungi kesehatan dan
kesejahteraan manusia, diperlukan
percobaan pada berbagai spesies
hewan yang utuh. Ini dilakukan
setelah pertimbangan yang seksama
karena jika layak, harus digunakan
metode seperti model matematika,
simulasi komputer, dan sistem in
Hewan yang dipilih untuk penelitian harus
sesuai spesies dan mutu, serta jumlahnya
hendaknya sekecil mungkin, namun hasil
penelitiannya absah secara ilmiah
Peneliti dan tenaga kerja lainnya harus
memperlakukan hewan percobaan sebagai
makhluk perasa, memperhatikan
pemeliharaan dan pemanfaatannya serta
memahami cara mengurangi
penderitaannya
Peneliti harus menganggap bahwa prosedur
yang menimbulkan rasa nyeri pada manusia,
juga menimbulkan rasa nyeri pada spesies
bertulang belakang termasuk primata
Pada akhir penelitian bahkan pada
waktu dilakukan percobaan, hewan
yang menderita nyeri hebat atau
terus menerus atau menjadi cacat
yang tidak dapat dihilangkan harus
dimatikan tanpa rasa nyeri
 Hewan yang akan dimanfaatkan
untuk penelitian hendaknya
dipelihara dengan baik, termasuk
kandang, makanan, air minum,
transportasi dan cara menanganinya
sesuai tingkah laku dan kebutuhan
biologik tiap spesies
 Prinsip 5F dan 3R

Freedom from :
Hunger and Thirst Replacement
Discomfort
Fear and Distress
Reduction
Pain, Distress and Injury Refinemen
to express natural behaviour

LABORATORY ANIMAL WELFARE

 “The 5 freedoms”
(pendekatan praktik)
Freedom from :
- Hunger and thirst (ready access to water
and diet
to maintain health)
- Discomfort (by providing a suitable
environment)
- Pain, Injury, and Disease (by prevention or
treatment)
- to Express natural behaviour (by providing
sufficient space, adequate facilities)
 “3R”
Replacement
a. Replacement relatif, yaitu tetap
memanfaatkan hewan percobaan sebagai
donor organ, jaringan, atau sel;
b. Replacement absolut, yaitu tidak
memerlukan bahan dari hewan, melainkan
memanfaatkan galur sel (cell lines) atau
program komputer.
 Reduction
Mengurangi pemanfaatan jumlah hewan
percobaan sehingga sesedikit mungkin
dengan bantuan ilmu statistik, program
komputer, dan teknik-teknik biokimia serta
tidak mengulangi penelitian dengan hewan
percobaan apabila tidak perlu
Refinement
Mengurangi ketidaknyamanan yang diderita
oleh hewan percobaan sebelum, selama,
dan setelah penelitian, misalnya dengan
pemberian analgetik
 KETERSEDIAAN ALTERNATIF
• Alternatif
- Hewan Kultur sel; in vitro; simulasi (replacement)
- Metode “ pain & distress”
invasive non invasive

Hierarki (Apes monkey dog/cat/pig/goat Rabbit Rodent

non Mammalia vertebrae Invertebrae Insect/Worms
Single Cell (Yeast, bacteri))

Laparotomi Biopsi perkutan + Fluoroscopy

Invasive Fluoroscopy, DEXA, MRI, PET

ACUAN/REFERENSI
(Jurnal, Penelitian Awal, dll)
 Handling, Care and
Use of Experimental
Animals for
Biomedical Research
understand the
potential hazard
when handling
experimental
animals used in
biomedical
research
safe exposure to
experimental
animals
Why is animal research important?

“Without the use and animals and

human beings, it would have been
impossible to acquire important
knowledge needed to prevent much
suffering and premature death not
only among humans but also among
animals”
Albert Sabin
Developer of the polio vaccine
Animal Testing

Animal testing, or animal research,

refers to the use of animals in
experiments.

It is estimated that 50 to 100

million animals worldwide— from
fruit flies and mice to
non-human primates — are used
annually and either killed during the
experiments or subsequently
1964 Regulation of cholesterol discovered
(Rat )

• 1968 Rubella vaccine developed (Monkey )

• 1970 Lithium approved (Rat, Guinea Pig )

• 1973 Animal social and behavior patterns

discovered (Bee, Fish, Bird )*

• 1975 Interaction between tumor viruses and

genetic material • discovered (Monkey, Horse
, Chicken, Mouse)*

• 1982 Treatment for leprosy developed

(Armadillo )

• 1984 Monoclonal antibodies developed

(Mouse )*
•1992 Laproscopicsurgical techniques advanced (Pig )
• 1995 Gene transfer for cystic fibrosis developed (Mouse,
Non-Human Primate )

• 1997 Prionsdiscovered and characterized (Hamster,

Mouse)*

• 1998 Nitric oxide as signaling molecule in cardiovascular

system discovered (Rabbit)*

• 2000 Brain signal transduction discovered (Mouse, Rat,

Sea Slug )*

• 2002 Mechanism of cell death discovered (Worm)*

• 2003 Non-invasive imaging methods (MRI) for medical

diagnosis developed (Clam, Rat)*

• 2004 Odorant receptors and the organization of the

olfactory system discovered (Mouse)* –

Source: Foundation for Biomedical Research, 2008 –

*Denotes Nobel prize winning research
 approximat
ely 100
million
vertebrates
are
experiment
ed around
the world
every year
Source: British Union for the Abolition of Vivisection
HAZARDS IN ANIMAL
HANDLING
 Types of Hazards
1. Physical Risks
ex. Bites, scratches
2. Hypersensitivity/Allergies
(LAA)
- animal hair, urine, saliva
of
animals
3. Zoonoses
- infection transmitted
from animals to humans
1. Physical Risks
• Bite/scratch
• Unrestrained
 Injecting or bleeding
animal sharps
 Improper
disposal

• Injecting or
bleeding
• Improper sharps
disposal
2. Hypersensitivity/(Laboratory
Animal Allergies (LAA)
Animal hair, Exposure:
saliva, urine
inhalation, direct skin
of animals
or eye contact,
percutaneous exposure
Manifestations:

allergic rhinitis or
conjunctivitis,
wheezing, cough, chest
tightness and atopic
dermatitis
3. Zoonoses
Diseases and infections, which are naturally
transmitted between vertebrate animals and
man.
Modes of transmission: feces, urine, saliva,
blood
Common routes of exposure to infectious
organisms:
 aerosol
 ingestion
 absorption through the skin, through mucus
membranes or skin wounds
 contact with bedding
• Mode of Transmission:
Direct or indirect contact
with skin lesions or
infected hair, or fomites
• Incubation Period: 4-10
days.
• Clinical Disease: The fungi
infect keratinized areas of Rat bite fever
the body. Signs include Streptobacillus moniliformis
round lesion of scaling skin,  Mode of Transmission: Animal
hair loss or breakage,
sometimes reddened and bite, direct contact with
crusting of infected skin. secretions of the mouth,
• Communicability: person to nose, eye of an infected
animal.
person when infective
lesions are present.  Incubation Period: 3-10 days.

 Clinical Disease: Initial bite

wound usually heals. Sudden
onset of fever, chills,
vomiting, headache and joint
Ringworm pains, rash.
Microsporum canis or
 Communicability: not directly
Trichophyton mentagrophytes transmitted from person to
person.
BIOSAFETY PRACTICES
IN ANIMAL HANDLING
Wash hands
after handling
animals and
before leaving
the animal
facility
Decontaminate
work surfaces daily
- general surface
disinfection 1%
Small Spill – 10%
Large Spill –
undiluted from
bottle
Microbial quality of all materials that
will be used
- cage
- bedding
- rodent chow
- drinking water
- feeding bottle
Disposal of carcasses and bedding
 TECHNIQUES IN
ANIMAL HANDLING
 ANIMAL QUARANTINE
hold the newly-arrived animals in a
separate area/cubicle
- to reduce the risk of introducing disease
- to observe the health condition of the
animals
- to acclimatize the animals :
environment and feeds
3-7 days
 carbol fuschin, methylene
blue, gentian violet,
malachite green and
trypan blue
GUIDE FOR THE CARE AND USE OF
LABORATORY ANIMALS:
 Animal Care and Handling Factors
1. Type of caging

 Cages should have

impervious surfaces
with minimal angles,
corners to:
- reduce accumulation
of dirt, debris and
moisture
- facilitate cleaning and
disinfecting
2. Number of
Animals per
Cage
- enough space
to turn around
and express
normal
postural
adjustment
- unobstructed
area to move
around and
rest.
The space allocations for laboratory animals are as follows :

a
To convert square inches to
square centimeters, multiply
by 6.45.
b
From cage floor to cage top.
c
To convert inches to
centimeters. multiply by 2.54.
d
In kg.
e
Larger animals might require
more space to meet the
performance standards
 Social animals should
housed in groups rather
individually provided that
such housing is not
contraindicated by the
protocol in question and
does not pose risk to the
animals.
3. Gentle Handling
Physical and Environmental Factors
 1. Diet and water

wholesome, fresh
rodent pellets (rats and
mice)
 free from contaminants
(microbial and
chemical)
adequate food
according to the
requirements of the
species
Water
Drinking water should
be available to animals
at all times unless
contraindicated by the
experimental protocol.
Water bottles should
always be replaced with
clean water everyday
2. Contact bedding
 Laboratory animals should be moved to
freshly cleaned cages at least once a
week (use gloves and lab gown to
prevent transmission of zoonotic
diseases)

 Frequency of changing the bedding will

depend on the following factors:

- Number and size of the animals in the

primary enclosure

- Size of the cage

- Urinary and fecal output

- Appearance and wetness of the

bedding
Bedding should be
changed 2 or 3 times
a week to prevent the
buildup of urea
Preferably, bedding
should be rice hulls
since some wood
shavings emit toxic
fumes
Environmental Enrichment

Achieved by providing:

- social opportunities

- nesting material

- nutritional rewards

- other species specific

requirements
Sunflower seeds under bedding to encourage normal
foraging and behavior
PVC or toilet rolls for shelter and refuge

Paper clips attached to wire tops to promote activity

and play
Social enrichment with group housing
Researchers using
experimental animals
are required to
accomplish room care
record to ensure that
animal care and
handling factors,
physical and
environmental needs of
the animals are
addressed. (form is
available at RCNS or
eLeap)
Research Manipulation Factors
1. Time of Manipulation/s

 placed in clean cages in a designated reception

area (Quarantine Area) separate from animal
holding room/s prior to introducing them to
other animals and prior to being included in
any research project.
 hold the newly-arrived animals in a separate
area/cubicle (Quarantine Area)

- to reduce the risk of introducing disease

- to observe the health condition of the animals

- to acclimatize the animals to the environment

3-7 days acclimatization period (before animals
can be used for research)
2. Experimental Stressors
- proper animal handling is important
- less stress in experimental animals
 3. Pain and Distress
How and by Whom are Levels of Pain and
Distress Assessed and Categorized?
Animals vary in their response to pain.

- species specific behavioral manifestations of

pain or distress should be used as an indicators
to assess pain in animals.
ex. vocalization, depression or other
behavioral changes, abnormal appearance or
posture and immobility.
- researchers and animal care personnel should
be very familiar with these species-specific,
physiologic and biochemical indicators of
animal wellbeing.
 Euthanasia

 Methods for Each Species

 Euthanasia will be carried out in such a way that it will avoid

animal distress. Other animals should not be present during
euthanasia and this will be performed by personnel who are
skilled and it will be performed in compassionate manner.

 Death will be confirmed by personnel who can recognize

cessation of vital sings in species being euthanized.

 Rodents – cervical dislocation or cervical/cranial concussion

(stunning). Thumb, first finger or metal object will be placed
against the back of the neck and pressed down against a
surface. Pressure will be applied to the neck and at the same
time tail will be pulled firmly and suddenly.

 Rabbits – cervical dislocation or cervical/cranial concussion

(stunning).
 Laboratory Animal
Handling Technique
- Mouse
- Rat
- Rabbit
Laboratory Animal Handling
Technique - Mouse
A. Blood collection from tail vein

B. Blood collection from orbital sinus

C. Blood collection from cardiac puncture

D. Blood collection from saphenous vein

E. Intraperitoneal injection

F.Subcutaneous injection

G. Oral Feeding


Blood Collection From Tail
in Mouse

For collection of small amount of blood

(Approximate 0.1 ml )
Tools for Blood Collection from Tail
75% alcohol
cotton ball for
surface
disinfection
Small plastic
bottle with 1/2
cm diameter
holes in both
ends as mouse
restrainer
Scissors

Pipetteman and
tips
Placing a mouse on a cage lid and grasping the loose
skin behind the ears by the thumb and forefinger
Push the mouse into the restrainer
Leave the tail of the mouse outside the cover of the restrainer
Amputate the tip of the mouse tail by scissors
Massage the tail and collect blood by pipetteman
Blood Collection From
Orbital Sinus in Mouse

Should apply anesthetic before blood

withdraw
A convenience and easy apply method for
blood collection in mouse
Collect amount up to 0.5 ml
Tools for Blood Collection from Orbital Sinus in
Mouse

75% alcohol cotton ball for surface disinfection

Hypnorm for general anesthetic

27 G needle with 1 ml syringe for injection

Glass capillary tube and vial for blood collection

Anesthetize a mouse by intraperitoneal injection of Hypnorm
Use a sharp end glass capillary tube to penetrate the orbital
conjunctiva and rupture the orbital sinus
Collect blood with a vial
Blood Collection From
Cardiac Puncture in
Mouse
For collect up to 1 ml of blood within a
short period of time
Must be performed under general
anesthetic
 Tools for Cardiac puncture in Mouse

75% alcohol cotton ball for surface disinfection

Hypnorm used as anesthetic

27G needle with 1 ml syringe for injection

24G needle with 3 ml syringe for blood

Anesthetize a mouse by intraperitoneal injection of
Hypnorm
Disinfect the thorax area with 75% alcohol cotton ball
Search for the maximum heart palpitation with your finger
Insert a 24G 1” needle through the thoracic wall at the point of maximum
heart palpitation
Withdraw blood slowly by your right hand
Blood Collection From
Saphenous Vein in
Mouse
This method is used of multiple
samples are taken in the course of
a day
It can also be applied on rats,
hamsters, gerbils and guinea-pigs
Tools for blood collection from
Saphenous vein in mice
75% alcohol cotton
ball for surface
disinfection
50 ml syringe tube
with small holes at
the end as restrainer
a scalpel and shaver
for remove of hair
24 G 1 “ needle for
release of blood
tips and pipetteman
for blood collection
Placing a mouse on a cage lid and grasping the loose skin
behind the ears with your thumb and forefinger
Place the mouse in the restainer
Pull out the leg and removed the hair by a assistant
Hair can also be shaved by using a small scalpel
The saphenous vein is seen on the surface of the thigh
Apply vaseline after disinfect the surface area to reduce
clotting and coagulation during blood collection.
Use a 24 G 1” needle to puncture the vein and release
blood from the saphenous vein
Use a Microvette or a pipetteman with tip to collect blood
from the saphenous vein
Approximate 100 microliters can be collected
Flex the foot of the mouse to reduce the flow of blood back
to the puncture site
A cotton ball is applied to the puncture site to stop
further bleeding
Intraperitoneal Injection
in Mouse

A common method of administering

drugs to rodents
Tools for Intraperitoneal Injection in Mouse

75% alcohol cotton ball for surface

disinfection
25G 1/2” needle with 1 ml syringe for
Place a mouse on a cage lid and grasping the loose
skin behind the ears with your thumb and forefinger
As soon as the mouse’s head is restrained, the mouse can
be picked up and the tail secured within your ring finger
and little finger
The injection site should be in the lower left quadrant of the
abdomen because vital organs are absent from this area. Only
the tip of the needle should penetrate the abdominal wall to
prevent injection into the intestine.
Subcutaneous Injection
in Mouse

The most common method for

immunology studies
Tools for Subcutaneous Injection in Mouse

75% alcohol cotton ball for surface

disinfection
25G 1 “ needle with 1 ml syringe for
Pick up a nude mouse and spin it’s tail to put it in a faint condition
Grasp the loose skin on the back of the mouse from ears along the legs
and restrain the legs with your ring finger and little finger
After disinfect the surface area, insert the needle in the
lateral side of the abdominal wall and push upwards to
the armpit of the mouse
Inject the substance slowly
A lump of injection substance can be seen through the
skin after injection
 Oral Feeding in Mouse

Gastric intubation ensures that all the

material was administered
Feeding amount limited to 1% of body weight
Tools for Oral Feeding in Mouse

A 18 G stainless steel, ball tipped

needle
a glove
Grasp the loose skin on the back of the mouse and restrain
it’s tail with your ring finger and little finger. Then, introduce
the feeding tube from the pharynx in to the esophagus
when the mouse is in the act of swallowing.
Common complications associated
with gastric intubation are damage
to the esophagus and
administration of substance into
the trachea. Careful and gentle
passage of the feeding needle will
greatly reduce these possibilities.
The anatomy picture showed the position of the feeding
needle tip inside the esophagus with the heart and
sternum removed.
Sexing mice - The distance between the anal and genital
orifices is greater in the male (left) compared to the female
(right).
Spesies SC IM IP IV PO

Mencit 05-1 ml 0,05 ml 1 ml 0,5 ml 1 ml

20-30g
Tikus 2-5 ml 0,1 ml 2-5 ml 1 ml 5 ml
100-200g
Hamster 2,5 ml 0,1 ml 1-2 ml 0,3 ml 2,5 ml
50g
Kelinci 5-10 ml 0,5-1 ml 10-20 ml 5-10 ml 20 ml
2,5 kg
Kucing 5-10 ml 1 ml 10-20 ml 5-10 ml 50 ml
3 kg
Anjing 10 ml 5 ml 20-50 ml 10-20 ml 100 ml
5 kg
Thank You!