PRACTICAL TECHNIQUES animals used for food and fiber are

exempted
AND PROTOCOLS OF - Requires the IO of the institution to appoint
ANIMAL RESEARCH an IACUC
- Compliance is ensured by unannounced
Why Do We Need to Discuss This? annual inspections (at a min) of each
registered institution
 You will be using / working with animals
in research or studying Principles of Animal Handling – 3RS of Russell
and Burch
 You need to have a basic understanding
The Principle of Humane Experimental Technique –
of the complex system that is meant to
The concept of the 3RS are cited in the regulations
ensure the protection of animals
and are used in policies as ethical guidance for the
 We are obligated by law to make sure
use of animals in research
everyone using animals on campus is
aware of the regulations
 Replacement – methods that avoid
 The safety of the society is also
using animals such as replacing animals
considered in animal research
with inanimate systems or replacing
animals such as vertebrates with
Basics on Animal Handling
animals lower on the phylogenetic scale
Laws and agencies to guide researchers in
 Refinement – modifications of husbands
handling animals
or experimental procedures to enhance
animal well-being and minimize or
Laws governing research on animals
eliminate pain and distress
 Foreign
 Reduction – strategies for obtaining
- The Animal Welfare Act, passed in 1966, is
comparable levels of information from
overseen by the United States department
the use of fewer animals or for
of agriculture
maximizing the information obtained
- Public Health Service policy was passed in
from a given number of animals (without
1985 and is overseen by the OLAW and
increasing pain or distress) so that fewer
NIH
animals are needed to acquire the same
scientific information
US Public Health Policy
- Defines “animal” as any live vertebrate  Respect – respect the welfare of animal
- Requires IO of institution to appoint IACUC used in the study
- Each institution has its own assurance
number on file with NIH Word of caution!
o You must provide your campus Investigators are discouraged from avocation
assurance number on your funding animal reuse as a reduction strategy. Reduction
application shouldn’t be a rationale for reusing an animal that
- Requires that institutions follow both of has already undergone experimental procedures,
these publications to receive funding especially if the well-being of the animal would be
o PHS policy on human care and use compromised
of laboratory animals
o Guide for the care and use of Local agencies enforcing RA 8485
laboratory animals
 Bureau of Animal Industry (BAI)
 Local - Ensures compliance by measuring that
RA 8485 (The Animal Welfare Act of 1998) – An act each institution’s assurance is resubmitted
to promote animal welfare in the Philippines and renewed every 3 years
- Rats (Genus Rattus), Mice (Genus Mus), - Not forced on fish and other marine
Birds, Frogs, and Fish are among these mammals since it is under BFAR that
currently exempted from the definition of doesn’t issue research permit
animal
- Horses and farm animals used in
biomedical research and teaching are
included in the definition of animal but
  Philippine Association of Laboratory - Establishments of a mechanism for receipt
Animal Science (PALAS) and review of concerns involving the care
- Accreditation is voluntary and is recognized and use of animals at the institution. The
as the highest standard of laboratory animal IACUC is authorized to suspend any animal
care act in order to protect animals welfare
- Triennial visits to ensure that the institutes - Submit reports of program evaluation to
meets regulatory compliance and ensures government agencies and to the institutional
high standards of laboratory animal officer (IO)
medicine
- Accredited by the AAALAC. Each campus 3. IACUC Protocols
maintains its own accreditation member Basis
- All protocols used by IACUC for student
 Institutional Animal Care and Use projects should be based on INTEL – ISEF
Committee (IACUC) guidelines
- Serves as the advisory committee on all the - IACUC protocols revolves on evaluating the
matters concerning the humane and ethical following :
care on research animals
- Investigates and ensures resolution to  Project objectives justification
compliance issues involving the care and - Basis of the study and reason for using
use of research and teaching animals animals
- Required to describe proposed research in
1. IACUC and the institution key terminology to represent public’s
 Institution officer (IO) is the institution interest
representative of the IACUC and is - Should include hypothesis, explicit goals,
tasked to assist when: potential contributions to human and / or
-An issue of non-compliance is voted to be animal health and the advancement of Bio
reportable by the IACUC medical knowledge
-All federal reports are submitted - Justify use of animals and why non – animal
-Financial resources are necessary to alternatives cannot be utilized
maintain compliance in the program or the - Justify choice of species and describe
facilities unique characteristics that each species,
stock, or strain has that are necessary for
 Qualifications of the member: your objectives
- Practicing scientist experienced in research - Requires approval of all procedures
involving animals performed in animals. This includes
- Attending veterinarian procedures that are not “experimental” such
- One member from a non-science as tall biopsies, post – procedural care, etc.
background from you school
- One public member to represent general  Experimental plan and design
interests in the proper care and use of - Describe all procedure and time intervals
animals between procedures. Include specific
information on how study durations and
2. Roles of the IACUC endpoints will be determined. Indicate how
- Ensure that the constitution is in compliance all procedures and compounds
with all the national laws administered will effect animal health and
- Protect the institutions assurance licenses ultimate disposition of animals
and accreditation so that the grant funding - Requires approval of all procedures
can be received performed on animals. This includes
- Review and approve proposed animal use procedure that are not experimental such as
(via protocols) and of proposed changes tail biopsies, post – procedure care, etc.
(via amendments) to animal use
- Regulate infections / centralized facilities  Category Definition
and laboratories approved for animal use - Place your animals into one of the following
- Regulate review of program requirements, three categories and justify the choice of
such as training, occupational health and category:
safety, grant verifications, etc.
 o USDA Category C – procedures of housing without prior to conduct
minimal momentary or no distress animal research
o USDA Category D – use of o Acknowledgement that failure to
appropriate anesthetics, comply with IACUC policies and
tranquilizers, or analgesics to procedures will jeopardize
alleviate pain, and / or distress accreditation with the BAI, and may
o USDA Category E – animals may lead to revocation of privileged to
experience unrelieved pain and / or conduct animal research
distress without intervention
Hazards in Animal Handling
 Search for Alternatives Thing you should know to protect oneself from
- Regulation la mandate you to describe how harm in animals
the lack of alternatives were verified for
each potentially painful / distressing  Bites / Scratch Injury in the Animal
procedure (USDA Category D / E) Facility
- Must be in the form of a narrative - If you are bitten or scratched by an animal
description including the date search was or if you have been cut it scratched by an
conducted, database searched, years instrument or piece of equipment that has
covered by search, keywords, or search been exposed to an animal or its bodily
strategies and number of references found fluids, then follow the basic first aid
precautions immediately
 Personnel Qualifications (QS) - Scrub the wound with antiseptic soap and
- The IACUC evaluation of training and flush the injured area liberally with water
qualifications of personnel intending to use - Contact you institutional care provider and
live animals in research or teaching is report the incident. Follow their instructions
required regarding treatment and / or proceed
o Understand the basic need of the directly
species utilized
o Use of proper techniques when  Bio hazardous Agents
handling animals - All Bio hazardous agents must be approved
o Select methods that minimize by the institutional Bio safety committee
distress in animals (IBC) prior to use
o Provide proper pre and post - All bio hazardous agents used in animals
procedural care to animals must be listed in the cage card
o Use aseptic surgical techniques - These agents must also be approved as
o Use anesthetics / tranquilizers part of the animal use protocol form prior to
appropriate for each species use
- The investigators are mandated to follow
 Authorization for Student the disposal directions given by IBC for
Researcher these agents
- You must confirm that information is - Potentially hazardous biological agents:
accurate and confirms with institutional and collective term for microorganisms,
regulatory policies bacteria’s, etc.
o Using animals in research
o That alternatives have been  Chemical Hazards
considered - All chemical hazards must be approved by
o The project doesn’t unnecessarily the SRC / RB prior to use
duplicates previous experiments - All chemical hazards must also be approved
o The personnel (QS) have agreed to as part of the animal use protocol from prior
participate and are aware of to use
approved procedures and are - Investigators are mandated to follow the
instructed human care, handling, disposal directions given by IRC – IRB for
and use of animals these chemicals
o You must confirm that no changes
will be made to procedures, care, or
  Infectious in Research Animals
- Often subclinical : means you cannot tell - Large animals
there is a problem by visual inspections o Chronic Procedure : 5 days
- Can inhibit induction of animal model o Acute procedure : 2 weeks ; 3 days
- Can increase difficulty interpreting results - Exceptions may be made with veterinarian
- Can increase variation in group (requiring approval
larger sample size) - Studies involving immune response,
- Can have zoonotic potential (means this hormone levels, or response to stimulus
could be transmitted from one species to should be given 72 hours to stabilize
another) o Corticosterone levels and immune
function affected by shipping
Protocols of Animal Usage
 Escaped animals
- Use of animals outside of the centralized - If animals escapes, inform IACUC
facilities (RR) is not permitted without - Laboratory rodents caught in traps or found
IACUC approval loose in animal rooms should be euthanized
o Lab use: locations that are approved and may be necropsied
for animal use of short duration.
Rats and mice may be taken to a  Special Husbandry Requirements
laboratory for no more than 24 - Special husbandry
hours. USDA covered species may o Special food
be taken to a laboratory for no more o Special cage, environment – “no
than 12 hours healers”
o Satellite housing : locations - Special husbandry cards are made specific
approved for housing of the animals to the PI request and placed on the animal
over 24 hours cages
- For any use of animal outside of the - Use of rodent post-operative monitoring
regulated research facilities, investigators card is required by ICUC policy
must provide justification to the IACUC in a - It is the investigation’s staff’s responsibility
protocol or amendment to properly monitor animal in the post –
- Satellite housing and areas where survival operative period for 3 days which includes
surgery is conducted must be approved by filling
the IACUC prior to use - Improper monitoring is an issue of
- Areas approved for animal use need to be compliance
inspected by IACUC semiannually
 Required Information on Cage Cards
 Animal Procurement - Investigator’s name
- Animal must be covered by an IACUC - Investigator’s phone number, both in the
protocol prior to ordering office and laboratory
- Animals must only be purchased through - Approved and active protocol number
approved vendors such as - Species, strain, or stock (if applicable)
o DOST - BAI tag number or other identification (if
o Veterinary clinic with IACUC applicable)
o Universities with IACUC - Contact person (individual respondent for
- Animal procurement from non – approved the animal) and extension
vendors will require submission of request - Emergency contact name and phone
and approval from the IACUC chair number
- Bio hazardous or radioactive agents used
 Animal Arrival and Acclimation
- All animals should be afforded a period of  Mouse Cage Population
acclimation / stabilization following shipment - For a small standard shoebox
- Stabilization periods vary depending on o Five adult animals
species, type of transportation and intended o One adult pair and one litter pups
use of animal must be weaned at 21 days of age
o Rodents : 2-3 days o One adult female and one litter pups
o Rabbits : 1-2 weeks must be weaned between 22-28
 days of age. No post - part I’m o Tail clip : tail vein puncture ; a piece of tail is
estrus breeding allowed excused and blood is harvested from the
o Two adult female with one to two severed tail vein
litters; no more than ten pups in a o Saphenous vein puncture : bleeding in
cage. They must be weaned at 21 surface of the thigh
days. Pups must be within one to o Submandibular bleeding: cheek bleeding on
two days of each other in cage. the mouse on the lower vein that drains the
eye.
 Euthanasia (Final Disposition)
- Must justify how euthanasia is performed  Restraining animals
and provide details: a secondary physical - Should be grasped at the tail, preferably the
method of euthanasia is required if an proximal third and lifted clear of its cage
inhalant is used (CO2, isoflurane, etc.) - Scruff grasped between thumb and
Authorization (on protocol) is required prior forefinger, do not let go of tail
to performing euthanasia. An approved
procedure includes the following: (AVMA  Subcutaneous Administration
Panel of Euthanasia) - Subcutaneous injection can be made into
o Appropriate method (CO2, the scruff of the neck. Care must be taken
Anesthetic OD) to direct the needle into the scruff and not
o Conventional methods require into the handler’s finger or thumb
justification (decapitation, cervical
dislocation)  Intra peritoneal Injection
- Note on CO2 Euthanasia - Can be made into the posterior quadrant of
o Must be performed via glass cylinder the abdomen
o Dry ice isn’t permitted
o All secondary physical methods of  Using Gavages
euthanasia is required if inhalant is - Liquid compounds may be administered
used directly into the stomach of mice and rats

Practical Techniques in Animal Research  How to administer oral gavages

- Following the roof of the mouth, advance
 Injection and Bleeding the needle into the esophagus and toward
o Injection : forcing of liquid into a part, as the stomach
subcutaneous tissues, the vascular tree, or - After the needle is passed to the correct
an organ length, the compound may be injected.
o Blood Collection: technique used to collect
blood. This is used for purposes of
diagnosis and can be administered prior
and after treatment

 Injection Techniques in Animals

o Oral Gavage : injection through throat of
animal using special gavage needle
o Subcutaneous : injection through
subcutaneous part of the skin of the animal
o Intra peritoneal : injection through the
abdomen

 Blood Collection Techniques In Animals

o Retro – orbital (intra orbital bleeding) :
puncturing the orbital sinus behind the eye
o Cardiac puncture : terminal ; inserting a
small gavage needle into the ventricle and
slowly drawing out blood