Experimental Animal User Training Core Topics Modules
List of Modules
Introduction
One of the resources provided by the CCAC to support the development and
implementation of an institutional training program by the animal care committee
(ACC) is a series of twelve web-based modules covering the general core topics for
all animal users and specific core topics for the Laboratory Animal/Teaching Stream
of the Recommended Syllabus.
These modules have been developed and produced with funding received from the
Canadian Institutes for Health Research and the Natural Sciences and Engineering
Research Council.
The modules were written by a team from the University of Saskatchewan, the
University of British Columbia and the CCAC Secretariat. Several scientists, animal
care professionals and representatives from the Canadian public and the Canadian
Federation of Humane Societies have reviewed the material extensively. Various
institutions across Canada conducted beta-testing of these modules.
Each module deals with a specific topic and specific goals. There are a few overlaps
between modules; but modules are independent from each other so ACCs have the
necessary flexibility to integrate one or more of these modules within their own
institutional training program as they see fit.
Questions are listed at the end of each module to help readers focus their attention
on key elements. The bank of questions and associated answers will be forwarded to
ACCs upon request ([email protected]) if they choose to use them for performance
evaluations of the competency of their animal users.
The following chart lists the twelve modules of the core topics. To view the
objectives of a particular module, click on the title of that module.
1.
Guidelines, Legislation,
and Regulations
2.
Ethics in Animal
Experimentation
3.
The Three Rs of Humane
Animal Experimentation
4.
Occupational Health and
Safety
5.
Research Issues
6.
Basic Animal Care
7.
Environmental Enrichment
8.
Basic Diseases and the
Animal Facility
9.
Pain, Distress and
Endpoints
10. Analgesia
11. Anesthesia
12. Euthanasia of
Experimental Animals
Glossary
Module 01 - Guidelines, Legislation, and Regulations
Introduction
The first written standards for the care and use of experimental animals in Canada were developed in
1961 by a committee of the Canadian Federation of Biological Societies - Guiding Principles on the
Care of Experimental Animals.
A few years later the Medical Research Council and the National Research Council undertook a study
to look at the question of how national standards for experimental animal care and use should be
implemented. The report came back recommending that the Canadian Council on Animal Care (CCAC)
be established, to provide guidance for all aspects of the care and use of experimental animals.
The Canadian Council on Animal Care (CCAC) was founded in 1968 as a standing committee of the
Association of Universities and Colleges of Canada. Following the recommendations in the report,
Canada opted for a peer review system based on guidelines. The system was selected to draw on the
strengths of many organizations to reach its goals. The Canadian Federation of Humane Societies was
included from the outset as a representative of the animal welfare movement in Canada. In this
system, the CCAC pioneered the establishment of local animal care committees responsible for the
ethical use of animals at their institutions. There are now more than 220 Animal Care Committees
(ACCs) across Canada.
Since 1968, the CCAC program has brought about high standards for experimental animal care and
use through education, voluntary compliance and a code of ethics. General and specific guidelines
have been written, revised and expanded in response to changes in scientific and ethical attitudes to
the use of animals in research, teaching and testing. Such "living" documents as the standards for the
CCAC program have been a model for exemplary animal care and use throughout the world, and have
been emulated in several countries.
Dr Harry Rowsell was commissioned by
MRC and NRC to develop a proposal for a
national program for laboratory animal
care standards in Canada. He became
the first Executive Director of the new
Canadian Council on Animal Care, a
position he held for 25 years. He was
made an Officer of the Order of Canada
for his incalculable contribution to the
establishment of ethically based animal
research spearheaded by CCAC.
Questions (self-test)
You will be presented with a number of questions that should provide you with an indication of the
scope of this module. They are not part of your evaluation, but are intended to raise some of the
issues that we will be presenting in this module.
Which of the following research animals come under the CCAC guidelines?
a.
b.
c.
d.
e.
Rabbits
Rats
Fish
Cephalopods
Monkeys
Which of the following groups are represented on the Council?
a.
b.
c.
d.
e.
Granting agencies
Human societies
Association of Universities and Colleges of Canada
Pharmaceutical industry
Animal care technicians
Which of the following should normally be represented on an Animal Care Committee to ensure that
the well-being of the animals is fully considered?
a.
b.
c.
d.
e.
f.
g.
Veterinarians
Scientists
Institutional members who do not use animals
Community members
Technicians
Students
Animal facility managers
In reviewing a protocol for the use of animals in a research project, which of the following should be
considered?
a.
b.
c.
d.
e.
f.
The overall objectives of the research
The numbers of animals to be used
The procedures to be carried out on each animal
Steps to control pain or discomfort during the study
The facilities where the animals are kept
The source of the animals
What is the Canadian Council on Animal Care?
The purpose of the Canadian Council on Animal Care is to act in the interests of the people of Canada
to ensure through programs of education, assessment and guidelines development that the use of
animals, where necessary, for research, teaching and testing employs optimal physical and
psychological care according to acceptable scientific standards, and to promote an increased level of
knowledge, awareness and sensitivity to relevant ethical principles.
The "council" is the governing body of the CCAC. It consists of representatives of a wide range of
national organizations with an interest in the care and use of animals in research, teaching and
testing.
•
•
•
•
•
•
•
Federal Granting Agencies
Federal Government Departments / Agencies Using Animals or Supporting Animal-Based
Research
National Voluntary Health Organisations
Institutionally-based National Academic Associations
National Scientific and Academic Associations
National Organisations Representing Pharmaceutical Companies
National Organisations Representing Animal Welfare and Animal Care
The more complete list is available here.
Supporting the work of the Council are five standing committees, with additional experts that serve on
specific committees (e.g., ad hoc subcommittees are established when new guidelines are being
developed or old ones revised).
The Council is supported by a secretariat (an office staff) in Ottawa, which coordinates the many
aspects of the national program of standards for the care and use of experimental animals.
At the inaugural meeting on January 30, 1968, the CCAC adopted the following statement of
objective: "to develop guiding principles for the care of experimental animals in Canada, and to work
for their effective application". These guiding principles contain the fundamental requirements for
humane and ethical use of animals in research, teaching and testing in Canada.
To carry out its mandate, the CCAC adopted a decentralised model in which responsibility for all
matters relating to the care and welfare of experimental animals resides within the institution through the animal care committee. The CCAC provides the national standards through the various
guidelines, and through its Assessment Program, evaluates the work of the animal care committees
and monitors compliance with the national standards. It is essential that the institution be committed
to the CCAC standards for physical infrastructure, animal care and protocol review. These aspects of
the CCAC program are discussed in more detail in following sections.
Guidelines of the CCAC
Since the first Guide to the Care and Use of Experimental Animals was published in 1968, the
guidelines of the CCAC have steadily evolved in response to new information or requirements that
address new research or ethical needs.
The most recent general guidelines document is the Guide to the Care and Use of Experimental
Animals Volume 1, 2nd Edition, 1993. This volume along with the currently approved CCAC guidelines,
is available on the CCAC website (www.ccac.ca). All guidelines are available in French and English,
and the second edition of Volume 1 and the guidelines on choosing an appropriate endpoint are also
available in Spanish.
It became clear during the 1990s that there was a need for more specific guidelines in certain areas,
and so the CCAC embarked on a program to publish individual guidelines on selected topics. The
development of these specific guidelines involves the establishment of small ad hoc subcommittees to
draft the guideline document. Once a draft is ready, there is widespread consultation throughout
Canada to achieve a consensus on the information in the guideline. For example, new guidelines are
posted on the CCAC website in draft form to allow people to comment on them. Thus as many people
as possible are encouraged to participate in the development of guidelines.
The list of specific guidelines documents currently approved includes:
•
Guidelines on: antibody production (2002)
•
Guidelines on: institutional animal user training (1999)
•
Guidelines on: choosing an appropriate endpoint in experiments using animals for research,
teaching and testing (1998)
•
Guidelines on: animal use protocol review (1997)
•
Guidelines on: transgenic animals (1997)
Guidelines are under development for the care and use of wildlife; for laboratory animal facilities; for
the care and use of fish; and for the care and use of farm animals.
In addition to the guidelines, the CCAC also publishes policy statements on a number of issues of
interest and concern, which help everyone involved carry out his/her responsibilities to conduct animal
based studies with scientific validity and with high ethical standards.
The Institutional Animal Care Committee
The Animal Care Committee (ACC) in each institution has a number of responsibilities relating to the
care and use of animals in research, testing and teaching. These responsibilities are detailed in the
CCAC publication entitled Terms of Reference for Animal Care Committees .
The following is an overview of the major responsibilities of an Animal Care Committee. However it is
recommended that research personnel review the full document.
Review and approval of all proposals to use animals:
The ACC must ensure that every proposal to use animals for teaching, testing or research has been
reviewed both for scientific merit (see the CCAC Policy on the Importance of Independent Peer Review
of the Scientific Merit of Animal-Based Research Projects (2000) ) and for compliance with accepted
ethical standards (see Ethics of Animal Investigation ). Each protocol must be the subject of an
annual review, and any amendments reviewed and approved.
Authority:
As defined in the CCAC Terms of Reference for Animal Care Committees, the ACC must have the
authority to halt any study that deviates from the approved protocol or where the animals are found
to be suffering excessive pain or distress that cannot be relieved. In the latter case, an animal may be
euthanized if the pain or distress cannot be relieved in any other way. Usually the veterinary staff is
given this authority on behalf of the ACC.
Ensuring standards for animal facilities and care:
The ACC must ensure that facility standards and the care of the animals are in accordance with the
CCAC Guidelines, and that a qualified person has been clearly designated to be responsible for the
day-to-day facility management and animal care.
Prevention and relief of pain and distress, and ensuring adequate veterinary care:
The ACC must ensure that adequate veterinary care is provided, and that appropriate procedures are
in place to ensure that unnecessary pain and suffering do not occur.
Ensuring the training and skills of all persons working with animals used in science:
The ACC must implement a program to ensure that all persons working with animals receive practical
skills training, in accordance with the CCAC guidelines.
Membership of the ACC:
To ensure that the well-being of the animals is fully considered, the ACC needs to have a membership
with a broad representation of interests and expertise. This broad representation is reflected in the
membership requirements found in the CCAC Terms of Reference for Animal Care Committees. A
properly constituted ACC must have:
•
•
•
•
•
•
•
Scientists and /or teachers experienced in animal care and use
Veterinarian(s) experienced in animal care and use
An institutional member whose normal activities do not involve animals
At least one person representing community interests and concerns who does not have any
links with the institution or with animal use for research, teaching or testing.
Technical staff involved in animal care and use
Student representation in academic institutions
Animal facility managers
The number of people from each category will depend on the size of the institution and its animal use
program. The ACC must have active support from the administration of the institution, including
adequate office and secretarial support to fulfill all its responsibilities both to the institution and to the
CCAC. Documenting all ACC actions and activities is very important.
An Animal Care Committee Meeting
Reporting to the Canadian Council on Animal Care:
The ACC is required to maintain certain documentation regarding the animal care and use at the
institution, and must provide the CCAC with annual reports of animal use, and other specific
information prior to a full CCAC assessment. Animal users can greatly facilitate this important process
by providing their animal use numbers promptly when requested to do so.
Protocol Review
The review of proposed animal use before it begins is one of the fundamental pillars of the CCAC
program and it is the most important responsibility of the ACC. The CCAC has a number of documents
and guidelines that assist the ACC in fulfilling this important responsibility. The following two CCAC
documents should be read as part of this module: Terms of Reference for Animal Care Committees;
and guidelines on animal use protocol review.
Before a request to use animals in research, teaching or testing can be approved, the protocol
reviewers should be able to satisfactorily answer a series of questions such as:
Do you understand why the study should be done?
The scientist proposing the animal use should have explained, in language easily
understood by every one of the reviewers, including the non-scientific members,
the potential benefits, for people or animals, arising from the study.
Are you convinced that animals must be used?
The scientist proposing the animal use should have convincing arguments that
there is no other way to obtain the information being sought in the study.
Has the proposal been independently reviewed for scientific merit?
If this has not been done, the ACC must take steps to ensure that a peer review
for scientific merit is undertaken. Approval to use animals requires that the use
is ethically acceptable and scientifically meritorious.
Has the concept of the Three-Rs been addressed?
The scientist proposing the study or teaching use of animals must indicate the
steps taken to refine procedures and to reduce or replace animals in the study.
Has the choice of animal species and model, and the number of animals
requested been justified?
Not all species are suitable for all studies. The number of animals requested
should fit the proposed experimental design. Are there too many animals or
perhaps not enough? (Statistical justification should be provided.)
Do you understand exactly what will be done to each animal and in what
sequence?
The description of the procedures should be clearly written in understandable
language. For example, volumes of injections or samplings, and their frequency
should be presented in the written protocol, along with a plan for animal
monitoring.
Are you comfortable that the expertise of the people carrying out the
procedures is optimal?
Will additional training or help be required to carry out the project?
Are the facilities for performing the study suitable?
Are the facilities appropriate for housing the species proposed? Will there be
environmental enrichment for the animals? Are surgical facilities available? What
about suitable anesthetic equipment?
Have the signs of pain, stress or distress been described?
Are there measures to relieve these signs, including euthanasia? Humane
endpoints should be identified, particularly when it is known that pain and
distress are likely to occur, but also when there is the possibility of inadvertent
animal injury or pain and/or distress.
Will euthanasia be carried out in an appropriate, approved manner?
Do the people involved have the necessary skills to perform euthanasia?
The questions should provide a sense of the role that members of an ACC have in evaluating the
acceptability of a proposal to use animals in research, teaching or testing.
Although members of the ACC must be able to easily understand all parts of an animal use protocol,
there are two sections of the protocol form that commonly present problems for reviewers - the lay
summary of the study, and the proposed numbers of animals to be used. These warrant a little
further explanation.
The lay summary of the proposed animal use is that part of the form that requires the investigator to
describe in simple terms why the study needs to be carried out. In this section the principal
investigator is required to tell the ACC how the study fits into a broader context, often a problem of
human or animal health. Even basic studies can be explained within a larger context. Although some
scientific language may be used, it should be possible for the principal investigator to avoid technical
terms, abbreviations and acronyms and provide a simple explanation.
The following is an example of a lay summary written in scientific/technical jargon, followed by a more
understandable description of the same project.
An assessment of the value of a vegetable oil organic emulsion on the gustatory features of laminates
of heat processed yeast-grain combinations, nitrated jambon, and bacterially processed lactogenic
products.
Does mayo improve the taste of ham and cheese sandwiches?
Many nerve cells that initially survive a stroke, die a few days later, when the
situation is stable. The reason for this late vulnerability lies partly in the
interaction of these surviving nerve cells with their surrounding supporting cells.
These supporting cells make up one half of the brain volume. After a stroke they
become even more numerous and they form a brain scar tissue in and around
the lesion that is caused by the stroke. Some of the supporting cells are
scavenger cells that seem to aggregate around surviving nerve cells and to exert
damage via neurotoxins. In addition, surviving nerve cells try to re-establish
connections with other nerve cells that were interrupted by the stroke. They
have elongations that grow through the scar tissue region in search of another
viable nerve cell. Scar cells repel these elongations and after a fruitless search
for another neuron, these nerve cells simply give up and initiate the "suicide"
program. However, some cells of the scar tissue have also a positive effect.
They release substances that help nerve cells to survive stress and damage. Our
research uses a rat model with which the beneficial properties of this tissue can
be used to help nerve cells to survive and to suppress the negative aspects of the
scar tissue.
This is an actual
lay summary
from a research
protocol
submitted to an
ACC. It is used
here with
permission.
A second problem is the justification of the number of animals requested for the study. The
ACC must be able to understand the experimental design, and the reasons for the number of animals
required for different components of the study. If the information is not adequate, the ACC will
usually request further information, resulting in a delay in the review process. Although the ACC is
concerned that excessive numbers of animals not be used, it is also concerned with too few animals
being used so that the results may not be interpretable, resulting in the need to carry out additional
studies.
Animal Facility Visits by the ACC
Inspecting all animal facilities at least once each year is an important responsibility of the ACC. These
inspections ensure that animal care or facility problems are identified and resolved. The ACC's facility
inspection reports are reviewed by the CCAC assessment panels when they visit the institution, and
this review allows an assessment panel to target problem areas. This approach enables the CCAC to
provide positive feedback and support to an institution while maintaining institutional responsibility for
quality control of its animal care and use program.
Developing and Approving Standardized Procedures (SOPs)
Written Standard Operating Procedures (SOPs) are common in many work areas. This is an excellent
mechanism for ensuring that work procedures are consistently done regardless of who performs the
work. Within animal facilities the implementation of approved SOPs for the care and use of
experimental animals and for facililty management helps ensure that the treatment of animals in all
phases of research, teaching and testing is of a high standard.
For animal care or use procedures that are essentially the same from one facility to another
throughout the institution, the ACC may develop and approve SOPs for those procedures. SOPs that
are specific to a research program (e.g., procedures involved in the production of transgenic mice)
may be developed by the research team and submitted to the ACC for approval.
A major advantage of approved SOPs is that they may be quoted within a protocol submission as an
indication to the ACC that the procedures will be carried out in an approved manner. Properly written,
approved SOPs provide an easily understood outline of what exactly will be happening to an animal in
a research protocol, thereby assisting the ACC in its review of the protocol, and reducing the work
required by protocol authors.
The Role of the Principal Investigator in Ensuring Responsible Experimental Animal Care
and Use
Each individual involved in a project using animals is responsible for the well-being of the animals.
The principal investigator has the added responsibility of fostering a responsible caring attitude
towards the animals in the conduct of the research. This extends to all aspects of the project so that
at the end of the day, no animal derived data has to be discarded because of lack of care or foresight.
This principle should also govern the actions of all the members of the research team. The principal
investigator must ensure that the team is following his/her example.
The CCAC Assessment Program
The CCAC assessments serve as the quality assurance program for the institution's Animal Care
Committee (ACC) and all aspects of an institution's animal care and use program. It is the primary
means whereby the CCAC receives assurance that the national standards - the CCAC guidelines and
policies - are being met. The Assessment Program is a peer review system, with collaboration among
the CCAC, the assessment panel and the institution, to ensure that animals being used in research,
teaching and testing are receiving exemplary care.
Two types of assessment may occur: a formal, announced full assessment of an institution's animal
care and use program, and unannounced or other special visits (usually by a CCAC Assessments
Director) to deal with specific areas of concern that need to be resolved.
Assessment Frequency
Institutions are formally assessed every three years. Institutions found to be in full Compliance for
two successive full assessments may be placed on a five-year cycle by the CCAC Standing Committee
on Assessments. Special visits may occur at any time and at any frequency required by the CCAC to
ensure that the level of animal care and use meets its standards.
The CCAC Assessment Panel
The members of an assessment panel are selected based on their expertise in animal care and use
and in the areas of research and teaching taking place at the institution. An assessment panel is
usually composed of a veterinarian, at least one scientist, and a community representative nominated
by the Canadian Federation of Humane Societies. These assessment panel members provide their
time voluntarily; only their expenses are covered by the CCAC budget.
The CCAC Assessment Process
(http://www.ccac.ca/en/CCAC_Programs/Guidelines_Policies/POLICIES/ASSESS.HTM)
Pre-assessment Documentation
When a formal assessment is due, the institution is asked to provide each panel member with all the
required information about the administrative organization, animal care personnel, veterinary care
program, animal care and use practices, and the animal facilities.
This documentation allows the panels to develop a clear idea of the functioning of all aspects of the
animal care and use program at the institution and permits more focused probing of areas of concern.
The Site Visit
When an assessment panel arrives at an institution, there is usually a meeting with the ACC, senior
representatives of the administration, animal users and care-givers. During this meeting the panel
explores various aspects of the animal care and use program and clarifies any areas of concern
identified in the pre-assessment documentation. Broadly, the assessment panel reviews five program
areas:
•
•
•
•
•
Functioning of the ACC
Animal holding facilities
Animal care and management practice
Veterinary care program
Continuing education and training
In any of these five areas, any recurring deficiencies might be symptoms of a problem that needs to
be identified and resolved.
Following the initial meeting, all locations at the institution where animals are held and used are
visited. Usually a senior technician working in the area and one or more of the scientists will
accompany the panel. The panel may ask to speak with individual scientists regarding their studies,
and must be given access to any protocol or SOP of interest.
An assessment panel at work.
Once the site visit is completed, the panel meets to formulate the recommendations that will be made
to the institution (usually to the senior administrator responsible for the animal care and use
program), particularly those of a major or serious nature. At this final meeting, the Chair of the panel
will present any recommendations to be made. This is an important meeting in that it should allow
the panel to clarify any possible misapprehensions gained through the pre-assessment documentation
or during the site visits.
Follow this link for CCAC Assessment Panel Policy (1999).
The Assessment Report
The final wording and recommendations contained in the assessment report are prepared at the CCAC
secretariat, then sent to the assessment panel and the CCAC Assessment Committee for final review
and approval. On its recommendation, the report is sent to the institution. The report is a
confidential document to the institution. However the institution may choose to release all or part of it
following written notification to the CCAC.
The assessment report usually contains recommendations to which the institution must respond within
a specified time period. Serious or Major recommendations relate to situations that impose an
immediate threat to the well being of the animals, and these problems must be corrected within a
short time frame (some may need immediate correction and others within three months of receiving
the report). Regular recommendations point to things that may not pose an immediate threat to the
animals but need to be corrected in due course and not left to deteriorate (e.g., facility maintenance).
A plan for the correction of these problems must be submitted within six months of receiving the
Assessment Report. See the Definitions of Recommendations made in CCAC Reports (1996).
Commendatory recommendations are given in recognition of excellent components of the animal care
and use program.
The Follow-Up
When an institution submits its Implementation Report, it is reviewed by this same Assessment
Committee and assessment panel. If the report is acceptable, the institution will be assigned a status
of "Compliance" or "Conditional Compliance" and given a certificate of Good Animal Practice®. If all or
part of an Implementation report are not accepted, further action is taken.
Compliance and Non-compliance
The CCAC Policy on Compliance and Non-compliance defines four statuses that can be assigned to an
institution: Compliance; Conditional Compliance; Probation, and Non-compliance.
The most serious situation would occur when an institution is placed in a status of "Non-compliance".
If no timely resolution of the situation is found, the CCAC policy states that the granting agencies will
be notified, and the granting agencies in turn have agreed to withdraw research funding from the noncompliant institution. See the CCAC Policy on Non-compliance. See also the Memorandum of
Understanding on the Roles and Responsibilities in the Management of Federal Grants and Awards
(MOU) Schedule 3: Ethical Review of Research Involving Animals.
http://www.nserc.ca/institution/mou_sch3_e.htm
Legislation in Canada Related to Experimental Animals
Federal Legislation and Regulations
A legal opinion commissioned by the CCAC in 1998, Legislative Jurisdiction over Animals Used in
Research, Teaching and Testing and an independent study commissioned by Health Canada in 2000,
The Protection of Animals Used for the Purpose of Xenotransplantation in Canada, both reach the
conclusion that under the Constitution Act 1867, the federal government does not have jurisdiction to
legislate with respect to experiments involving animals as this is a provincial jurisdiction. However, the
federal government is not totally absent from the field of animal welfare. The three areas in which the
federal government has taken action are under the criminal law power, the health power and the
spending power.
The Criminal Code of Canada
Section 446 and 447 of the Criminal Code protect animals from cruelty, abuse and neglect. This
section of the Criminal Code has been under review for several years.
The Health of Animals Act
The Health of Animals Act (1990) and its regulations are aimed primarily at protecting Canadian
livestock from a variety of infectious diseases that would threaten both the health of the animals and
people, and Canadian trade in livestock with other countries. This act is used both to deal with named
disease outbreaks in Canada, and to prevent the entry of unacceptable diseases that do not exist in
Canada.
The Spending Power
The other mechanism through which the federal government has lent its support to the humane
treatment of animals is not strictly speaking legislative in nature, but in many respects it is one of the
most powerful instruments available to the federal government for setting national standards. The
federal government's power to provide for grants subject to conditions imposed on the recipients, be
they provincial governments or individual or corporate recipients, may take a variety of different
forms. One form is that of the conditional federal grant or contract. This manifestation of the federal
power is what currently underpins the imposition of CCAC standards on facilities receiving funding
from the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research
Council. Where the government itself awards a contract on an academic or non-academic institution,
clause A9015C of Public Works Standard Acquisition Clauses and Conditions Manual imposes
conditions related to the care and use of experimental animals in public works and government
services.
Provincial Legislation and Regulations
While all of the provinces have legislated in the area of animal welfare in some form or another, only
certain provinces have specifically occupied the field of animals acquired and used for research,
teaching and testing purposes. These are Alberta, Manitoba, Ontario, New Brunswick, Nova Scotia and
Prince Edward Island.
One of the general concerns about the "voluntary" nature of the national CCAC program has been that
some private and provincial government units have not subscribed to the CCAC program, and thus
may not have external assessment of the care and use of animals. Many private and provincial
government units have embraced the CCAC program, recognizing the scientific and public relations
benefits that a monitored animal care and use program brings. In an attempt to provide a more
universal oversight of animals in research, five of the six provinces listed above amended the
regulations to their respective legislation to make reference to CCAC standards.
Click on the province's name for information on provincial legislation.
Alberta
Control of animal experimentation in Alberta is provided by the Universities Act and in its regulations.
Similar to the system developed by the CCAC, although much more limited in its application, the
system put in place in Alberta requires the establishment of Animal Welfare Committees in each
university in the province. It also requires the Director of the Animal Industry Division of the
Department of Agriculture, Food and Rural Development to conduct an annual inspection of the
facilities where, at the universities, experimental animals are housed and research activities occur. In
addition, an annual report on the results of the inspection visits must be submitted to the Minister.
Finally, the conditions governing the care and use of animals are set out in the regulations made
under the Act that were amended in November 2000 to make reference to CCAC standards.
(http://www.agric.gov.ab.ca/ministry/acts/regs/ar221-2000.html) - link no longer available
Back to Province selection
Prince Edward Island
In Prince Edward Island, the Animal Protection Regulations made under the Animal Health and
Protection Act provide that the conditions governing the care of animals used for medical or scientific
research shall be those contained in Volumes 1 and 2 of the Guide to the Care and Use of
Experimental Animals published by the CCAC. These regulations accordingly extend the application of
the general principles set out in the Guide to all institutions that use animals for research in the
province. As a result, it extends the control system developed by the CCAC to all such institutions.
(http://www.gov.pe.ca/infopei/onelisting.php3?number=57455) - link no longer available
***Note, this is the URL for the Act, regulations to the Act are not found at URL
Back to Province selection
Manitoba
Under the Animal Care Act of the province of Manitoba, no one may cause suffering to an animal.
However, this prohibition does not apply to the "accepted activities" listed in the Act as long as these
activities are carried out in accordance with a standard or code of conduct, criteria, practice or
procedure specified as acceptable in the regulations. The use of animals for research and teaching is
an accepted activity within the meaning of the Act. Furthermore, according to the Animal Care
Regulations, Volumes 1 and 2 of the Guide to the Care and Use of Experimental Animals as well as the
CCAC policy statements and guidelines impose standards, criteria, practices or procedures that are
classified as acceptable for the purposes of the Act. As a result, all institutions that use animals for
research and teaching purposes in Manitoba must submit to the control system put in place by the
CCAC. Failing this, any suffering caused to an animal in a research or teaching program constitutes
an offence under the Act. Moreover, the Animal Care Regulations require that animals reared or used
for research or teaching be kept in accordance with the CCAC policy statements and guidelines. As a
result of this obligation, the CCAC's system of control covers the breeders of experimental animals
operating in Manitoba. http://web2.gov.mb.ca/laws/statutes/ccsm/a084e.php
Back to Province selection
New Brunswick
The situation in New Brunswick is unique. The law governs the use of animals for experimental and
other scientific purposes only indirectly. In this sense, it can hardly be said that such use is subject to
regulation in the province.
Section 18(1) of the Society for the Prevention of Cruelty to Animals Act provides that "[a] person who
has ownership, possession or the care and control of an animal shall provide the animal with food,
water, shelter and care in accordance with the regulations." Anyone who violates this duty or fails to
comply with it commits an offence. However, under section 4(2) of Schedule A to the General
Regulation - Society for the Prevention of Cruelty to Animals Act, no one may be found guilty of such
an offence as long as he or she has complied with the CCAC Guide to the Care and Use of
Experimental Animals. Although it does not make the control system developed by the CCAC
mandatory in the province, this exception has the effect of encouraging research institutions and those
who breed and transport experimental animals to comply with this system and submit to it.
http://www.gnb.ca/acts/acts/s-12.htm
Back to Province selection
Nova Scotia
In Nova Scotia the power to inspect and monitor research laboratories belongs to the Nova Scotia
Society for the Prevention of Cruelty (SPC). This power extends to the breeders and suppliers of
animals for experimental purposes since the Society's power of inspection and supervision is not
limited solely to the facilities listed in the legislation. By means of regulations, the SPC may prescribe
conditions governing the housing and care of animals kept for the purposes of sale, research or
breeding. However, the conditions laid down may not conflict with the standards contained in the
codes of practice recommended for the care and housing of farm animals published by Agriculture and
Agri-Food Canada or the Agri-Food Research Council of Canada. Nor may they contradict the CCAC
guidelines. The regulations even indicate that no prosecution may be brought against a person who
complies with these guidelines. Although it does not make the system of control developed by the
CCAC mandatory in the province, this provision nevertheless offers an incentive to comply with it.
Furthermore, the Governor in Council of the province has the power to exempt any research
conducted in accordance with a control system approved by the CCAC from the requirements laid
down by the SPC. However, this power has not yet been exercised.
http://www.gov.ns.ca/legi/legc/statutes/animalcr.htm
Back to Province selection
Ontario
The Ontario legislation, namely the Animals for Research Act, is unique in Canada in that it creates a
system of control based on the registration of research facilities and the issuance of licences for supply
facilities. In Ontario, therefore, all research facilities that use animals in their work must be
registered. Among the provisions of the Animals for Research Act, one should note the duty to
establish an animal care committee, the responsibilities and powers of which are similar to those
required under the CCAC system, and the requirement for any operator of a research facility to submit
to the person designated by the Minister of Agriculture, Food and Rural Affairs a report respecting the
animals used in the research facility for research. The regulation Research Facilities and Supply
Facilities, also provides minimum standards for the housing and care of animals and the regulation
Transportation, prescribes the conditions for transporting animals used or intended for use by a
research facility. http://192.75.156.68/DBLaws/Statutes/English/90a22_e.htm
Back to Province selection
Saskatchewan
In Saskatchewan's Veterinarians Act, an exemption is given to researchers using animals at a
university as long as an Animal Care Committee that included a veterinarian has approved the
protocol. http://www.qp.gov.sk.ca/documents/English/Statutes/Statutes/V5-1.pdf
Back to Province selection
Other Interests Affecting Experimental Animal Care and Use
The media have an interest in research in general, and biomedical research in particular. A
publication in a journal may stimulate interest from the media and a discussion of animal use in
research may ensue. Institutions and investigators should be prepared, through a carefully thoughtout institutional communications plan, to explain their use of experimental animals, including the
procedures and safeguards in place to ensure that the animals were not subjected to any unnecessary
pain and/or distress.
Animal protection groups are always concerned about animals and how they are used. The positions
taken may be extreme or moderate, ranging from the position that humans have no right to use
animals for our own purposes to positions that accept some use of animals but condemn unnecessary
pain and suffering. These concerns for the well-being of experimental animals should be treated with
respect and not necessarily viewed as obstructive. Maintaining a dialogue with all persons interested
in animals is an approach that is fundamental to the CCAC philosophy.
Sample Questions
Which one of the following consequences may result after the assignment of a status of "Noncompliance" to an academic institution ?
a.
b.
c.
d.
e.
Loss of grant support to the offending area
Loss of grant support to the institution
Legal action against the institution
Legal action against the head of the non-compliant area
Expulsion from the CCAC program
How does the institutional ACC ensure investigator compliance with CCAC guidelines ?
a.
b.
c.
d.
e.
Investigators must report animal use to the ACC annually
Visits to animal facilities by the ACC
Reports from veterinarians to the ACC
Annual review of protocols
All of the above
Reference to the guidelines of the CCAC is incorporated into which of the following ?
a.
b.
c.
d.
e.
Federal legislation
Legislation in all provinces
Legislation in some provinces
Both 1 and 3
None of the above
Module 02 - Ethics in Animal Experimentation
Module Objectives
The objectives of this module are:
•
•
•
to introduce readers to basic notions in ethics
to identify the socio-historical basis of the debate on animal experimentation
to define levels of ethical questioning in animal experimentation
Science and Ethics
At the start of the twenty-first century, it is obvious to the public as well as to the scientific community
that the scientific enterprise routinely begs a host of ethical questions. In the area of animal-based
research, these can include questions such as:
•
•
•
•
Is there anything inherently wrong with transfering human genes into other species?
Is the pursuit of knowledge enough to justify carrying out experiments involving pain and/or
distress to an animal?
When primates are no longer needed for research, should they be destroyed humanely or
retired to a primate sanctuary?
If research involves dogs, is it better to use purpose-bred laboratory dogs or unclaimed strays
from a pound?
The informed public expects scientists to have thought through these and other issues. To do this,
scientists need to see the ethical issues not as someone else's field, not as peripheral to the scientific
enterprise, but as an essential element of being a scientist (Monamy, 2000).
Basic Notions in Ethics
The word philosophy, derived from the Greek literally means "love of wisdom." In keeping with its
roots, The Cambridge International Dictionary defines the word philosophy as "the use of reason in
understanding such things as the nature of reality and existence, the use and limits of knowledge and
the principles that govern and influence moral judgment." As an academic discipline, philosophy’s
chief branches include logic, metaphysics, epistemology, and ethics.
Although "ethics" is an academic discipline in its own right, it is useful for scientists to understand the
concepts used in ethical discussions. Just as a discussion of business ethics should involve business
people, so a discussion of ethics in science should actively involve scientists, as scientists bring an indepth knowledge and data, necessary to inform decision making. Discussion of any of the issues listed
at the beginning of this module would benefit from an understanding of the scientific data associated
with the issue.
Ethics is derived from the Greek ethos meaning custom, people, the predominant community spirit.
Within that community spirit, morality is the distinction between right and wrong. The field of ethics,
also called moral philosophy, involves developing, defending, and recommending concepts of right and
wrong behavior. There has been a tendency for scientists to view themselves and their work outside
this realm; however, increasingly science is being seen as part of society rather than apart from it. For
example, the Canadian Institutes of Health Research is mandated by Parliament to promote, assist,
and undertake health research that meets the highest standards of ethics. It now has an ethics
secretariat and ethics directors associated with each of the Institutes.
Philosophers divide "ethics" into three distinct but related kinds of inquiry: metaethics, normative
ethics, and applied ethics.
Metaethics, also known as analytical ethics, investigates where our ethical principles come from, and
what they mean. For example: Are ethical principles merely social inventions? Do they involve more
than expressions of our individual emotions? Answers to these types of questions focus on issues of
universal truths, the will of God, the role of reason in ethical judgment, as well as the meaning of
ethical terms themselves.
In the context of animal experimentation, a metaethical question is the role of reason in motivating
moral actions.
Two sides of the historical debate in this area are:
1.
2.
only emotions can motivate people to act morally (Hume); and,
the opposing stance; true moral action is motivated only by reason when it is free of emotion
and desire (Kant).
Current thinking tends to favour a rationalist approach and focusses on the reasoning and
argumentation process that takes place when making moral choices. According to Baier, proper moral
decision making involves giving the best reasons in support of one course of action versus another.
Normative ethics, also known as substantive ethics, involves a more practical task which is to arrive
at moral standards or norms that regulate right or wrong, good and bad for evaluation and decision.
Applied ethics is the philosophical examination of problems in private and public life that are matters
of moral judgment. By using tools of metaethics and normative ethics, discussions in applied ethics try
to resolve controversial issues such as abortion, environmental concerns, the rights of animals, and
the morality of animal experimentation. It is in the area of applied ethics where scientists have the
most to contribute, recognizing that philosophy is not just about analysis and clarification of moral
dilemma, but can also be used to seek answers.
The Socio-Historical Basis of the Debate on Animal
Experimentation
Concerns about the use of animals in science have existed for almost as long as animals have been
used to better understand the workings of the human/animal body. In order to understand the basis
of some of today’s attitudes to the use of animals and the philosophical debate, it is useful to have an
appreciation of the history of animal-based research and the underlying ethical attitudes.
The detailed history of animal based research has been outlined in many publications. Here only a few
key landmarks are given, in order to give a background to the time-frame for the debate of the use of
animals in science. Alongside are given some of the key ethical attitudes about the use of animals. It
should be clear that almost from the outset that scientists have concerned themselves with
discussions of the "rightness or wrongness" of the use of animals, and in considering what conditions
should be placed on the use of animals for scientific purposes.
TIME-LINE (Panoramic version - Printable version)
Based Research and the Key Moral Statements.
: Chronology of Landmarks in Animal
In 1989, the American Medical Association Council on Scientific Affairs published an impressive list of
medical advances made possible through research using animals including, among others: studies on
autoimmune deficiency syndrome, behavior, cardiovascular disease, cholera, haemophilia, malaria,
muscular dystrophy, anaesthesia,nutrition, and the prevention of rabies. Such research resulted in
subsequent benefits for humans and non-human health. Further lists of medical milestones during the
past century can be seen on the Research Defence Society website and Americans for medical
progress.
With World War I, the focus on antivivisection shifted; benefits to human health through animal
research were welcomed by the public, and in addition, for those who had witnessed the human
suffering as a result of the war, consideration of animal pain seemed "faintly ridiculous" (Ryder,1989).
After World War I, groups with an interest in the well-being of animals used in science were formed
such as the Universities Federation for Animal Welfare (UFAW). UFAW commissioned a philosopher
and a microbiologist, William Russell and Rex Burch to write The Principles of Humane Experimental
Technique (1959), a guide which pioneered the notion of the Three Rs that became a uniting focus
both for the animal welfare and the scientific community worldwide, including in Canada.
The time-line above could be filled with many other examples and should be overlaid with
considerations of the shift of societal values over time. The role of religion, the shift away from
creationist philosophies, the movement to end slavery, to give women the vote; the shift from a rural
to an urban-based economy, etc. are all relevant in the context of an evolving societal ethic of animal
use in science. The time-line can be used both as a brief overview and to pinpoint where the genesis
of some of the thinking of today’s philosophers originated.
The Nature of Science and the Emergence of Bioethics
In parallel with the emergence of physiology, the school of thought called "Positivism" developed
which shaped ideas about the nature of science. Positivism resulted from Auguste Comte's (17981857) attempt to create a clear distinction between the study of the material world and other
branches of human thought such as theology and metaphysics. Science, as seen by the Positivists, is
concerned only with what we can observe. It asks purely empirical questions: what, where, when, how
much? Within this system, ethical questions — good and evil, right and wrong, should and should not
— have no obvious place. Positivism helped to reinforce the distinction between the empirical and the
ethical. However, that distinction expanded into the much broader view that science should not, or
cannot, concern itself with ethical questions at all — that science is an island of pure, empirical
investigation, unattached to ethical values.
By the end of the Second World War, this view was actively being challenged. At that time,
experiments were being carried out, some of them lethal, on human beings who had been imprisoned
and then forced to serve as subjects solely on the basis of race, religion, or mental development.
Other experiments focussed on designing weapons of mass destruction. Even after the war, there
were renowned scientists who conducted painful or harmful experiments on human subjects. These
were clear cases where no one could portray scientific research as a disinterested search for
knowledge, unrelated to ethical values or social agendas. In the wake of such tangible examples,
many scientists found it necessary to reconceptualize their roles to incorporate both the empirical and
the ethical issues inherent to science.
By 1975, the American Association for the Advancement of Science declared:
"It is often said that science is ethically neutral and value-free. Such statements, in our opinion, are
seriously misleading and in some respects quite false. It is, of course, obvious that a scientific
discovery, once published ... can be used in exceedingly diverse ways, with consequences that may be
good or bad, or commonly a complicated mixture of both. The activities of scientists and technologists,
however, are conditioned and directed at every turn by considerations of human values. This is true
over the whole range of activity, from the most basic research to the applications of science in
technology." (cited in Monamy 2000)
Marshall Hall's Principles
1.
2.
3.
4.
5.
No experiment should take place if the necessary information could be gained by observation.
Only experiments that would result in the fulfillment of clearly defined and attainable aims
ought to proceed.
Unnecessary repetition of an experiment must be avoided — particularly if reputable
physiologists had been responsible for the original experiment.
All experiments must be conducted with a minimum of suffering.
All physiological experiments should be witnessed by peers, further reducing the need for
repetition.
(cited in Monamy 2000)
Toward a Coherent Ethic of Research Involving Laboratory
Animals
At present there is no widely accepted comprehensive moral theory pertaining to research involving
laboratory animals. Ethical theories for animal-based research have lagged behind those of human
medical ethics, partially because of the focus on human research ethics following the experiments
during World War II, but also because concern for non-human animals did not and still does not fit
well with the dominant intellectual paradigms driving the development of the field of bioethics.
The 1970s and 1980s saw increased interest in the use of animals among moral philosophers.
Australian philosopher Peter Singer’s Animal Liberation (1975) together with Richard Ryder’s Victims
of Science (1975) and Tom Regan’s The Case for Animal Rights (1983) were published. Because these
publications were both accessible to the lay public as well as firmly rooted in ethical theory they
attracted the attention of opponents of animal research as well as academic philosophers. Reviving
Bentham’s utilitarianism (1789), Singer argued for the liberation of animals based on equality of
consideration of "interests" and their capacity to suffer, and claimed moral status for animals on that
ground. Singer has been criticized by other philosophers as a preference utilitarian for his approval of
the use of less sentient animals. Ryder based his considerations more on the ability of animals to
experience pain, an extension of the concerns expressed by the physiologists Boyle, Hooke, and Lower
as well as the English essayists Pope and Johnson. Another moral view, supported most strongly by
Tom Reagan involves animal "rights." The beginnings of this theory can be seen in Primatt’s extension
of the principle of justice beyond the human sphere. Other philosophers, such as Frey, Wren etc. have
argued for the interests of individual species, and for the right to use animals in research.
The distinction between those who recognize rights in animals and oppose research and those who opt
for animal welfare and permit or endorse humane research may be useful, but it does not accurately
reflect the positions taken by leading contemporary philosophers. Some of those who advocate animal
rights, such as Jerrold Tannenbaum, support the humane use of animals in research. Others, like
Singer, do not claim rights for animals, but are strongly opposed to research involving animal
subjects. The aim here is not to engage in a lengthy discourse of the various philosophical
standpoints. Interested readers are encouraged to consult the reference list accompanying this
module for a more in-depth understanding of current philosophical theories. In particular, Monamy
2000, Smith and Boyd 1991, and Tennenbaum 1999 provide a synthesis of the current philosophical
discussion concerning the ethics of animal-based research.
Moral Stewardship
In the absence of a universal ethic of animal experimentation, animal welfarists, both within science
and without, have plotted a different course of action recognizing that animal researchers have a role
to play as moral stewards. To a certain extent this view can be said to be based on the approach of
Albert Schweitzer (1875-1965), Nobel Peace Laureate, medical practitioner, and doctor of philosophy
— i.e., to cause pain or death when it can be avoided is wrong. In addition, it signifies the beginnings
of a move towards an ecological ethic, where the preservation of a greater whole is seen as important,
occasionally at the expense of individual animal lives. In this context, animal experimentation is
viewed as a "necessary evil," which is justifiable as long as those who conduct the experiments are in
tune with their moral obligations — to society and to the animals in their care (Monamy, 2000). The
CCAC position statement Ethics of Animal Investigation, published in 1989 expresses these concepts
for the CCAC. Building on principles first outlined by Marshall Hall; it also enshrines the Three Rs into
the CCAC system.
This is the convergence point for 2,000 scientists, veterinarians, animal care technicians, students,
community representatives and animal welfare organizations representatives participating in the CCAC
system of ethical review and oversight for the care and use of animals used in science in Canada since
1968.
Applied Ethics in Animal Experimentation: Defining Levels of
Ethical Questioning
As outlined at the beginning of the module, there are genuine societal debates about animal use that
need to occur outside the boundaries of the CCAC system of ethical review and oversight for the care
and use of animals in science. For example, questions such as:
•
•
•
•
Should animals be used in research?
Do we as a society want xenotransplantation as a medical procedure?
Should marine mammals be kept in captivity?
Should society permit stem cell research involving fusion of human-mouse embryos?
The involvement of scientists in these debates is critical to ensure that appropriate scientific data is
used to inform the debate. However, scientists also need to be aware that not only scientific
knowledge will be engaged and other societal inputs may result in a prohibition of certain areas of
animal-based research. (For example, an 18-month Canadian consultation to answer the question
"Should xenotransplantation proceed?" led to the conclusion that scientific knowledge is not
sufficiently advanced to answer two of the key issues: disease transmission, and the balance between
immunosupression and the genetic modification of the source organ to prevent rejection – so it should
not proceed until further research.)
When we have the answers to these types of questions, or rather when we have some understanding
of where we stand as a society on these issues, at this time, in this place, then we are able to engage
in the process of developing guidelines which accept as the societal norm that animals are going to be
used for research, teaching and testing; or that xenotransplantation should only proceed under a set
of prescribed conditions; or that it is necessary to keep some marine mammals in captivity to engage
the public in concern for the marine environment.
The CCAC guidelines development process provides a framework under which the activity can take
place, based on a willingness to do our best, taking into consideration all the information available.
Scientists have a key role to play here in ensuring that the guidelines are based on sound scientific
evidence.
Institutional animal care committees (ACCs), whose functioning is described in more detail in the
Guidelines module, make ethical decisions on individual projects involving animal use. ACCs,
composed of scientists/teachers, animal care personnel, personnel who do not use animals, and
community representatives, function as a microcosm of society, using the guidelines and policies of
the CCAC and their own expertise, experience, values, and common sense to reach decisions about
what animal-based work should be allowed to proceed and under what conditions.
Scientists have a crucial role to play in ensuring responsible experimental animal care and use, and in
fostering a caring attitude towards animals in the conduct of their research. Beyond overseeing the
appropriate conduct of their own projects, the role that scientists play on ACCs is essential. Scientists
provide ACCs with informed views on the need for animal use in science, and exchange views with all
other members of the committee, including those with informed views on animal welfare and
community representatives, to arrive at decisions that balance costs to animals with expected benefits
for humans and animals. ACCs strive to reconcile public demands for medical, scientific, and economic
progress with demands that animal welfare and integrity be protected.
The CCAC Assessment Program is peer review-based, and depends on the active involvement of
scientists on CCAC assessment panels, sharing their expertise and experience with the members of
the institution being assessed. Scientists also play a crucial role on the CCAC Assessment Committee,
a standing committee that reviews all CCAC assessment reports and institutional implementation
reports, and makes final decisions on the CCAC status of each institution in the Program.
A key element of the CCAC system is the involvement of the public in all of the CCAC’s activities,
namely in establishing ethical standards through guidelines development, in ethical decision-making at
the level of each institutional ACC, in providing sound judgment on each CCAC assessment panel, and
in providing a public perspective on the CCAC Council. This integrated approach is essential to ensure
that an external perspective is actively provided to all discussions and decisions on animal care and
use in science, and that those who conduct the experiments are in tune with their obligations to the
animals in their care, as well as to the other members of society.
References
1.
Monoamy, V. 2000. Animal Experimentation: A Guide to the Issues. Cambridge University
Press.
2. The Cambridge International Dictionary (http://dictionary.cambridge.org).
3. The Internet Encyclopedia of Philosophy (http://www.iep.utm.edu/).
4. A Dictionary of Philosophy, Thomas Mautner, Blackwell 1996.
5. Ryder,R.D. 1989. Animal Revolution: Changing Attitudes Towards Speciesism. Oxford: Basil
Blackwell.
6. Russell, W.M.S. and Burch, R.L. 1959. The Principles of Humane Experimental Technique.
London:Methuen.
7. Singer, P. 1975. Animal Liberation. New York:Avon Books.
8. Ryder, R.D. 1975. Victims of Science: the Use of Animals in Research. London: Davis-Poynter.
9. Regan, T. 1983. The Case for Animal Rights. Berkeley: University of California Press.
10. Bentham,J. 1789/1970. An introduction to the principles of morals and legislation. In Burns,
J.H. and Hart, H.L.A (eds). The Collected Works of Jeremy Bentham, Vol. 2,I. London:Athlone.
11. ILAR Journal.1999. Bioethics in laboratory animal research. V40(1)
12. Smith, J.A. and Boyd, K.M. (eds.) 1991. Lives in the Balance: the Ethics of Using Animals in
Biomedical Research. Oxford: Oxford University Press.
Sample Questions
Which one of the following statements best describes the philosophical common ground shared
between individual involved in developing CCAC guidelines ?
a.
b.
c.
d.
e.
only emotions can motivate people to act morally
true moral action is motivated only by reason
reasoning and argumentation process is key to making moral choices
moral standards are grounded in individual approval
social approval of moral value does not change over time
In the absence of a universal ethic of animal xperimentation, which one of the following approach is in
use in Canada by participants in the CCAC Program ?
a.
b.
c.
d.
e.
moral stewardship
utilitarianism
beast-machine
anthropocentric
positivism
Module 03 - The Three Rs of Humane Animal
Experimentation
Module Objectives
The objectives of this module are:
•
•
•
•
To
To
To
To
discuss the Three Rs as they were defined by Russell and Burch in 1959
introduce the concept of alternatives in research, teaching and testing
discuss the potentials and limitations of alternatives
consider examples of alternatives and how they may be used
Preface Questions
Two leading questions are posed here to stimulate the reader, and frame the importance of
considering the Three Rs principles in striving to meet our ethical responsibilities for conducting
humane animal experimentation.
The questions posed represent two of the more contentious issues in animal based research, teaching
and testing: the numbers of animals being used, and the pain and suffering being experienced by
these animals. The answers are presented as arguments for animal use, in terms of what is done to
the animals, and in terms of how many are used. Do you think any of the answers are justified?
Introduction
The question of pain and distress in animals used for research, teaching and testing has concerned the
general public and thoughtful researchers for a long time. It was this concern, together with increasing
use of animals in fundamental and applied research, that motivated W.M.S. Russell and R.L. Burch to
examine how decisions should be made about such use of animals.
The Three Rs stand for Reduction, Replacement and Refinement. In the book The Principles of
Humane Experimental Technique, published in 1959, the authors Russell and Burch proposed that all
research using animals should be evaluated to see if the Three Rs could be applied. They recognized
that while the replacement of animals as research subjects was a desirable goal, considerable gains
could be made in humane science through reducing the numbers of animals used and by refining the
techniques that were applied to animals. Over the past 40 years the Three Rs have become widely
accepted ethical principles to be embedded in the conduct of animal based science.
Many agencies responsible for setting standards for the care and use of experimental animals,
including the Canadian Council on Animal Care, require investigators toconsider the implementation of
the Three Rs during the design of experiments that will use animals. The principal investigator must
consider the question of whether animals are needed or not and if an animal must be used, then the
investigator is required to consider the Three Rs in detail. The protocol submitted to the Animal Care
Committee should outline the rationale for using animals and list the databases that were searched to
confirm that there are no alternatives to animals.
The word "alternatives" came into use after 1978 following the publication by David Smyth, a
physiologist and President of the UK Research Defence Society, of Alternatives to Animal Experiments.
In this book, Smyth provided a Three Rs definition of alternatives: “ All procedures which can
completely replace the need for animal experiments, reduce the numbers of animals required, or
diminish the amount of pain or distress suffered by animals in meeting the essential needs of man and
other animals." Although there have been repeated attempts to limit the term “alternatives” to
replacement, it is in the broader context that alternatives will be discussed in this module, as originally
intended.
What is Meant by the Terms "Replacement" "Reduction" and
"Refinement"?
As has been noted, the word alternatives is used to describe any change from present procedures that
will result in the replacement of animals, a reduction in the numbers used or a refinement of
techniques that may reduce or replace animals or reduce the pain, stress or distress of the animals.
Replacement often means the use of an inanimate system as an alternative (e.g., a computer model
or program, a mannequin). It can also mean the replacement of sentient animals (usually vertebrates)
with less sentient animals (usually invertebrates such as worms, bacteria, etc). It also includes the
use of cell and tissue cultures. The cells must come from somewhere and often this means animals.
Reduction means a decrease in the number of animals used previously with no loss of useful
information. This may be achieved by reducing the number of variables through good experimental
design, by using genetically homogeneous animals or by ensuring that the conditions of the
experiment are rigorously controlled.
Refinement means a change in some aspect of the experiment that results in a reduction or
replacement of animals or in a reduction of any pain, stress or distress that animals may experience.
The establishment of early endpoints for intervention in a study that has the potential to cause pain or
distress is an example of refinement.
Few of the alternatives completely fulfill the definition of the R category in which they are placed. For
example, although the use of tissue cultures will replace many animals, some will be required as a
source of cells. In the following sections on Replacement, Reduction and Refinement, examples of
each will be given.
Satisfying the Replacement Principle
In this section, we will consider replacement as it pertains to three different areas of research,
teaching and testing.
General Principles Concerning Replacement in Research and Testing
Cell cultures, bacteria and inanimate models cannot be used to study processes as they would occur
within the context of a whole, live organism. Thus a culture of heart cells is not comparable to heart
cells in situ, as it cannot reveal the interactions between all the various heart cells as they are
normally situated within a whole heart, nor those with the nervous, endocrinologic and immune
systems that normally affect them, nor the effects of blood flow and pressure and of the many other
factors and signals that exist in a live, whole organism.
Behavioral responses cannot be studied in simple cultures of cells. The behavior of simple organisms
(e.g., bacteria, nematodes) could be studied, however it would be very difficult to extrapolate the
relevance to more complex organisms. Along the same line, it would be impossible to study species
specific and sex specific phenomena.
In cases where specific processes, either cellular or molecular, need to be looked at or used in
isolation, replacement alternatives such as cell/tissue/organ cultures or bacterial cultures become
excellent tools. Some of the variability factors that complicate intact animal research are reduced
when cell cultures, bacteria, etc., are used. These include factors such as light, sound, latent
infections, etc. Of course, if totally inanimate alternatives are used, variability of this type should not
be a factor at all.
Where fresh cell lines are required, it should be possible to get many more cultures and therefore
experiments from each animal than if the whole animal was used for the study. If the alternative is
inanimate (e.g., a computer) there may still be a need to use a small number of animals to get data to
feed into the computer. The quality of that data needs to be excellent or it becomes a case of garbage
in and garbage out.
Biological systems are known for their complexity and their ability to behave in an unexpected manner
with the production of artefacts. A much simpler system such as a cell line is not so likely to produce
artefacts, as long as the cells are maintained in the appropriate milieu.
A corollary to the artefact problem is the simplicity with which the environment of the cells may be
altered and in a manner that could not be repeated in the intact animal. It is easy for example, to
alter the pH, the ion content, the oxygen level etc. of the growth medium to study the effect of these
changes. The repeatability of the studies should be much greater when there is good control of all the
potential variables.
The cost of using alternatives is likely to be less than the cost of using intact animals although this
may not be inevitable. The costs of computers, software, cell/tissue/organ culture equipment, etc.,
may exceed the costs of animals.
Replacement in Research
Basic research. Animals have been used extensively to study fundamental principles in biology.
Usually investigators tried to use animals where there was a similarity between the animal's
physiology and biochemistry and the human's. It is recognized that many of the more fundamental
processes are common to a wide range of organisms including invertebrates.
The alternatives. The use of lower, less sentient animals, particularly invertebrates is considered to
be an acceptable means of replacing higher animals as research subjects. The nematode,
Caenorhabditis elegans, is widely used to study basic neuronal function. This organism has 302
neurons in its nervous system and so it is reasonable to study the function of each neuron and its
interaction with other neurons. In a similar vein, geneticists have used fruit flies for many years.
There are other important replacement alternatives in research: one of the most common and useful
ones is the replacement of rodent-based methods by in vitro methods for monoclonal antibody
production.
Replacement in Safety and Efficacy Testing
The use of animals for safety and efficacy testing new products has increased greatly over the past
forty years or so. Companies producing the products, regulatory agencies and consumers want to be
sure that the products are safe to use. While medical treatments make up the greatest bulk of these
products, just about anything we use must be proven to be safe, for example, the cars we drive and
the products we use, including household cleaners, pesticides, cosmetic products, etc. Once upon a
time baboons were used in crash tests. The alternatives, instrumented mannequins (crash test
dummies), provide much more precise information than did the animal model.
Public concerns for safety of products drove the need for increased testing, and public concerns about
how animals are used in safety testing are now driving the need to seek alternatives.
One of the major challenges for the proponents of alternative methodologies for testing new
compounds has been to prove that they are as effective as the animal based tests they are intended
to replace. Two organizations created to ensure sound scientific validation and subsequent acceptance
by regulatory agencies of proposed alternatives to animals in testing are the European Centre for the
Validation of Alternative Methods (ECVAM) in Italy and the Interagency Coordinating Committee for
the Validation of Alternative Methods (ICCVAM) in USA.
Although regulatory agencies throughout the world have been cautious about accepting these
alternatives, progress continues to be made. As of 2002, there are three in vitro tests accepted by the
European regulatory agencies and three by the USA regulatory agencies and there are several more
being evaluated.
Replacement in Education and Training
Practical Skills Training
Learning skills, from simple techniques such as blood sampling to complicated surgical procedures
such as laparoscopic surgery, are an important part of the training of medical and veterinary
personnel. Animals continue to be used in this training. However, some skills such as suturing
techniques may be developed without using animals. Discarded placentas may be used to practice
microsurgery techniques.
The alternatives. There are now inanimate models that can be used to practice procedures. The
Koken rat, for example, will allow a student to practice tail vein injections many times before it is
attempted on a live animal. Mannequins and computer-based technologies are available to allow
surgeons to practice laparoscopic surgeries. The acceptance of these inanimate objects for training
comes when the touch and feel of the training is similar to that experienced when using a living
organism.
Education
Animals have been used extensively for teaching and demonstration of biological principles. In recent
years, there has been a significant reduction in the numbers of animals due to the adoption of
alternatives.
The alternatives. A wide range of materials may be substituted for animals in teaching. Audiovisual
aids and computer-based programs allow the student to see the effects of manipulating various organ
systems. Many of the computer programs are interactive, allowing the students to participate in the
'experiments'. For example, an interactive program on anesthesia allows the student to assess the
depth of anesthesia, to calculate the dose and route of different anesthetic agents, etc.
Satisfying the Reduction Principle
Literature searches are vital in preventing unnecessary duplication of experiments. Some duplication
of studies is required to ensure that the results from one study are reproducible by other investigators
in different laboratories. However, it is not necessary to repeat studies over and over again.
There are several ways in which an investigator may attempt to reduce the number of animals
required in a study. It is important to ensure that appropriate numbers of animals are used, both the
experimental animals and the controls. This means that the statistical design of the study should be
carefully evaluated before the study starts. Perhaps a statistician should be consulted. Good
experimental design with proper data collection and analysis will minimize the number of animals
required.
A well trained research team extending from the principal investigator to the animal care technicians
will ensure that all procedures related to and peripheral to the study will be standardized. It is
important that the team members are trained in their specialty and additional expertise brought on as
needed. For example, if the project requires a particular surgical procedure for which no one has been
trained, an experienced surgeon should assist. Training in all procedures applied to the animals should
be done before the project starts.
For teaching laboratories using animals, the success of the laboratory session is greatly increased if
trained instructors rather than untrained students set up the animal preparations.
One cause of large group sizes comes from the variability that can occur when the conditions of the
experiments are poorly controlled. Large group sizes may be reduced if, for example, a genetically
homogeneous population of animals is used, or the animals are not subject to intercurrent diseases, or
the husbandry conditions are stable. The issue of variability is considered in more detail in Module#4
Research Issues. In that module, the influence of various parameters (e.g., sound, light, infections,
procedures, etc.) on the precision of experimental results is discussed.
Control animals may represent up to 50% of the animals in a study. The investigator should try to
minimize the number of control animals. Using one control group with several test groups rather than
one control group for each test group may do this. If a particular procedure is used repeatedly in a
laboratory, there will be a historical record of controls for that procedure. For a study using the
procedure, it may be possible to use a very small number of controls and show that they fall within
the historical limits of the controls, rather than use a full complement of controls.
Targeted animal models. In the past, it was difficult to find animal models that accurately mimicked
human conditions like many cancers. There were animal models of breast cancer but the cause and
the biological behaviour of the cancer differed from that in the human. Thus treatments for the animal
model were not necessarily applicable to humans.
The alternative. The development of immune compromised animals meant that cells of human origin
could be grown in animals without the need for immune suppression of the host. Now the behaviour
and treatment of the tumour in the animal model could reflect the situation in the human. Such
precisely targeted animal models will result in an overall reduction in animal use through a reduction
in the variability of the model and the increased usefulness of the results.
Genetically modified (GM) animals (transgenic, knockout and mutant) represent alternatives that
promise to provide more relevant results for human disease understanding. Initially there may be little
reduction or replacement because the production of foundation stocks of GM animals still requires
large numbers for breeding. The refinement of results from the GM animals should lead to a more
rapid advance in the understanding and treatment of human diseases with the use of smaller numbers
of animals.
Satisfying the Refinement Principle
Refinement has been the least glamorous of the Three Rs because it produces the least obvious
changes in animal use if numbers are the most important statistic. The refinement of techniques has a
significant role to play in both the reduction and replacement of animals in research, teaching and
testing. Refined techniques will result in less variability and improve the outcome in terms of results
obtained. For example, the introduction of new and safer anesthetic agents together with better
training of investigators in their use has reduced the number of anesthetic deaths. The refinement of
statistical analytical techniques has allowed investigators to use fewer animals without losing
significant information.
Refinement has its greatest impact in the reduction of pain and distress in animals. Appropriate use of
anesthetics, analgesics and other therapeutic measures are very important refinement measures in
invasive studies. The refinement of husbandry, particularly by increasing the complexity of social and
physical environments, has improved the well-being of research animals. The establishment of
scientific and appropriate endpoints for many studies (e.g., vaccine testing) has meant that animals
have had to suffer less without affecting confidence in the results.
There are many examples of refinements that have made a difference both to the animals (in terms of
minimizing pain and distress), and the results of scientific investigations.
Husbandry. In the past research animals were often singly housed in cages or pens that provided
very little substrate or space for normal behavioral activities. Most research animals are social in
behavior and isolation is stressful for them.
The alternatives. Most animals may be kept in social groups in complex environments that allow
them to behave in a normal manner. There are many reports documenting the beneficial effects of this
type of husbandry. For example, rats living in a socially and physically complex environment develop a
thicker cerebral cortex, with more dendritic connections compared with those that are kept in
isolation. Young rabbits that were kept in small cages developed skeletal abnormalities because they
were unable to hop and run during the time when their muscles and bones were maturing.
Alternatives to previously used blood sampling techniques. The retro-orbital sinus of some
small species (particularly rodents) was a convenient site from which to collect fairly large samples of
blood. The procedure had risks (e.g., the eye could be damaged, especially if samples were taken
repeatedly), and was painful. Several alternatives have been developed, including blood sampling
from the tail vein, the saphenous vein and the jugular vein. Although some skill is required to perform
these efficiently, the risk of causing severe damage to the animal is greatly reduced.
Experiments that cause severe suffering or death. For studies involving vaccine testing,
infectious diseases, tumours, organ rejection, etc., the endpoint for the animal may in the past have
been death from the disease. As an animal approaches death, it stops eating and drinking and rapidly
becomes dehydrated, and except in a small number of instances, death can be predicted to occur
within a short period of time from the point at which the animal stops eating and drinking.
The alternatives. When an experiment is expected to cause severe suffering or the death of an
animal, endpoints should be established to limit the extent of the suffering and to anticipate death. If
possible, pilot studies should be used to demonstrate the earliest point at which the scientific goals are
reached so that the experiment can be terminated before the animals suffer. At a minimum, the pilot
studies should be used to determine which clinical signs are most appropriate to indicate that the
endpoint has been reached or when the death of the animal becomes inevitable.
Toxicity testing. The LD50 test was required by regulatory agencies as an assessment of toxicity of
new compounds. The LD50 is the dose that will kill 50% of the animals. Many animals were used to
accurately find this dose although its relevance to human toxicity has not been established.
The alternatives. A number of refinements to toxicity testing have been developed and have become
accepted as OECD guidelines. For acute toxicity testing the fixed dose procedure (Tg420); the acute
toxic class method (Tg423) and the up and down procedure (Tg425) have now been accepted by
OECD member countries. Fewer animals and earlier endpoints are part of the refinements. The LD50
test (Tg401) has now been withdrawn and regulatory agencies from the OECD member countries are
required to accept data generated using one of the three alternative guidelines. In addition,
recommendations from ICCVAM have been published describing how in vitro data may be used to
select the starting dose for the test, further limiting the numbers of animals needed and increasing the
predictivity of the data.
Summary
The use of animals in research, teaching and testing is not a right but a privilege. It is incumbent upon
every researcher to ensure that privilege is not abused. Even though animals are, in most cases, bred
for research, that does not mean that we may use as many as we like in whatever way we like. Each
animal is an individual and should be treated as such. We must be careful that they are not subjected
to needless pain or suffering. Excessive numbers should not be used just because they are there. They
should not be used at all if an equally suitable model system could be used to obtain the same results.
Every possible step must be taken to reduce or prevent pain and suffering.
By evaluating each research, teaching or testing protocol for adherence to the principles of the Three
Rs, we will replace, reduce and refine the use of animals in science.
References
Norina Database http://oslovet.veths.no/NORINA/
European Resource Centre for Alternatives (EURCA) http://www.eurca.org/
The Principles of Humane Experimental Technique W.M.S. Russell and R.L. Burch Methuen & Co. Ltd.
London 1959. (Special Edition publ. by Universities Federation for Animal Welfare, 1992)
http://altweb.jhsph.edu/publications/humane_exp/het-toc.htm
Balls, M., Goldberg, A.M., Fentem, J.H. et al (1995) The three Rs: The way forward: ECVAM Workshop
Report 11. ATLA 23: 838-866 http://altweb.jhsph.edu/publications/ECVAM/ecvam11.htm (link no
longer available)
Rispin, A., Farrar, D., Margosches, E. et al. (2002). Alternative Methods for the Median Lethal Dose
(LD50) Test: The Up-And-Down Procedure for Acute Oral Toxicity. ILAR 43(4):233-243.
Smyth D. (1978) Alternatives to Animal Experiments 218pp London: Scolar Press
Sample Questions
The word "alternatives" within the context of the Three Rs means:
a.
b.
c.
d.
e.
Using animals of lower sentiency
Using inanimate models
Using fewer control animals
Using safer anaesthetics
All of the above
Which one of the following statements regarding the use of animals for research and teaching is
correct?
a.
b.
c.
d.
e.
The use of animals in research, teaching and testing is a right.
Researchers may use as many animals as they wish in any way they wish.
The number of animals used should be based on cost of the animals.
Mice and rats are prey species and do not feel pain, so use of analgesics is unnecessary.
The use of an animal in research, testing or teaching should be based entirely on the
suitability of the animal for that project.
A researcher is very concerned about reducing pain and/or distress in his research animals. Which one
of the following will not accomplish this goal?
a.
b.
c.
d.
e.
Providing an enriched environment
Establishing humane endpoints
Using analgesics post-operatively
Isolating animals from one another
Using pilot studies for early detection of goals
Module 04 - Occupational Health and Safety
Occupational Health and Safety in Experimental Animal
Facilities
Purpose
The purpose of this module is to present information on working safely with animals in the course of
research, teaching or testing.
Objectives
Upon completion of the module, the reader will be able to:
•
•
•
•
•
•
•
•
Understand the ways common laboratory animal species react, or defend themselves, if a
procedure causes pain, or they perceive their safety to be threatened
Describe the pertinent aspects of safe handling and performance of manipulations
Describe the procedure for reporting animal related injuries
Identify the proper waste disposal procedures in animal facilities
Define zoonoses, and give examples of animal infections that can be transmitted to humans
Outline the levels of biohazard control, and methods used to minimize biohazard risks
Describe the sources of animal allergy
Describe the procedures necessary to minimize exposure to animal allergens
Index of topics:
•
•
•
•
•
•
•
Introduction
Physical Hazards Associated With Experimental Animal Care and Use
Safe Waste Disposal Practices in Animal Facilities
Biological Hazards of Working with Experimental Animals - Zoonoses
Biohazards as Part of Research Programs
Laboratory Animal Allergy
Chemical and Radiation Safety
Introduction
Working safely with experimental animals encompasses not only the people and the animals, but also
the facilities, equipment, and the procedures we use. It also encompasses the community in which we
each live. We must each practice safe working habits to ensure that any health risks in our working
environments never "leak" into the community because of faulty procedures or carelessness. The
principal investigator must assume responsibility for ensuring that personnel working on the project
are aware of any risks to health and safety.
Policies and programs required under provincial Occupational Health and Safety laws and regulations
are implemented by each institution. Such institutional programs must support a safe working
environment in animal facilities as well.
Physical Hazards Associated With Experimental
Animal Care and Use
Avoiding Physical Injuries
Many tasks in animal facilities require moderate to heavy physical labour, and performing these tasks
may expose personnel to risks from moving heavy equipment (strains), slippery floors, electrical
hazards when washing, noise, etc. Each person must exercise due caution when performing such
tasks.
Although the importance of understanding basic animal behaviour in the human/experimental animal
interaction to avoid injuries can be emphasized here, it cannot replace the skills that are learned by
working directly with the animals. Skilled animal care technical staff will already have the right
attitudes and approaches towards animal handling and manipulations. They will also have the practical
skills to do so safely and humanely. For others, some of the material presented here can serve as a
useful introduction to handling animals safely in an experimental animal facility.
To work safely with an experimental animal a person should:
•
•
•
•
•
•
•
•
understand basic animal behaviour in relation to their interactions with people during handling
appreciate the "flight zones" typical of a species
understand how to communicate with the animal
use appropriate restraint techniques
use restraint equipment properly
identify any animals that may be unpredictable
wear appropriate protective clothing and equipment
maintain appropriate vaccination status
Basic Animal Behaviour Related to Handling and
Manipulations
The flight zone is an animal's "personal space". The size of the flight zone varies with the tameness of
the animal, and other animal-related factors. Completely tame animals have little or no flight zone and
a person can touch them. An animal will begin to move away when the person enters the edge of the
flight zone. When the person is outside the flight zone, an animal (or group of animals in a herd) will
turn and face the person while maintaining a safe distance.
It is probably safe to say that when animals are in small cages or pens, all human "intrusions" are
inside the animal's flight zone. Therefore, it is very important to condition the animals to regular
handling to reduce the apprehension and stress imposed by human presence.
When an animal is apprehensive (e.g., about being picked up), aggressive (e.g., about to attack), or
defensive (e.g., protecting itself, or its young in the case of a mother), its posture and other
behavioural signs can give clues about its state and possible intentions. In many mammalian species
the "warning" posture includes lowered head, ears down or back, and in the smaller animals, mouth
open in a snarl.
By carefully observing the animal's behaviour while approaching it, injuries such as bites and
scratches can be avoided.
Communicating With the Animal
Your voice, your touch, your smell, are all part of an animal's knowledge about you. To establish a
two-way familiarity before a project starts, the people who will be handling or restraining the animals
should talk to, touch, and regularly handle each animal. The conditioning period after transport to the
laboratory (usually one or two weeks) is an excellent time to begin. Consistency in handling each
animal is important. Most laboratory animals learn very quickly who their regular handlers or
caretakers are, and accept the handling without undue stress.
Using Appropriate Restraint Techniques
Different species defend themselves in different ways. For example, a mouse, rat, hamster or dog may
bite, a rabbit may struggle furiously and kick or sometimes bite to try and escape, a cat may scratch
(with intent!) or bite; a cow or horse may kick. The approach to restraining the animal, including any
equipment used for restraint, is to prevent the animal from taking such action while ensuring it is
safely and humanely held. Although the correct approach to handling and restraint can be understood
from printed and audio-visual materials, practice is essential.
Appropriate handling and restraint methods have been developed for most laboratory animal species.
Skills in the appropriate handling and restraint methods should be attained BEFORE the research
project starts.
The handling and restraint of non-human primates require special training, equipment and facilities.
CCAC Guide, Volume 1, 2nd Ed. 1993. Chapter VIII Occupational Health and Safety.
Use of Restraint Equipment
For some procedures such as intravenous injection in a rabbit or blood sampling in a cow, restraint
devices or equipment are useful adjuncts to the handling, and help ensure that the procedure can be
done safely for both the animal and the person. Correct use of such restraint devices will help avoid
unnecessary stress or injury to the animal during the procedure. Conditioning the animal to accept the
restraint device is important to minimizing the risk of injury both to the animal and to the handler.
Use of Chemical Restraint
The safe handling of some species either in the laboratory, (e.g., some non-human primates) or in the
field, may require the use of "chemical" restraint. Chemical restraint is the use of sedatives or
anesthetics to control an animal's activity and thereby allow certain procedures to be done with
minimal stress to the animal. Some of the drugs discussed in the analgesia and anesthesia sections of
this course are useful for chemically restraining animals in circumstances where physical restraint
represents a serious risk of harm to the animal or the handler, or is not feasible (e.g., many wild
species).
Wearing Appropriate Protective Clothing
Protective clothing suitable for the handling to be done should be worn at all times; laboratory coats,
coveralls, gloves, masks, boots (e.g., steel-toed for working with cattle), etc. As noted above, the
handling of non-human primates is a special situation that requires special protective clothing.
Identifying Problem Animals
Any animal known to be difficult to handle should be so identified to all who might be working with it
(e.g., weekend staff, veterinarian). As an old veterinarian once said, "I've never been bitten by a
"biting" dog, but I've been bitten by lots of dogs that didn't bite".
Immunization of Staff
Tetanus Vaccination
To minimize the risks associated with infections arising from any penetrating wounds such as animal
bites or needle sticks, all persons working in laboratory animal facilities should maintain their tetanus
vaccination status.
Rabies Vaccination
All persons at risk of exposure to rabies from any animals that may be infected, should consider
vaccination for rabies. Any animals brought into experimental animal facilities that might have been
exposed to rabies should be considered risks. Generally this refers to any domestic animals housed
outdoors (including farm or fur animals), random source dogs and cats, and any wild animals.
Institutions may require staff to have rabies vaccination as a condition of working with such species.
Other Vaccinations
Depending on the species handled (e.g., non-human primates) other immunizations may be
recommended as part of a health and safety program.
Appropriate records on the vaccination status of all employees should be maintained by the institution.
Animal-Related Injuries, Management and Reporting
Any animal-related injury that may be serious should be handled by the usual emergency medical care
system. Apply the appropriate first aid, call an ambulance or get the injured person to a hospital
emergency department.
Any minor injuries or incidents (e.g., a laboratory mouse or rat bite) should be handled by the
appropriate first aid, and documented. Institutions usually have a procedure for documenting all
injuries, including minor ones, in case complications develop later. The mechanism may be as simple
as filing an incident report form.
Safe Waste Disposal Practices in Animal Facilities
Work in animal facilities commonly involves use of sharp instruments. All sharp items (e.g., needles,
scalpels, capillary tubes, etc.) must be handled safely, and placed in designated sharps containers for
disposal as per institutional policy. Needles should never be recapped and re-used.
Animal Waste Disposal
All animals, animal wastes and related materials should be disposed of as per institutional policy.
Institutions commonly have a protocol defining proper disposal of all animal carcasses or organs. For
example, this might involve collection of all such materials for incineration or other safe disposal.
Disposal of non-contaminated waste (dirty bedding, feed, etc.) may differ from institution to
institution. Adherence to animal facility waste disposal policies will minimize the risks to the
community.
Biological Hazards of Working with Experimental Animals
Zoonoses
Definition: The CCAC Guide defines zoonosis as a disease of animals that may under natural
conditions be transmitted to humans. What this really means is a disease that it is communicable
between animals and humans.
The list of potential zoonoses related to working with animals in research, teaching or testing is quite
long, and numerous books have been written on the subject. (See Appendix VII Zoonoses of Volume 1
of the CCAC Guide to the Care and Use of Experimental Animals) However, in reality the risks are very
low when dealing with the common small laboratory animal species in the laboratory. There are
several reasons for this low risk. Firstly, commercial suppliers of laboratory animals have done an
excellent job of producing disease-free animals. As well, institutions generally have developed good
occupational health and safety programs that include active veterinary monitoring and care programs.
The risk of exposure to zoonotic diseases is greater for those who work with experimental animals
from random sources (including cats, dogs and most livestock), and for field researchers studying wild
animals in their habitat. Working with non-human primates in the laboratory is a special case because
of the many zoonotic concerns.
A few of the most common zoonoses in each of these areas of animal research will be presented as
examples. For more information on zoonoses, and for more information about specific disease
organisms, the Material Safety Data Sheets (MSDS) for individual organisms published by Health
Canada Office of Laboratory Security can be consulted.
Routes of Exposure
Common routes of exposure to infectious organisms are:
•
•
•
•
aerosol (inhaling the organisms)
ingestion (swallowing the organisms)
absorption through the skin, through mucus membranes or skin wounds
injection (accidental, in research)
The use of appropriate equipment, including personal protective equipment appropriate to the route of
exposure for a particular infectious organism, and appropriate practices, will minimize the risk of
exposure.
Zoonoses Associated with Commercially Produced Laboratory Animal Species
As noted above, the risk of exposure to a zoonosis while working with common small laboratory
animals that are commercially reared is very small. One example is presented here: Rat bite fever.
Rat Bite Fever
•
•
•
•
•
•
•
Organism name, and synonyms: Streptobacillus moniliformis, a Gram-negative bacteria.
Synonyms: Rat-bite fever, Haverhill fever.
Reservoir: Rats. Commensal in the mouth and pharynx.
Mode of Transmission: Animal bite, direct contact with secretions of the mouth, nose, eye of
an infected animal.
Incubation Period: 3-10 days.
Clinical Disease: Initial bite wound usually heals. Sudden onset of fever, chills, vomiting,
headache and joint pains, rash.
Epidemiology: Uncommon in North America.
Communicability: Not directly transmitted from person to person.
Zoonoses Associated with Random Source Laboratory Animal Species
Ringworm
Ringworm is a fungal infection of the skin that can occur in a wide range of animals including humans.
•
•
•
•
•
•
•
Rabies
Organism names, and synonym: Microsporum spp., Trichophyton spp., fungal organisms.
Synonyms: Ringworm, dermatomycosis, tinea.
Reservoir: Most domestic and wild animals, and humans. May be latent in hair of some
species.
Mode of Transmission: Direct or indirect contact with skin lesions or infected hair, or fomites
(brushes, clippers, etc.).
Incubation Period: 4-10 days.
Clinical Disease: The fungi infect keratinized areas of the body - hair, skin and nails. Signs
include round lesion of scaling skin, hair loss or breakage, sometimes reddened and crusting
of infected skin.
Communicability: Communicable from person to person when infective lesions are present.
Diagnosis and Prevention: Monitoring for typical signs, confirmed by skin scrapings and
culture. Many treatments are available.
Rabies can infect any mammal, including humans. Purpose-bred laboratory animals are not a likely
source of rabies. However wild animals, animals obtained from random sources, or livestock, may
carry rabies. Many institutions have rabies vaccination policies for at-risk personnel.
•
•
•
•
•
•
•
•
•
Organism name, and synonym: Rabies - a rhabdovirus, Rabies, Hydrophobia.
Reservoir: Wild and domestic animals (e.g., dogs, cats, foxes, coyotes, skunks, racoons) and
bats.
Livestock and rodents may be secondary hosts if infected by a biting animal.
Mode of Transmission: Most commonly by a bite which introduces the virus from the saliva of
a rabid animal. May be airborne in caves inhabited by infected bats.
Incubation Period: Usually a few weeks, but may be up to a year or longer. The virus
propagates in nerves. Thus the site of the wound (distance from the brain), presence of
nerves at the wound, etc., influence the incubation period.
Clinical Disease: Once clinical signs appear, the clinical course is short - usually less than 10
days with death due to respiratory paralysis. Signs include apprehension, behavioural
changes, spasms of swallowing muscles, delirium, weakness progressing to paralysis.
Epidemiology: Worldwide distribution with some rabies free areas. All mammals susceptible.
Communicability: Infected animals shed virus for a few days before clinical signs appear. From
then until the death of the animal, it is infectious.
Diagnosis and Prevention: Pre-exposure immunization of all individuals at high risk (those who
will handle animals, including laboratory workers, veterinarians and other animal handlers)
should be used. The human diploid cell vaccine (HDCV) is currently used. Post-exposure
treatment includes immediate first aid by generously flushing the wound and washing with
soap and/or antiseptics, and providing post-exposure treatments as directed by the physician
(e.g. rabies immune globulin, and vaccination).
Zoonoses Associated with Farm Animal Species
Q Fever
Q Fever may be contracted from working with sheep or goats, particularly ewes during lambing. The
placenta and fetal fluids contain high levels of the organism in infected ewes.
•
Organism name, and synonyms: Coxiella burnetii, an intracellular bacteria. Synonyms: Q
Fever, Query fever, Rickettsia
•
•
•
•
•
•
•
Reservoir: Most common in sheep, cattle, goats. Infected domestic animals usually
asymptomatic but shed massive numbers of organisms at parturition, in the placenta and
placental fluids.
Mode of Transmission: Direct contact with infected animals and their birth fluids; inhalation of
organisms in dust from contaminated premises; wool from sheep.
Incubation Period: Usually 2-3 weeks.
Clinical Disease: Sudden onset fever, chills, headache, weakness, malaise, severe sweats;
pneumonia, usually self-limiting. Chronic infection mainly involves endocarditis. Up to half of
infections are asymptomatic.
Epidemiology: Worldwide distribution. Occurs in laboratories using sheep for research; cases
in research staff, exposed hospital patients.
Communicability: Direct transmission from person to person very rare.
Prevention: Use of appropriate protective clothing including masks. Serologic monitoring of
ewes of limited value. Appropriate sanitation procedures. Vaccination available.
Zoonoses Associated with the Non-human Primates
As noted earlier, non-human primates are a potential source of many zoonotic diseases, and special
facilities, equipment and procedures are required to work with them safely. The zoonosis discussed
here - Herpes B infection - is the one that most people will have heard about.
Herpes B virus Infection
Herpesvirus simiae (B virus) causes a fatal ascending encephalitis in humans infected from old world
non-human primates. The disease in the host non-human primates is usually mild or asymptomatic.
Rhesus monkey infected with Herpesvirus
simiae. Note the ulcers in the inner
mucosal surface of the lower lip.
•
•
Organism name and Synonym: Herpesvirus simiae, a DNA herpesvirus. Synonyms: B virus,
Monkey B virus, Simian B disease.
Reservoir: Common in old world monkey of the macaque group (most common in rhesus and
cynomolgus macaques), both wild and captive colonies.
•
•
•
•
•
•
Mode of Transmission: Latent infection in macaques with periodic episodes of oral lesions and
shedding of virus in saliva. Transmission after a bite or direct or indirect contact with infected
saliva or tissues. Laboratory infections from infected tissues can occur. Aerosol exposure
minimal.
Incubation Period: 3 days to a month.
Clinical Disease: Acute, usually fatal, ascending encephalomyelitis; fever with headache,
lesions at site of exposure. Death in 1 day - 3 weeks after onset of symptoms in over 70% of
cases.
Epidemiology: Occurs in veterinarians, laboratory workers and others handling old world
monkeys or tissue cultures derived from these species.
Communicability: Transmission from person to person rare.
Prevention: Appropriate use of protective laboratory clothing (long sleeved lab coats, face
shields or surgical masks and goggles or glasses), proper methods of restraint.
Zoonoses Associated with Wild Animals and Field Studies
Hantavirus Infection
Deer mouse - Peromyscus sp. The main rodent reservoir for
Hantavirus infection in humans.
•
•
•
•
•
•
•
•
Organism name, and synonym: Hantavirus, an RNA virus in the Bunyavirus family. Synonyms:
hantavirus, hantavirus pulmonary syndrome (HPS), Sin Nombre Virus (SNV) in North America.
Reservoir: Wild rodents such as Peromyscus (deer mouse) and Microtus species in the
Americas.
Mode of Transmission: Inhalation of the virus in the dust from areas where infected rodent
excreta (urine and feces) are present is the most common route. Rodent bites may transmit
the disease.
Incubation Period: Average two to four weeks but may be shorter or longer.
Clinical Disease: Hantavirus Pulmonary Syndrome (HPS) is characterized by a sudden onset
fever, pain, vomiting, and onset of respiratory distress and prostration. Mortality rates are
high despite symptomatic treatment.
Epidemiology: Occurs throughout much of North America including the western provinces.
Communicability: Not thought to be communicable between persons.
Diagnosis and Prevention: Use of personal protective equipment to avoid inhaling the dust
particles with virus, and other direct contact in high risk areas should be used. Field biologists
and persons working in previously "contaminated" buildings are at risk. Guidelines for
protection against infection and for decontaminating facilities are available at this web site.
Exposure Control Plans
Any circumstances that present particular risks of zoonotic infections should be identified before the
risks are encountered. This includes immune compromised states (e.g., HIV infection, anti-rejections
drugs or steroids, pregnancy, etc.).
Provincial Occupational Health and Safety regulations commonly define a requirement to develop a
written "exposure control plan" for workers required to handle, use or produce an infectious material
or organism or likely to be exposed. Responsibility for this rests with the employer. Such a written
plan includes: identifying workers at risk, routes of infection, signs and symptoms of disease,
vaccination, engineering controls, personal protective equipment, personnel training, safe work
practices and procedures, dealing with accidents, and investigating accidents.
Biohazards as Part of Research Programs
When experiments are planned that will involve biohazardous agents, both the institutional
occupational health and safety office, and Health Canada, Office of Laboratory Security Laboratory
Biosafety Guidelines must be consulted. Material Safety Data Sheets (MSDS) are available for the
individual organisms in the risk groups.
Biosafety Guidelines and Levels of Containment
Please note: the information provided here will give the reader a general understanding of the levels
of biohazard control required to work with biohazardous agents safely in animal facilities. It is not
intended to be definitive or complete.
Quote from the Health Canada Laboratory Biosafety Guidelines,
"The attitudes and actions of those who work in the laboratory determine their own safety, and that of
their colleagues and of the community. Laboratory equipment and design can contribute to safety only
if they are used properly by people who are genuinely concerned and knowledgeable about safety
issues."
Biohazards are rated at four levels with a risk group associated with each level. Containment levels
refer to the physical requirements and risk groups refer to the pathogenicity of the organisms.
Biosafety Level 1 is required to manage the lowest risk and Biosafety Level 4 is required to manage
the highest risk to human or animal health.
Biosafety Level 1
Risk Group 1 infectious agents are biological agents that are unlikely to cause disease in healthy
workers or animals (low individual and community risk).
Facilities required to contain risk group 1 organisms - Containment Level 1: No special facilities,
equipment or procedures are required. Standard well-designed experimental animal and laboratory
facilities and basic safe laboratory practices suffice. Hand-washing facilities must be provided.
Disinfectants must be properly used.
Biosafety Level 2
Risk Group 2 infectious agents are pathogens that can cause human or animal disease but, under
normal circumstances, are unlikely to be a serious hazard to laboratory workers, the community,
livestock, or the environment (moderate individual risk, limited community risk). Laboratory
exposures rarely cause infection leading to serious disease; effective treatment and preventive
measures are available and the risk of spread is limited.
Risk Group 2 infectious agents include, for example: E. coli; many salmonella; some fungi like
ringworm; California encephalitis viruses; human herpes simplex viruses; many influenza viruses;
Transmissible Gastro-enteritis of swine; Mouse Hepatitis Virus; and a few parasites.
Facilities, equipment, and procedures required to contain risk group 2 organisms at Level 2:
Laboratory separated from other activities, biohazard sign, room surfaces impervious and readily
cleanable. Equipment should include an autoclave, certified HEPA filtered class I or II biological safety
cabinet for organism manipulations, and personal protective equipment to include laboratory coats
worn only in the laboratory, gloves worn when handling infected animals. All contaminated material to
be properly decontaminated.
Biosafety Level 3
Risk Group 3 infectious agents are pathogens that usually cause serious human or animal disease, or
which can result in serious economic consequences, but do not ordinarily spread by casual contact
from one individual to another (high individual risk, low community risk), or that can be treated by
antimicrobial or antiparasitic agents.
Risk Group 3 pathogens include bacteria such as anthrax, Q Fever, tuberculosis, and viruses such as
hanta viruses, Human immunodeficiency viruses (HIV - all isolates), eastern and western equine
encephalitis viruses.
Facilities, equipment and procedures required to contain risk group 3 organisms include: Specialized
design and construction of laboratories, with controlled access double door entry and body shower. All
wall penetrations must be sealed. Ventilation system design must ensure that air pressure is negative
to surrounding areas at all times, with no recirculation of air; air exhausted through a dedicated
exhaust or HEPA filtration system. Minimum furnishings, all readily cleanable and sterilizable
(fumigation). Laboratory windows sealed and unbreakable. Backup power available.
Equipment must include an autoclave, certified HEPA filtered class II biological safety cabinet for
organism manipulations, and a dedicated handwashing sink with foot, knee or automatic controls,
located near the exit. Personal protective equipment should include solid front laboratory clothing
worn only in the laboratory, head covers and dedicated footwear, gloves worn when handling infected
animals and appropriate respiratory protection, depending on the infectious agents in use.
Exit procedures should include showers, depending on infectious agents used and manipulations
involved. All animal wastes to be disposed of as contaminated laboratory materials. All activities
involving infectious materials to be conducted in biological safety cabinets or other appropriate
combinations of personal protective and physical containment devices.
Laboratory staff must be fully trained in the handling of pathogenic and other hazardous material, in
the use of safety equipment, disposal techniques, handling of contaminated waste, and emergency
response. Standard Operating Procedures must be provided and posted within the laboratory outlining
operational protocols, waste disposal, disinfection procedures and emergency response. The facility
must have a medical surveillance program appropriate to the agents used, which includes serum
storage for all personnel working in the containment laboratory and an accident report system.
Biosafety Level 4
Risk Group 4 infectious agents are pathogens that usually produce very serious human or animal
disease, often untreatable, and may be readily transmitted from one individual to another, or from
animal to human or vice-versa directly or indirectly, or by casual contact (high individual risk, high
community risk).
Risk Group 4 infectious agents are all viruses, such as, Ebola viruses, Herpes B virus (Monkey virus),
Foot and Mouth Disease.
Containment Level 4 is the highest level of containment and represents an isolated unit that is
completely self-contained to function independently. Facilities are highly specialized, secure with an air
lock for entry and exit, Class III biological safety cabinets or positive pressure ventilated suits, and a
separate ventilation system with full controls to contain contamination.
Only fully trained and authorised personnel may enter the Level 4 containment laboratory. On exit
from the area, personnel will shower and re-dress in street clothing. All manipulations with agents
must be performed in Class III biological safety cabinets or in conjunction with one-piece, positivepressure-ventilated suits.
The following table summarizes the biosafety levels.
Biosafety
Level
Infectious Agents
1
2
unlikely to cause
can cause human
disease in healthy
or animal disease
workers or animals but unlikely to be
a serious hazard
low individual and
community risk
3
4
cause serious
human or animal
disease but not
ordinarily spread
by casual contact
cause very serious
human or animal
disease, often
untreatable and
transmitted
high individual
risk, low
community risk
high individual
risk, high
community risk
E. coli, California
encephalitis
viruses, many
influenza viruses
Anthrax, Q Fever,
tuberculosis,
Hantaviruses,
Human immunodeficiency viruses
Ebola viruses,
Herpes B virus
(Monkey virus),
Foot and Mouth
Disease
Level 1
Level 2
moderate
individual risk,
limited community
risk
effective
treatments
available
Examples of
infectious agents
in this risk level
Facilities
standard welldesigned
experimental
animal and
laboratory facilities
plus:Separate
laboratory, room
surfaces
impervious and
readily cleaned,
biohazard sign
specialized,
secure, completely
self-contained unit
plus:Controlled
access double door with specialized
ventilation, fully
entry and body
monitored; air lock
shower, air
entry and exit,
pressure must be
negative at all
times, no
recirculation, HEPA
filtration, backup
power
Safety Equipment
Procedures
handwashing
facilities,
laboratory coats
Level 1 plus:
Level 2 plus:
autoclave, HEPA
filtered class I or
II biological safety
cabinet, personal
protective
equipment
Autoclave, HEPA
filtered class II
biological safety
cabinet, personal
protective
equipment to
include solid front
laboratory
clothing, head
covers, dedicated
footwear, and
gloves,
appropriate
respiratory
protection
basic safe
laboratory
practices
use of personal
protective
equipment
laboratory coat
worn only in the
laboratory, gloves,
decontamination
Staff fully trained,
written protocols;
showers, wastes
disposed of as
contaminated, use
of biological safety
cabinets, personal
protective devices
Class III biological
safety cabinets,
positive pressure
ventilated suits
access only to
certified staff,
rigorous
sterilization /
decontamination
procedures
Allergies to Laboratory Animals
Laboratory animal allergy (LAA) may be the most prevalent occupational hazard facing people working
in experimental animal facilities. Surveys have revealed that up to 44% of people working with
laboratory animals develop allergies to one or more species, and they usually become allergic within 3
years of first exposure (range; 1 month to 9 years).
Allergic reactions can be classified according to the site of the reaction: upper respiratory; lower
respiratory; skin; generalized, anaphylactic.
In any individual, several symptoms may occur. The upper respiratory symptoms are the most
common - up to 80% of affected people experience symptoms such as itchy, runny nose and eyes,
and sneezing. About 20-30% of affected people experience lower respiratory symptoms, some
progressing to occupational asthma. There is shortness of breath due to bronchoconstriction and
airway mucus production. Asthma may become life-threatening if not treated. About 40% of
laboratory animal allergic people experience skin reactions upon contact with the animal or the
allergens. Much more rare, fortunately, is the acute generalized reaction (anaphylaxis) requiring
emergency treatment. There are only a few documented cases of anaphylactic reactions to laboratory
animal bites (e.g., rat bites).
Almost all species of common laboratory animals can trigger an allergic reaction. Allergies to the rat,
rabbit, mouse, guinea pig, cat and dog are the most common.
The animal allergens are mostly small molecular weight proteins such as albumen. These proteins
occur in the serum and tissues, but also in the saliva, urine and skin dander. When animals groom
themselves, the salivary proteins also end up on the skin, and on the dander particles that flake off
and become aerosolised.
Risk Factors for Becoming Allergic to Laboratory Animals
The risk factors for becoming allergic to laboratory animal allergens include atopy, smoking, gender
and intensity of exposure.
There is a correlation between atopy (an inherited, familial tendency to develop some form of allergy
such as hay fever, asthma, eczema) and the potential for developing LAA, and a stronger positive
correlation between atopy and development of lower respiratory symptoms (asthma). Preemployment health screening may be useful to identify atopic individuals.
Smoking reportedly does not increase the risk of developing LAA, but if a smoker does develop LAA,
they are 1.5-3 times as likely to get the lower respiratory symptoms (asthma).
Males are more likely to be atopic than females (47% vs 37%) and so more likely to develop LAA.
There is a strong correlation between the intensity of exposure to the allergen, and the severity of
symptoms. However, any allergen exposure, even very low levels, will trigger symptoms in allergic
individuals.
Factors Affecting Animal Allergen Levels in Laboratory Animal Rooms
Ventilation and Relative Humidity
Directional room ventilation, negative flow laminar ventilated cage racks, or ventilated racks assist in
reducing particles in room air. Low relative humidity results in higher dust and allergen levels. A
relative humidity of 50-65% significantly reduces the quantity of allergen being aerosolized.
Type of Bedding
Studies have shown that sawdust/wood chip bedding results in higher levels of aerosolised allergen in
rodent rooms than corncob bedding. Use of processed paper products and absorbent pads result in
lower levels of aerosolised allergens.
Cleaning and Sanitation Practices
A high level of cleanliness results in reduced levels of allergens circulating in laboratory animal rooms.
Animal Room Tasks Associated with Exposure to Allergens
All commonly performed animal room tasks result in significant exposure to airborne allergens and
dust. Cage cleaning (and waste dumping), animal care procedures (feeding, watering, etc.), animal
manipulations (e.g., handling, injections), and general room cleaning all result in significant levels of
airborne allergens.
Reducing Exposure to Allergens
There are several approaches to reducing exposure to laboratory animal allergens. Housing rodents in
filtered cages and ventilated cage racks, use of ventilated waste dumping stations and laminar flow
hoods for animal manipulations, will all help minimize exposure to laboratory animal allergens.
Maintaining a high level of cleanliness, and using a bedding type that minimizes aerosol dust particles
will also help minimize exposure to laboratory animal allergens.
The appropriate use of personal protective equipment such as good quality particulate masks and
gloves can significantly reduce exposure to animal allergens. Such equipment should be provided for
all staff required to work in high exposure areas. As well, good personal hygiene (regular hand
washing, showering, etc.) should be practised.
Institutional Responsibilities
There are several institutional responsibilities to minimize the impact of laboratory animal allergies.
These include education programs for staff, health monitoring of at risk persons, improved engineering
standards for ventilation and relative humidity, and provision of appropriate personal protective gear.
Education programs that cover topics such as symptoms, risks, defining risk zones and tasks, proper
use of personal protective equipment, and health counselling for affected and at-risk staff, are very
important.
Allergies to animals are nothing to sneeze at.
Chemical Safety
Experimental animal facilities routinely contain various chemicals such as detergents, disinfectants,
anesthetics, tissue preservatives (e.g., formalin). Most staff will be familiar with safe work practices
for use of these chemicals. A laboratory animal facility should be following the Canadian Workplace
Hazardous Materials Information System (WHMIS), which consists of labelling chemicals, provision of
material safety data sheets (MSDSs) and employee education programs. A detailed discussion of all
the chemicals used in experimental animal facilities, their hazards and safe use is beyond the scope of
this module.
Radiation Safety
Institutions will already have a program in place to ensure work with ionizing radiation, including
isotopes injected into animals as part of their research use, is done safely. Training and licensing of
users and facilities are mandated. A detailed discussion of the use of radiation in experimental animal
facilities, their hazards and safe use is beyond the scope of this module.
Remember: YOU are the primary person responsible for working safely in your laboratory animal
facility!
Reference Material
Canadian Council on Animal Care. Guide to the Care and use of Experimental Animals, Volume 1, 2nd
Ed. 1993. Chapter VIII Occupational Health and Safety.
National Research Council. 1997. Occupational Health and Safety in the Care and Use of Research
Animals. ILAR, CLS, NRC. National Academy Press, Washington, DC 154p. Available for reading online
at this web site.
Sample Questions
Which one of the following best describes the flight zone of an animal?
a.
b.
c.
d.
e.
The
The
The
The
The
distance an animal will run from an unfamiliar person.
furthest distance an animal will stray from its den or burrow.
area around an animal that it keeps free from predators or people.
distance an animal will run when startled.
distance a bird will fly when it perceives danger
Up to date vaccination against tetanus is important in which one of the following situations?
a.
b.
c.
d.
e.
When
When
When
When
When
using needles or scalpels.
performing behavioural studies with fish.
working with tissue culture specimens.
examining specimens from human origin.
performing field work with rodents
Level 3 biocontainment is required for studying pathogens with which one of the following
characteristics?
a.
b.
Infect only people
Are spread only by direct contact
Module 05 - Research Issues
On the Importance of Planned Routines in Animal Care and
Use Practices in Research
Topics
•
•
•
•
•
The use of animal models and the selection of an animal model for biomedical research
Identifying the many variables that influence the way the selected animal model will respond
in an experiment
The importance of controlling all the non-experimental variables, including the many protocol
variables, and of monitoring and recording the variables
Examples of how people involved with a research project - the animal care staff, facility
managers, research technicians, investigators - can affect the way the selected animal model
responds in an experiment
An outline of the importance of ensuring that each team member fulfils his/her responsibilities
for controlling the variables that may alter the animal model's response.
Learning Objectives
Upon completion of the module, the team members should be able to:
•
•
•
•
•
Identify the various aspects of an animal model that are part of its "definition"
List the variables - the non-experimental factors - that can influence the animal model's
response
Recognise the importance of accounting for all the variables in the experimental design, and
controlling those variables (the non-experimental factors)
For principal investigators and graduate students: develop a checklist of the variables that can
affect their research program
Describe and accept responsibility for their role in ensuring the successful conduct of the
experiment
Module Format
For the purposes of this module, imagine a group of experimental animals as the stars of a show being
recorded for public presentation (the publication). All the work by the crew, behind the scenes, is
aimed at making sure that the show goes on without a hitch the FIRST TIME it is staged. There are
to be no re-takes. This is consistent with the ethical principles contained in the "Three Rs" of humane
animal experimentation: Reduction; Refinement; Replacement.
Following the identification of many of the variables that can affect how an animal responds in a
research program, the roles various members of the "crew" have in making sure all the factors that
can influence the model's performance are accounted for and controlled, will be presented. Then the
"show" can go on without a hitch.
The responsibilities of the various members of the biomedical research team in controlling both the
non-experimental and protocol variables are listed here in introduction:
Responsibilities of the Principal Investigator
•
Consider all pertinent non-experimental variables
•
•
•
•
•
•
Outline need for controlling pertinent variables to all team members
Ensure that all experimental variables are controlled or conducted using Standard Operating
Procedures (SOP)
Ensure monitoring and recording of controls on variables (through SOP)
Ensure animal health quality is being checked before purchase
Ensure health monitoring is being carried out according to facility rules
Observe all facility SOP established to limit disease introduction
Responsibilities of the Graduate Students, Post-doctoral Students and Research Technical Staff
•
•
•
•
Monitor and record controls on all variables
Conduct all experimental procedures according to Standard Operating Procedures (SOPs)
Employ skilled animal handling and manipulation techniques
Observe all facility Standard Operating Procedures established to limit disease introduction
Responsibilities of the Facility Manager
•
•
•
•
•
•
Understand demands for controls on non-experimental variables for all research underway
Ensure consistent facility environmental operations
Ensure high level of animal care training and expertise
Ensure animal health quality before purchase
Ensure health monitoring is done according to facility rules
Implement Standard Operating Procedures for all animal facility operations
Responsibilities of the Animal Care Facility Staff
•
•
Conduct all daily animal facility routines in a consistent manner, according to Standard
Operating Procedures
Conduct all animal care handling and manipulations in a consistent, gentle, humane way
Responsibilities of the Laboratory Animal Veterinary Staff
•
•
To advise on and ensure health status of all animals
To effect procedures that will maintain animal health quality
The following questions should provide an indication of the scope of this module. The student should
consider the questions before clicking the link to the commentary.
Rats should be housed in small groups:
(a) to reduce the number of cages and minimise per diem charges
(b) to ensure feed is not wasted
(c) to allow grooming by other rats in the cage
(d) to provide companionship for a highly social animal
(e) to minimise the stress each animal is experiencing
Relative humidity (RH) in an experimental animal room should be maintained in the range
of 40-70% RH because:
(a) this is the most comfortable level for the animals
(b) micro-organisms have the lowest survivability in the air at that level of humidity
(c) air borne allergens are lower at this level than when it is drier in the room
(d) it prevents condensation on metal doors, feeders and other equipment
Animal Models in Biomedical Research
Although the term "animal model" is commonly used, a definition may help clarify the context of the
term. The American National Research Council Committee on Animal Models for Research and Aging
drafted the following definition: "An animal model for biomedical research is one in which normative
biology or behaviour can be studied, or in which a spontaneous or induced pathological process can be
investigated, and in which the phenomenon in one or more respects resembles the same phenomenon
in humans or other species of animals." Animal models used in biomedical research, particularly those
used in the study of diseases and other conditions in humans, can be grouped as follows:
Spontaneous models - often called "natural" models. These include naturally occurring animal
diseases or conditions that correspond to the same diseases or conditions in humans. Diabetes,
hypertension, arthritis, immune deficiencies are just a few examples. Many hundreds of animal
strains/stocks with inherited conditions have been characterized and conserved. The Jackson
Laboratory holds one of the largest repository of these valuable animal models in mice (
http://www.jax.org/).
Experimental models. Experimental models are models in which a condition or disease is
experimentally reproduced by the scientist. Examples include producing diabetes using the chemical
streptozotocin to damage the insulin producing cells in the pancreas; using a chemical carcinogen to
produce a certain type of cancer; producing a stroke model through surgery.
Genetically modified models are a special group of induced animal models, involving manipulation
of the animal's genetic code to produce the condition that the scientist wants to study. Genetically
modified animals may carry inserted foreign DNA in their genome, or have genes replaced or removed
("knock-out" models). These models can help scientists study the genetic basis of disease,
susceptibility and resistance, etc.
Negative models. Some animals are resistant to a particular condition or disease. Examining why
this is the case may provide answers to questions about disease resistance and its physiological basis.
Orphan models. Orphan models are conditions appearing naturally in an animal, for which there is
no known human counterpart. Historically scrapie in sheep was such a model, but now is useful as a
model for the human spongiform encephalopathies that are of so much concern (eg., BSE, "mad cow
disease" and CWD, chronic Wasting Disease in deer).
Choosing the Appropriate Animal Model
Before an animal model is chosen the principal investigator must consider alternatives to the use of
live animals in his/her experiment. In line with the CCAC Guidelines on Animal Use Protocol Review,
protocol forms should include a declaration that the principal investigator has considered all nonanimal alternatives before making the decision to use animals in his/her research.
The most obvious choice of animal species and model for a specific research program may be the
same model used by other researchers for the same research. However, with ever increasing numbers
of animal models available including new spontaneous mutations, and genetically modified animals
constantly being developed, the investigator must consider all factors when selecting the best model
for his/her research.
Some of the factors that will influence which animal model the investigator selects are:
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Appropriateness of the model or organ system for the proposed study
Genetic aspects of the model
Natural vs. experimentally produced models. Both natural models and induced models of
disease are useful depending on the objectives of the study.
Responses of the animal to procedures
Environmental aspects important to that particular animal model
Background information available on the animal and specific model
Species availability
Numbers needed, according to statistically appropriate design
Age and sex needed
Life span of the animal model
Size of the animal model
Cost of both the animal model, and its ongoing care
Facilities required to house the chosen model appropriately
Husbandry expertise. Some models require not only special housing, but also special care.
Among these factors are scientific considerations as well as purely practical ones. All are important to
the success of the research program.
Once the most appropriate animal model for the research program has been chosen (whether it is a
specific mouse strain that will require specialised care and housing, or a conventional farm animal
such as a pig), the research scientist should review the many influences (the non-experimental
factors) that might potentially affect the outcome of the study. Those factors should be identified and
accounted for at all stages to ensure they do not increase the variability or adversely affect the
outcome of the experiment.
The many non-experimental factors that can influence the response of the animal model in an
experiment can be grouped as follows:
•
•
•
•
Animal factors
Physical / environmental factors
Husbandry, animal care, and handling factors
Research manipulation factors
Each group of factors will be discussed in this module, along with suitable examples to emphasise
their importance.
Suggested project for research scientists,
graduate students and post-doctoral
students upon completion of the material in
this module:
Develop a master list of non-experimental
variables that could influence your research. Use
this list to review the controls placed on each
experiment.
Establish a log book to record/document all the
pertinent non-experimental variables.
Animal-Related Factors that Influence the Response of the
Animal Model in an Experiment
There are many animal-related factors that influence the response of the animal model in an
experiment, and thus must be accounted for in the preparation and design of the study. Some of
these are listed here:
•
•
•
•
•
•
•
•
•
Genetic make-up
Inbred vs. outbred vs. mutants vs. genetically modified
Age, sex, reproductive status
Microbial flora
Biological rhythms
Presence of stress/distress
Diseases
Overt disease
Latent (subclinical or silent) infections
•
Genotype-related conditions
A number of animal factors are pre-selected by the investigator: the age, sex and reproductive state
of the animal, and its metabolic state (e.g., young and rapidly growing, adult, aged). The health
status must also be considered when selecting the source of the animals.
Of the many factors that can cause unknown or undesirable complications to the response of the
selected animal model, disease states are very significant. Disease organisms, overt or latent
(subclinical or silent), can significantly interfere with valid experimental results. Three examples are
presented here:
Example 1: Infection causing disease
Example 2: "Ordinary" bacteria causing disease in immune deficient models
Example 3: Silent infection affecting research results
Example 1: Infection causing disease
SDAV (sialodacryoadenitis virus) infection in rats is a highly infectious disease with a short clinical
course that is usually non-fatal. Most rats recover quickly with subsequent immunity to re-infection.
Rat with SDVA
Interference with research: SDAV infection alters several factors of the immune system and thereby
may interfere with research. There is inhibition of phagocytosis, reduced interleukin-1 production by
pulmonary macrophages, and depletion of epidermal growth factor. Because of the site of the
infection, it could also complicate studies involving the eye, salivary glands, lacrimal glands and
respiratory tract.
Example 2: Ordinary bacteria causing disease in immune deficient models
Immune deficient or immune compromised models (e.g., nude or severe combined immunodeficiency
(SCID) mice) are very susceptible to ordinarily non-pathogenic organisms such as Pasteurella spp. or
Staphylococcus spp. Special caging and care procedures are vital to minimizing such infections.
Nude mice
Inadvertent infections in these special models can not only cause serious disease, but they will also
interfere with the animal's immune response during research.
Example 3: Silent infection affecting research results
Mouse parvovirus (MPV) is a clinically silent infection. In mice with normal immune systems the
infection lasts up to 6 months, while in immunodeficient or immunosuppressed mice, infection and
shedding of the virus may be lifelong. The virus requires rapidly dividing cells to replicate, and grows
in gut mucosa with shedding of the virus in feces of infected mice.
MPV persistently infects the lymphoid tissue, particularly the T cells and causes changes in immune
system responses, either stimulating or suppressing depending on the studies being done. Infected T
cells may not grow properly when cultured in vitro. Parvoviruses may also interfere with cancer
research; T cells from infected mice may have diminished cytolytic capacity.
From these three examples, it should be obvious that infections, even subclinical infections causing no
overt disease signs, can significantly affect the results obtained in a study. The efforts of many people,
at many levels, to eliminate such infections from the laboratory animal models, have contributed
significantly to minimising this unwanted variable in biomedical research that uses animal models.
Avoiding Infectious Disease in Laboratory Animals
VAF (Virus Antibody Free - free of specific viruses) status of rodents was developed to avoid research
complications caused by infections. Actions that managers of laboratory animal facilities and
researchers themselves can take to limit risk of infections ruining research include: ensuring the
health quality of animals before purchase; conducting health monitoring according to facility rules;
observing all facility SOPs established to limit disease introduction. Investigators must share the
responsibility of ensuring the disease free status of their animal models.
Knowing the health status of the animals and documenting it, are a very important part of the records
that must be kept by the principal investigator, as part of defining the animal model in the
publications arising from the research.
Physical and Environmental Factors
Room Temperature
Small laboratory animals respond to temperature variations by changes in behaviour (e.g., shivering,
huddling) and metabolic rate (including increased food consumption if they require more body heat
production). These changes could affect several metabolic processes including drug metabolism. Daily
animal room temperature fluctuations should therefore be limited (by good heating/ventilation design)
to +/- 2oC. Daily temperature fluctuations should be monitored and recorded. For some studies it may
be necessary to measure cage (microclimate) parameters, and not just room environment.
Relative Humidity
Animal room relative humidity should be maintained at 55%+/-15%. This is important for several
reasons: for the welfare/comfort of the animals (and the staff); for minimising disease spread by
reducing the viability of airborne microbes; for allergen reduction. Prolonged relative humidity (RH) of
less than 40% can cause ringtail in young, unweaned rats, and result in respiratory irritation.
Ringtail in a young rat.
Ventilation
Recommended air exchange rates in animal rooms are 15-20 air changes per hour. Such high rates,
compared to human office or laboratory spaces, are necessary to remove animal generated heat,
ammonia, carbon dioxide, and airborne particles (dust and allergens). Static microisolator cages may
contain high levels of ammonia and carbon dioxide despite good ventilation rates in the room itself. Air
pressure gradients from animal rooms to corridors, or between zones in an animal facility are also
important for containment of micro-organisms.
Within a laboratory animal holding room there can be significant variations in ventilation, and levels of
ammonia and CO2, as well as air flow. Randomising the location of animals in different treatment
groups in a rack or in the room may help to avoid bias in the results due to these factors
Lighting
The lighting cycle for the animals has several aspects; the day/night cycle, the intensity of the lights,
and the wavelength. Timer control of day/night cycle is necessary to maintain a consistent diurnal
rhythm in the animal's metabolic state. The intensity and wavelength are also important to animals.
Albino rodents in particular experience retinal damage when room light intensities are above 300 Lux.
The sudden onset of lights in the morning affects some hormone levels - effects that may last for
several hours. The use of dusk/dawn lighting systems, which gradually change light intensity between
dark and light phases is encouraged.
For studies where the light levels might influence the research results, randomising the location of
animals in different treatment groups on a holding rack may be useful in avoiding a light-induced bias
in the results obtained.
Light "Pollution" Can Alter Tumour Growth Rates
In a study reported by Dauchy, et al., in the journal Laboratory Animal Science (1997), light
contamination during the dark phase significantly altered the growth of a tumour (Morris hepatoma) in
rats and changed some metabolic factors. In this experiment, the light "pollution" was a very low light
level from a light in a hallway shining through a window into the animal room. This study reinforces
the need to examine all factors that might cause variability, including the actual levels of light in the
animal room, to ensure that the results obtained are meaningful.
Effects of diurnal, light-contaminated dark phase, and constant light on estimated tumor
growth and carcass weights in Buffalo rats bearing hepatoma 7288CTC.
Daucy, et al., 1997.
Noise
The impact of noise on the behaviour or responses of laboratory animals in biomedical research has
received too little study. The fact that loud "buzzer" noises may induce seizures in young rodents (this
has been used to create a model of audiogenic seizures) is well known. Both intensity and sound
frequency are important. Rodents and some other animals are particularly sensitive to ultrasonic
frequencies, ones that we may not even be aware of since they are beyond the range of the human
ear. Low frequency and other noises, for example from nearby construction, may also disturb the
animals.
Feeds and Water
Unless otherwise specified by the investigator, SOPs in most animal facilities include provision of
regular (perhaps certified) feeds. Water is usually municipal water with perhaps some treatment in the
animal facility. If special dietary or water requirements are needed for the research project, the
researcher must inform the animal facility management and laboratory animal technical staff.
Laboratory Animal Bedding
Unless otherwise specified by the investigator, SOPs in most animal facilities include provision of
regular bedding materials for that facility. Any special bedding requirements have to be specified by
the principal investigator.
The phenomenon of resins in softwood bedding (e.g., cedar shavings) activating some of the hepatic
enzyme systems (P450 enzymes) is well known. This may complicate results if the experimental
outcome is related to hepatic enzyme activity.
Animal Care and Handling Factors
Animal Stress
Stress from many different sources can affect the animal's physiology, biochemistry and behaviour.
Sources of stress in the handling and care of the animals include transportation, and the handling and
manipulations done by animal care and research staff, and of course the procedures done as part of
the research itself.
Transportation Stress
Laboratory animals are rarely used in the same location where they are raised, so usually they are
transported to the facility where the research will be done. A number of studies have shown that
animals experience varying degrees of stress as a result of the transport, and that it takes some time
upon arrival at the research facility to return to a normal physiological state. Eating, drinking and
growth tend to return to normal levels in about a week after delivery to the new location. Some subtle
physiological and immunological changes may last longer. A common recommendation is to allow
laboratory animals at least one week conditioning after transport to the research facility.
Housing Factors
Caging
The amount of space per animal, and the number of animals per cage may influence an animal's
response in an experiment. Identical caging should be used for all animals in a study, to ensure that
the space per animal and number of animals per cage remains consistent within a study.
Studies have shown that the number of rodents per cage affects the stress level (either isolation or
crowding), and their growth. As a general rule small rodents should be housed in small groups to
minimise stress, and for social enrichment. If the research requires individual housing, this should be
scientifically justified to the animal care committee.
Environmental Enrichment
The value of providing an enriched environment in improving the well-being of the animals, must be
emphasised. In the context of controlling research variables however, it must be noted that any
changes (improvements) to the cages will have behavioural, physiological and anatomical effects,
some of which result in permanent changes to the animal. (Module 10 provides a more in-depth
discussion on environmental enrichment.) Therefore any improvements or enrichments should be
uniformly and consistently provided to all animals for the duration of an experiment. Changes in the
cage's physical environment should only be made following consultation with the investigator.
Routine (Daily/Weekly) Animal Care and Husbandry
The daily routines of the animal care staff may have a profound impact on the way an animal
responds in an experiment. As a general principle all animals should be handled the same way, and
the same time of day. Handling must be gentle, and consistent. Most animals quickly become familiar
with their regular caretakers, and their stress level rises when unfamiliar people handle them.
The kind of handling each animal receives may in fact alter its behaviour or physiology, and thereby
affect its response in a study. One example of this comes from a handling study conducted in pigs.
Hemsworth and co-workers published a paper in 1986 describing a study in which the responses of
pigs to three kinds of handling; pleasant handling, unpleasant handling and minimal handling, were
compared. The pigs exposed to pleasant handling approached people more quickly (not surprising).
The females receiving pleasant handling were different with respect to age at first estrus and sexual
receptivity when bred, and had a significantly higher pregnancy rate than the other two groups.
Although not a statistically significant results, the males receiving pleasant handling reportedly had
larger testicles.
Hemsworth PH, Barnett JL and Hansen C. 1986.
While it is interesting to speculate whether these results can be correlated with human teenage
behaviour, they do emphasise the point that how the animals are handled affects their behaviour,
physiology, and anatomy. Consistent handling, by animal care staff, by research technicians, and by
the investigators, will ensure that this source of variability is minimised.
The weekly routine in any animal room must also be understood by the investigator. In many
facilities, animal cages are cleaned once or twice weekly on specific days. Taking samples just after
the cages have been cleaned may result in altered results because of the disturbance of the animals at
that time. In addition, it must be noted that weekend and holiday animal care routines are generally
different than regular week day routines. This may alter the animals' responses on weekend days if
sampling is scheduled then.
Applying Basic Animal Behaviour in Your Research Project
An animal learns from experience what will be happening to it when it is handled. Animals very quickly
learn handling routines and procedures. Assuming the handling is competently done, not only will an
animal's stress level be reduced, it will be much more likely to accept the manipulation being done.
During the conditioning period before an experiment begins, animals should be exposed to the
routines that will be part of the study. This is particularly important if the study will involve special
restraint, use of devices such as collars or jackets containing emerging catheters (e.g., rats with
tethers for brain recordings; sheep in a metabolism crate). Familiarizing the animal to manipulations
or restraint BEFORE a project starts is important for both welfare and scientific reasons.
Rewards for good behaviour are an excellent way to enhance a cooperative attitude in an animal.
Rewards such as "gummi-bears" or Fruit Loops are enjoyed by rats. Other examples are, giving a dog
a dog biscuit after blood sampling, giving a cat a treat, giving a non-human primate a special treat. Of
course these would need to be acceptable to the research project.
Research Manipulation Factors
In addition to the many non-experimental variables that must be considered and controlled to ensure
that the fewest animals need to be used and that the results are valid, there are the many research or
protocol variables that the investigator and all research personnel must consider, and control. For
some studies such as the surgical induction of cerebral ischemia (stroke) in a rodent model, there may
be many variables to control; duration and depth of anesthesia, body temperature during surgery and
during recovery, duration of the application of the ischemia, timing of the analgesia, post-operative
care and monitoring, etc. All of these may influence the outcome of the ischemia.
It is recommended that for each specific research procedure a Standard Operating Procedure (SOP) be
written and observed by everyone involved, to standardise as much as possible each and every animal
manipulation.
Time as a Research Variable
The normal biological diurnal rhythms in an animal's biochemistry and physiology alter its responses
depending on the time of day that treatments are applied or samples are taken. Thus efforts should be
made to take repeated samples at the same time of day, every day.
The duration of the manipulation(s) for each animal should also be maintained as consistent as
possible. Biochemical and hematological changes start happening when an animal is taken from its
cage. Studies have shown that some of these changes last for minutes to hours and will be reflected in
the results obtained. For each animal in all treatment groups including control groups, time to
sampling (from removing the animal from the cage) should be consistent.
The influence of a familiar attendant working in the animal room (left), cage movement (middle), and
1 min ether anasthesia (right) on heart rate in 1 rat, and the packed cell volume, blood Haemoglobin,
serum total protein content, serum potassium and sodium concentration of 3 to 8 animals per mean
and standard deviation. Significance of differences between control and stressed animals are : ***P <
0.001; **P < 0.01; *P < 0.05.
Gaertner K, Buttner D, Dohler K, et al. 1980.
Summary
In this module we have tried to emphasise the importance of "routines" in all aspects of research with
animals. By doing so, the research scientist accomplishes both scientific and ethical goals - using the
fewest animals to generate valid, reproducible scientific data.
The responsibilities of the various members of the biomedical research team in controlling both the
non-experimental and protocol variables, as presented at the beginning of the module, are reviewed
here in summary:
Responsibilities of the principal investigator
•
Consider all pertinent non-experimental variables
•
•
•
•
•
•
Outline need for controlling pertinent variables to all team members
Ensure that all experimental variables are controlled or conducted using Standard Operating
Procedures (SOPs)
Ensure monitoring and recording of controls on variables (through Standard Operating
Procedures)
Ensure animal health quality before purchase
Assure health monitoring is done according to facility rules
Observe all facility SOPs established to limit disease introduction
Responsibilities of the graduate students, post-doctoral students and research technical staff
•
•
•
•
Monitor and record controls on all variables
Conduct all experimental procedures according to Standard Operating Procedures (SOPs)
Employ skilled animal handling and manipulation techniques
Observe all facility SOPs established to limit disease introduction
Responsibilities of the facility manager
•
•
•
•
•
•
Understand demands for controls on non-experimental variables for all research underway
Ensure consistent facility environmental operations
Ensure high level of animal care training and expertise
Ensure animal health quality before purchase
Ensure health monitoring is done according to facility rules
Implement Standard Operating Procedures for all animal facility operations
Responsibilities of the animal care facility staff
•
•
Conduct all daily animal facility routines in a consistent manner, according to Standard
Operating Procedures
Conduct all animal care handling and manipulations in a consistent, gentle, humane way
Responsibilities of the laboratory animal veterinary staff
•
•
To advise on and ensure health status of all animals
To effect procedures that will maintain animal health quality
Quality Animal Care = Quality Science
References - Research Issues
CCAC. 1993. Chapter III. The Environment. In: Guide to the Care and Use of Experimental Animals.
Vol 1, 2nd Ed. Ottawa: CCAC. pp 21-29.
Dauchy R, Sauer, L., et al. 1997. Light Contamination During the Dark Phase in "Photoperiodically
Controlled" Animal Rooms: Effect on Tumor Growth and Metabolism in Rats. Laboratory Animal
Science 47 (5) 511-518.
Dell RB, Hollerhan S and Ramakrishnan. 2002. Sample Size Determination ILAR Journal 43(4) 207213. http://dels.nas.edu/ilar_n/ilarjournal/43_4/v4304Dell.shtml
Donnelly H. 1995. Chapter 11. Quality in laboratory animals. In: Laboratory Animal - An introduction
for Experimenters. Tuffery AA. Ed. Chichester: Wiley & Sons. pp181-203.
FBR. 1987. The Biomedical Investigator's Handbook. Washington: Foundation for Biomedical Research.
86 pp.
Festing MFW and Altman DG. 2002. Guidelines for the Design and Statistical Analysis of Experiments
Using Laboratory Animals. ILAR Journal 43(4) 244-258.
http://dels.nas.edu/ilar_n/ilarjournal/43_4/v4304festing_b.shtml
Gaertner K, Buttner D, Dohler K, et al. 1980. Stress response of rats to handling and experimental
procedures. Lab Animal 14:267-274.
Gaines Das RE. 2002. Role of Ancilliary variables in the Design, Analysis and Interpretation of Animal
Experiments. ILAR Journal 43(4) 214-222.
http://dels.nas.edu/ilar_n/ilarjournal/43_4/v4304Gaines_das.shtml
Hemsworth PH, Barnett JL and Hansen C. 1986. The influence of handling by humans in the
behaviour, reproduction and corticosteroids of male and female pigs. Applied Animal Behavioural
Science. 15:303-314.
Johnson PD and Besselsen DG 2002. Practical Aspects of Experimental Design in Animal Research.
ILAR Journal 43(4) 202-206. http://dels.nas.edu/ilar_n/ilarjournal/43_4/v4304Johnson.shtml
Lipman NS and SE Perkins. 2002. Chapter 29. Factors that May Influence Animal Research. In:
Laboratory Animal Medicine, 2nd Ed. Fox JG, Anderson LC, Loew FM, Quimby FW, Eds. Orlando:
Academic Press pp 11434-1184.
Lawlor M. 1990. The size of rodent cages. In: Guidelines for the Well-Being of Rodents in Research.
SCAW. Pp.19-27.
Morton DB and Townsend P. 1995. Chapter 13. Dealing with adverse effects and suffering during
animal research. In: Laboratory Animal - An introduction for Experimenters. Tuffery AA. Ed.
Chichester: Wiley & Sons. pp 215-231.
National Research Council. 1991. Infectious Diseases of Mice and Rats. NAS. Washington: National
Academy Press. 397p.
Shaw R, Festing MFW, Peers I, et al. 2002. Use of factorial designs to optimize animal experiments
and reduce animal use. ILAR Journal 43(4) 223-232.
http://dels.nas.edu/ilar_n/ilarjournal/43_4/v4304Shaw.shtml
Sample Questions
A researcher has ordered some transgenic mice from an out-of-province commercial supplier. Which
one of the following best describes how the mice should be handled upon arrival at the research
facility?
a.
b.
c.
d.
e.
They should be examined carefully and placed into quarantine for at least a week.
They should be microchipped so they can enter the research project immediately.
They should be held in a barrier facility for one week with all the other mice.
Blood should immediately be collected from each mouse for disease screening.
They should immediately be placed into breeding groups for maximum productivity.
Which one of the following statements concerning a latent virus infection in a mouse is most important
in a research setting?
a.
b.
c.
d.
e.
It
It
It
It
It
cannot be detected since there are no signs of disease.
does not affect the health of the mouse.
may alter biochemical or immune functions.
is transmitted to mice from rats.
only occurs in young less than 1 month old.
Why is it important to have a ventilation rate of 15 or more air changes per hour in an animal room?
a.
b.
c.
d.
e.
To
To
To
To
All
remove heat generated by the animals.
remove carbon dioxide exhaled by the animals.
remove ammonia from the urine and feces in the cages.
remove airborne particles.
of the above
The principal investigator is responsible for all but which one of the following?
a.
b.
c.
d.
e.
considering all pertinent non-experimental variables;
outlining the need for controlling pertinent variables to all team members;
considering replacement, reduction and refinement alternatives;
ensuring consistent facility environmental operations;
ensuring animal health quality before purchase.
Module 06 - Basic Animal Care
Module Objectives
The objectives of this module are to describe:
•
•
•
•
•
•
•
animal facility security
animal facility zoning and traffic flow as it relates to disease control
basic animal facility operations
basic sanitation procedures
basic animal care procedures
individual animal identification
animal experiment identification
The information will be presented as a "virtual tour" of a hypothetical multi-purpose laboratory animal
facility.
Tour of Animal Care and Services Unit
Hello, Professor. Welcome to the Animal Care and Services (ACS) unit. We're pleased to give you a
tour of our laboratory animal facilities.
I brought out a floor plan to get you oriented before we do the tour. As you can see from the colours
on this floor plan, our animal facility is divided into zones, and we have entry rules for each area - no
exceptions. Most of these rules have to do with minimizing the risk of our animals becoming
contaminated with any unwanted infectious diseases. We are in the offices, labs and services area,
right here. This zone has the offices, lockers, showers, loading dock, storage rooms for feed, bedding
and extra caging, and a couple of labs.
Image courtesy of Dr. Ernest Olfert, University of Saskatchewan.
On the left here is the conventional animal wing. "Conventional" means the animals housed here are
not under any special housing requirements. These animals are in good health based on veterinary
examination but we don't do complete virus or health screens for animals housed in this area. One
wing of this area houses our larger conventional animals - we only have some resident dogs and pigs
in at the moment. They're in larger multipurpose rooms in floor pens. We house our dogs, pigs and
sheep in floor pens. The hoofed animals get bedding, and those pens are cleaned out two or three
times a week.
When we have cats in, they're group-housed in rooms with all kinds of amenities, like cat trees,
perches, scratching posts, litter boxes, cat toys, and so on. The dogs are in fairly roomy dog pens, but
if they're going to be here long term, we make sure they get out of their pens for exercise and play
with each other and the technicians as often as possible during the week. Actually that exercise and
play time is very important to the dogs. While they're out they also get their grooming and handling.
As part of that they get dog biscuit treats to eat.
Bioexclusion Zone
Through this double set of doors on the right is our cleanest zone. It holds our VAF rodents, immune
deficient models, and transgenic strains. "VAF" stands for virus antibody free. That means all the
rodents housed here have been tested for a group of viruses and don't show antibodies to those
viruses. They are shipped to us from the commercial suppliers in filtered crates and are supposed to
be virus free when we get them. But we do some testing during their quarantine period.
Image courtesy of Dr. Ernest Olfert, University of Saskatchewan.
Before entering this zone everyone has to do a complete change of outer clothing in this entryway. We
supply the clothing and footwear for inside the area. We encourage researchers to let our trained
animal technicians do most of the treatments and sampling, but if you or your graduate students are
going to come and go, we'll supply all the coats, gloves, masks, head caps and booties.
At the far end of this zone is a smaller area that is our highest bioexclusion zone. Everything going
into that area is sterilized before it is brought in. Except the people of course, hahaha. Everyone has
to shower in the anteroom entry suite to get in there. That's where we keep some of the most
sensitive transgenic and knockout rodents and the ones undergoing genetic manipulation. We've got
special filtered ventilation and air pressure systems to help make sure unwanted microbes don't get
in. We also put the animals in either static microisolator cages or in ventilated cage racks.
To give you an idea of how carefully our technicians have to work with microisolator cages or
ventilated racks to make sure no contamination happens, here's a couple of SOPs describing the
procedures step by step. "SOP" stands for Standard Operating Procedure. For almost all of the
standard things we do around here, we try to be as routine as possible, so that every job is done the
same standard way every time. And we've written that down, as "Standard Operating Procedures". I
can give you copies of any of our facility SOPs if you want. These can also help if you're doing a study
according to the Good Laboratory Practice regulations for drug studies.
Standard Operation Procedure: Mouse Microisolator Cage, or Individual Ventilated Cage,
Change (Extracted from an actual SOP).
Why all the precautions in the bioexclusion zone? Well, those mice are extremely valuable. One
researcher there has almost a year plus more than $70,000 invested in developing her transgenic
strain, so we do everything we can to make sure no disease gets in and destroys the research.
Although we don't have any gnotobiotic animals right now, if we did they would be housed in that area
too. We have two isolators that have just been set up to start a project next week. They're completely
sealed plastic bubbles capable of containing gnotobiotic and axenic (germ free) animals. The air goes
in through HEPA filters, and the food and water is sterilized and then put into the isolator bubble
through disinfectant dip tanks.
HEPA filters? That stands for High Efficiency Particulate Air filters. What that means in real terms is
that such a filter will be at least 99.97% efficient at removing all particles in the air down to a size of
tenths of a micrometre. That gets most microbes too, so the air coming through is almost "sterile". So
you can imagine they're pretty tight filters, and they need a reliable, strong air pressure to draw the
air through.
Once the animals are in the isolator, we handle them through gloves sealed into the plastic isolator
walls. That sure is a lot of extra work, but in the last two studies we didn't have any breaks either in
the isolator or in the procedures, and so the animals' germ free status was preserved.
Biocontainment Zone
On the other side of the building is the biocontainment zone. Projects that use biohazardous agents
have to be done there. We can only work with Level 2 biohazardous agents in this facility. Those
animals are housed in ventilated cages, and there's a biological safety cabinet in three of the rooms.
Like the VAF area I mentioned, there are special procedures to come and go into this zone. Everyone
working in there has to use procedures set out in our biocontainment SOPs. They put on surgical
gowns, booties, caps and masks before they go in through those double doors, and they leave those
all behind when they exit. The ventilation system is set up to "contain" any infectious agents, which
means that the air pressure is lowest inside the animal rooms, and it's drawn out of there through
filters to the outside. We've got an autoclave in there so we can sterilize any stuff coming out,
including the bedding and animal tissues if we have to.
OK, ready to go on our tour? Let me get you a lab coat while you put on these disposable booties, and
we can start. While we're getting ready I'll give you a run-down on our security system.
Image courtesy of Dr. Jim Love,
University of British Columbia
The doors to the animal area are always locked. Our staff have pass cards and ID codes to get in.
We'll have to issue you a card when your research starts. Here, put on this visitor ID card, and I'll sign
you in. After you.
Rat Room
Image courtesy of Dr. Jim Love, University of British Columbia.
Let's go down the left corridor to the corner. We can peek into this rat room. That is Ms Thera Peutick,
one of four animal care technicians who cares for the animals in this wing. Right now she's changing
cages, moving the rats from dirty cages to clean ones. That's done twice a week here. In this wing
cage changing is on Mondays and Thursdays, and in the next wing it is on Tuesdays and Fridays.
Usually that takes about an hour per room, but the rats, like most other rodents, take a while to settle
down again after cage changing, so you might think about that when you plan your sampling times.
Image courtesy of Dr. Ernest Olfert, University of Saskatchewan.
The dirty cages go out that other door in the room, see there on the opposite side of the room from
where we're standing. That leads to the back alley, or "dirty" corridor where all the dirty cages are
stacked. All these plastic cages get wheeled down the hall to the rack washer, a walk-in, truly
industrial strength washer. With the detergents we use and the high temperature in the washer (the
cage washer is programmed so that the water temperature must reach 83°C (180°F) for at least 10
minutes, for the whole wash cycle to run), the cages come out very, very clean. But in this zone the
cages are not sterilised each time like they are in the cleanest bioexclusion area.
Image courtesy of Dr. Ernest Olfert,
University of Saskatchewan.
The bedding Thera is putting into the cages is a processed paper chip product. Actually, there are
quite a few choices for bedding: wood chips of a couple of sizes; corn cob particles of a couple of
sizes; processed paper chips; and a few other types. We settled on this one because it is a fairly big
particle size, and the rats and mice seem to prefer that. But the bedding does more than just make it
comfortable for the animals. It helps absorb the urine and feces and slows down the build-up of
ammonia in the cages.
Did you know that some researchers have studied which kinds of bedding and other materials that lab
rats and mice prefer in their cages? Imagine setting up an experiment where you can ask the rats
what they like. Well, you can't actually "ask" them, but I guess by giving them certain choices in a
controlled way, you can find out which choice they like the most by how much time they spend there.
And it turns out that rats and mice prefer bedding of a fairly big particle size, so they can push it
around and make nests.
Rodents also like to have a place inside the cage to go into or maybe hide. That's what those other
plastic things are inside the cages. We're really sold on giving all the animals something extra in their
cages to "enrich" their environments - that's part of our overall cage enrichment plan. And the CCAC
(Canadian Council on Animal Care) really promotes this too. It maybe doesn't seem like much, but we
do our best. We also put some shredded paper in the cages so the rats and mice can make nests with
that material.
We try to treat each animal the same way on a study to minimize study variables, and that includes
the enrichment items.
There's a maximum number of animals allowed per cage for all our animals. The CCAC set down some
minimum space per animal recommendations, but we think of those as absolute minimums. We try
not to put that many animals in a cage and work with the researcher to set up numbers of animals in
each cage that fit with the experimental design. Besides, with the plastic condo in there, it takes up a
bit of space too. But it does add some vertical space inside the cage so maybe that helps a bit.
Have a look at the ceiling lights in the animal room, through this window. In this wing the lights are on
a 12-hour (6:30 am to 6:30 pm) automatic schedule. Would that be OK for your project? In all the
animal rooms we now have the brightness of the lights at a maximum of 300 Lux at cage level. That's
a lot less bright than most of us are used to in our labs or offices, but that's necessary to avoid
causing retinal damage to albino animals, and many of our rat and mouse strains are albinos.
The lights in the animal rooms are turned on and off by the automatic timer clock. We can set that to
any particular time your research needs. We don't have light timers that slowly turn the lights up, but
I wish we did. I read that when the lights suddenly come on, it upsets the animals and some of their
hormone levels change for a few hours after that.
Food and Water
In most areas we use ordinary city tap water for the animals. We use water bottles in some rooms,
and have automatic watering systems on racks in other rooms. If you need special control over the
water your animals get, we have distilled water outlets in each area - actually it's reverse osmosis
water. For some of the immunodeficient animals we put a few drops of hydrochloric acid in the water
to bring the pH down to 2.8. That's to reduce the chances of bacteria like Pseudomonas from
contaminating the water bottles or sipper tubes.
We keep all the feeds in a proper storage room according to manufacturers' recommendations, close
to the loading dock. And we use all the pelleted feeds within six months of milling to make sure
there's no loss of nutrient value. In the animal rooms we just keep enough feed in those covered
plastic containers, for daily use. For researchers who need sterilized diets for their animals, we can get
either irradiated or autoclavable feed. Our facility prefers the irradiated bags, because they come
sterilized, and we can disinfect the outside of the bags properly. The heat and steam of autoclaving
destroys some of the feed's nutrients such as vitamin K, and so we have to buy special autoclavable
diet, or add extra vitamins later.
Image courtesy of Dr. Ernest Olfert, University of
Saskatchewan.
We routinely use certified rat and mouse chow and get it in at least once a month. If you will be
needing any special diets for your animals make sure to let us know well ahead of time, OK? When we
get back to the office I'll give you a sheet with the ingredients and analysis of the diet we're using, for
your master experimental log book.
Image courtesy of Dr. Ernest Olfert,
University of Saskatchewan.
All the cages have lab chow available all the time and the animals are free to eat whenever they
choose. Our standard feed is a certified chow for all the rodents and rabbits, but we can get whatever
you need for any animals. The lab chow supplies all the nutrients the animals need, and the company
certifies it to be the same ingredients no matter when we buy it, so if your study lasted two years you
could be assured that the diet would be consistent all the way through.
Sometimes researchers need more strict controls over what is in (or what is not in) the feed. Then
they have to go to semi-synthetic or even synthetic diets where the whole diet is made up of refined
chemical ingredients. There are a number of companies that make those diets up for researchers.
Animal Identification
To make sure animals never get mixed up, we have ID cards on each cage, and in some studies the
animals get tattooed, micro-chipped, or ear-punched to make sure they are properly identified. I've
got more details about each of these ID methods. Here's a brochure for you to read. Once you've
decided on a method of identifying your rats, let us know and we can order the stuff for you.
Image courtesy of Dr. Ernest Olfert, University of Saskatchewan.
Here's a sample of our regular cage card. The CCAC recommends that every cage card should have at
least the following information on it about the animals and the experiment: arrival date, source
(supplier's name) type of animal, breed or strain, sex, age and/or weight, number in the cage, name
of investigator, and the protocol number. There's also room on the card to mark down your
treatments. Some of this information can be embedded in a bar code to facilitate record keeping.
However, enough information should still be printed on the card for the technicians to read.
Image courtesy of Dr. Ernest Olfert,
University of Saskatchewan.
Sometimes we put a bigger card with the same information on the room door or wall, if that works out
better, for floor pen animals for example.
Image courtesy of Dr. Ernest Olfert, University of Saskatchewan.
Click this link for a look at an Animal ID Brochure.
Procedures Room
These next two rooms are our procedures rooms, right down the hall from the animal rooms. You are
free to use one of them for all your weighing, sampling, or other treatments. None of the
manipulations should be done in the animal holding room itself. That's one of the CCAC guidelines. We
have some commonly used equipment here, but you may have to bring your own special equipment.
It's best to book the room ahead of time once you know your schedule.
Image courtesy of Dr. Jim Love,
University of British Columbia.
Image courtesy of Dr. Jim Love,
University of British Columbia.
We also have a fully equipped surgery in our facility, and a couple of highly skilled staff to help with
the anesthesia, assist the surgery, and provide monitoring after the surgery. That area can also be
used for euthanizing animals at the end of a study to do tissue or blood collections.
Down this hall towards the loading dock, are the feed and bedding storage rooms.
Now let's go back to the office. You can take your booties off here, and put them in that garbage can.
I'll take your lab coat, thanks.
Ordering Animals
How can you order animals when you're ready to start your project? Well, ordinarily we get rodents
shipped in once a week from the commercial suppliers. If the strain you want is on your protocol, and
the animal care committee office has approved it, we can place the order any time you're ready.
Usually the animals are shipped the following week after we've placed the order - at least for the
commonly used strains. The suppliers crate them in filtered cardboard or plastic shipping crates with
some sort of moist food inside. They put only so many animals in each crate depending on how big
they are. When they get to the airport we get a call and drive out and pick them up and bring them to
the animal facility. Sometimes they're consigned to a shipping company and then they're delivered
here by the company. On arrival the crates are inspected and checked to make sure they contain what
we ordered. Then they either go directly into the assigned animal rooms if they're going into the
conventional area for short term studies, or they are held in the quarantine area till they're checked.
We've got an SOP for that too. The veterinary staff does the health screening of the incoming animals
and the regular testing of all the longer term colonies in this facility. That cost is included in the per
diem charges.
Although you probably didn't think about this, we also have waste disposal SOPs. All animal carcasses
or tissues are collected and saved frozen till they can be taken for incineration. We make sure that this
material is NEVER thrown into the ordinary garbage.
Here we are back in the meeting and lunch room. Would you like a coffee?
End of Tour
This is a photo of all the staff taken last summer. We're very proud of our staff. Through the CALAS
training program they've all gotten one certificate level or another. CALAS stands for the Canadian
Association for Laboratory Animal Science - our national professional association that sets out training
programs, and tests and certifies laboratory animal technicians. The CALAS training along with their
experience working with all kinds of animals, makes it easy for me to say that the animals here are
always in expert hands. Plus, the staff come in every day of the week and of the year including
Christmas and New Year's to check the animals and do any treatments or other monitoring.
Sample Questions
Any milled diet such as chow or pellets should be used within which one of the following time periods?
a.
b.
c.
d.
e.
Two months of ordering
Four months of storage at the animal facility
Six months of milling date
Nine months if stored in a cool room
One year if stored in the freezer
Which one of the following best describes the ventilation regarding the anteroom and containment
room of a level 2 biohazard containment area?
a.
b.
c.
d.
e.
The air pressure is negative in the anteroom and containment room with respect to the
corridor outside the room.
The air pressure is positive in the anteroom and containment room with respect to the corridor
outside the room.
The air is HEPA filtered prior to entering the anteroom and containment room.
The air inside the anteroom and containment room is controlled by extra fans.
The air pressure in the containment room is neutral across the anteroom to the corridor.
Module 07 - Environmental Enrichment
Topics
The objectives of this module are:
•
•
•
•
To
To
To
To
introduce the reader to the concept of environmental enrichment.
discuss the elements involved in environmental enrichment
place the effects of environmental enrichment within the research effort
provide examples of environmental enrichment
As a start to this module, there is a short exercise to introduce some of the areas to be covered and to
encourage the reader to think about how we keep experimental animals and where we might need to
make improvements. In this exercise, we will try to compare the well-being of a wild and a laboratory
mouse, without delving too deeply into the husbandry or lives of either. Let your own thoughts or
perceptions about the two animals guide your answers. There are no right or wrong answers but
there may be some unexpected outcomes. The scores are not important, as you will see in the
commentary.
Wild Mouse versus Laboratory Mouse
Measuring Well-being
One of the ways we can look at our treatment of laboratory animals is to compare them with their wild
counterparts. As background information and perhaps to introduce some bias, you should know that
in the wild, mice live about three hundred days or so while outbred laboratory mice will live about six
to seven hundred days. If a young wild mouse is brought into the laboratory, it will live for about six
hundred days or so. Below, you are asked to compare the wild and laboratory mice using the Five
Freedoms as set out by the UK Farm Animal Welfare Council. The scale shown goes from minus 5 to
plus 5 and should be used to indicate your idea of how each parameter affects the well-being of each
mouse.
Using a piece of paper, give a score for each mouse in each of the categories, either plus or minus.
You may feel that you do not know enough about either mouse to judge properly but you should
allocate a score based on what you think each mouse experiences. Consider the laboratory mice to be
living in an animal facility and that they are not part of an experiment.
If you want to print off a paper copy of the following scales, print out Appendix D . You can also print
off this page of the website in order to complete this activity.
The Five Freedoms
Wild
Freedom from hunger and thirst
Freedom from discomfort
Laboratory
Freedom from pain, injury and disease
Freedom to express normal behaviours
Freedom from fear and distress
Total
A second system of looking at the well-being of the two mice considers in more detail some of the
parameters that affect their lives. The scoring system is the same as for the five freedoms.
Wild
Food
•
•
•
Quality
Variety
Availability
Water
•
•
Quality
Availability
Activity
•
Social
o
o
•
Variety
Stress
Physical
o
o
Variety
Extent
Laboratory
Environment
•
•
•
•
•
•
Climate
Variation
Extremes
Space
Size
Complexity
Health
•
•
•
Infections
Injuries
Deficiencies
Total
Commentary
When such tests are done with groups of volunteers, every possible result occurs. Either both tests
show the wild mouse to be better off or both show the laboratory mouse to be better off. Sometimes
one test shows the wild mouse to be better off and the other the laboratory mouse to be better off.
Occasionally the results show that the mice have equivalent levels of well-being.
A closer examination of the results shows that wild mice are better off in some respects than
laboratory mice and worse off in others. For example, the environment of the wild mouse is much
more varied but the availability of good quality food is less than for the laboratory mouse. The
difference in final scores may be just a reflection of how much better or worse a particular parameter
seems to be to us. So these tests are highly subjective; we may be displaying our anthropomorphic
views on the lives of laboratory or wild mice or we may have been influenced by some pre-existing
knowledge about them. Many people are surprised by their results. There is often an admission that
even though the laboratory mouse came out as better off in the test, that maybe the wild mouse was
better off, at least for the shorter life they live.
This should make us wonder how the mice view the importance of various aspects of their daily life.
In the tests, we gave each parameter the same weight. But maybe a complex environment is worth
much more than the quality of water. Maybe living a life with all its varied ups and downs for three
hundred days is much better than an uneventful life of seven hundred days.
For the purpose of this module, these tests introduce a number of different ideas with regard to
environmental enrichment for laboratory animals. In particular, if we look at those categories in each
test where the laboratory mouse was less well off than the wild mouse, we realize that we have room
for improvement. Admittedly, the tests are inaccurate and we may have overemphasized or
underestimated the extent of problems, but the fact that we think about them is important both to the
animals and to the studies they are used in.
Introduction
Much of our knowledge on the effects of environmental enrichment comes from studies on rats and
mice. The most common source of information comes from our observation of the wild counterparts
to our domestic and laboratory animals. We recognize that wild animals have certain behaviours that
are commonly performed. All young animals run and jump and play and this is important for the
development of strong muscles and bones, for good coordination, and for developing social skills and
relationships. They also learn discipline from adults and young animals that get out of line are likely
to be punished. Wild animals of all ages must deal with both threats and deprivations (e.g.,
predators, parasites, a lack of food or a poorly balanced diet, poor shelter against miserable weather,
excessive cold or heat, etc.). Laboratory animals are sheltered from these problems but are they
better off?
Experimental animals were traditionally kept in caging which provided little or no social or physical
stimulation. The use of such caging was justified on the basis of reduction of disease spread, ease of
sanitation, prevention of fights between animals, easy recognition of illness through measuring food
and water intake, etc. However, at the time, little consideration was given to the behavioural and
psychological well-being or the stress induced by social isolation and physical deprivation. It is
recognized now, that the well-being of animals is greatly improved if they are provided with
opportunities for interaction with each other and their environment. Furthermore, there is an
increasing volume of literature denoting the deleterious effects of impoverished environments on
experimental results. (See article by William M.S. Russell at this link)
Although the term "environmental enrichment" is used to describe efforts aimed at improving the
living conditions for animals, the move is really from a very impoverished environment to a less
impoverished environment. It is unlikely that the level of complexities encountered by wild
counterparts will ever be achieved within the laboratory. Furthermore, it is possible that the wellbeing of an animal will not be increased by our ideas of increased complexity in its environment.
Nevertheless, the wild species are often taken as the norm against which the environment of the
captive animal is measured. Some argue that the wild and laboratory animals are no longer the same
behaviourally, but most wild behaviours are seen in the laboratory animal.
The presence of a normal range of behaviours and the absence of abnormal behaviours or stereotypies
is a reasonable indication that the animal is coping with its environment. To make such judgements,
we must be able to recognize normal and abnormal behaviours. Those species that are prey animals
in nature, seldom reveal that they are hurting in any way as this would be an invitation for predation.
Another approach to evaluating well-being is to use the Five Freedoms of the UK Farm Animal Welfare
Council. These freedoms were defined to give guidance to farmers on the goals of husbandry.
However, the freedoms are easily adapted to other animals and have been accepted by various groups
including the World Veterinary Association and Humane Societies.
The five freedoms are:
1.
2.
3.
4.
5.
freedom from hunger and thirst (by ready access to fresh water and a diet to maintain full
health and vigour)
freedom from discomfort (by providing an appropriate environment including shelter and a
comfortable resting area)
freedom from pain, injury and disease (by prevention or rapid diagnosis and treatment)
freedom to express normal behaviour (by providing sufficient space, proper facilities and
company of the animal's own kind)
freedom from fear and distress (by ensuring conditions and treatment which avoid mental
suffering)
The freedoms are general enough to allow them to be used for any animal species and to allow for
interpretation related to particular species. They must be applied carefully with an understanding of
the biology of each species and care must be taken to avoid using our own ideas as animal standards.
For example, it is customary to keep newly weaned piglets at a temperature of about 27° 0 C, day and
night. However, when the piglets had the opportunity to control the temperature themselves, they
preferred a temperature of about 29°0 C during the day and about 15°0 C at night.
Terms like "discomfort" make us think about the animal's living conditions and while we tend to think
of the extremes of heat and cold, or wet and dry, there are grades of discomfort in between the
extremes as we know from our own experience. A cool room is uncomfortable if we do not wear
enough clothes and a hairless animal without any means of building a nest or others to huddle with
may be uncomfortable at the normally recommended temperatures in the animal facility.
We can assume, given our present knowledge, that the health, nutrition and general environment
needs of the common laboratory animal species are being met in present day laboratory animal
facilities. The major challenge for us is to provide them with social and physical opportunities to live
and behave in a normal manner. To do that we must have some knowledge of what a particular
animal needs based on understanding their preferences. All animals require social interactions
although for some this interaction is intermittent and occurs only at breeding times. Most wild animals
occupy their days in the search for food and water. The threat of predation is a fact of life for many
small animals, including those in the laboratory where we are the predators. To be frightened without
having any means of protecting yourself is a stressful experience. Lack of space or structure to
exercise or play, in the case of young animals, is detrimental to bone and muscle development and
maintenance.
The major factors to be considered then are:
•
•
•
•
Opportunities
Opportunities
Opportunities
Opportunities
to socialize or not
to occupy time during waking hours
to hide
and structure for exercise
Opportunities to Socialize or Not
Totally isolated animals are known to be different from group-housed animals. This is seen even with
pet animals like dogs. A dog that lives with a family and has little interaction with other dogs behaves
differently than a dog that has been well socialized with other dogs. But not all animals want to live
together and it may be undesirable to have groups with both sexes present. Aggression may be a
problem with some species and strains but this may be reduced or prevented if the environment is
sufficiently complex. Sight barriers may be enough to break aggressive activities within a group.
There should be the possibility for an animal in a group to socialize or not. This is often difficult to
arrange in a small cage but even a small cage may be broken up into separate areas. For example,
the use of small igloos in a mouse cage allows some to be in the igloo and some outside.
Opportunities to Occupy Time During Waking Hours
Searching for food, gathering nesting materials, play, travel, exploration, etc., are all activities that
help pass the time. The environment of many cages does not allow much of this to occur. The highly
nutritious food provided allows animals to consume their daily needs with very little effort. Monkeys
that work for their food waste less than those with easy access to it. While commercially available
foods are processed to contain all the elements that an animal requires, they may be low in
palatability and certainly in variety. It is possible to vary the diet for most experimental animals
without imposing yet another variable on the study by providing small amounts of treats. These
treats provide additional tactile, olfactory, and taste stimuli.
Cage equipment, nesting material, etc., allows the animal to interact with and in some cases
manipulate their environment. The equipment or material in the cage should be appropriate for the
animal's behavioural needs. Perches for birds, for example, should be the correct size or in a variety
of sizes so that the birds can pick the most comfortable one for them.
Animals are sometimes given toys to play with but the toys should have some relevance to the animal
or it will soon be neglected. Some animals, e.g., rats, may not like new toys, especially if they have
no apparent function for the animal and the toy may be buried. The unpredictability of another animal
may provide the majority of the diversion required to prevent the development of stereotypies that
often develop when there is nothing to do. Rats have been shown to work harder to gain access to
another rat than to gain access to their favourite toy.
Exploratory behaviour is an important component of the daily routine for many young animals,
particularly rodents. This activity is often studied in rats and it is recognized that there are clear
behavioural differences between rats that have had the opportunity to explore, for example, in a
complex environment and those that have been reared in a simple environment. Rats that have been
reared in the standard rat cage will stand up and look out when the top is removed. They will rarely
attempt to leave the cage. On the other hand, rats that have lived in a complex environment will take
the opportunity to explore the room if the cage is left open.
Opportunities to Hide
Most laboratory animals appreciate a place to hide, whether it be from cage mates, people or
unexpected noises. Even within gregarious species, individuals may require a place to get away.
Thus, where possible, animals should have a place within the cage where they feel safe. This may be
a dark corner or it may be a sight barrier (e.g. tubes, overturned containers), which allows them to
look for the cause of their alarm without revealing themselves. Chickens preferred sight barriers
made of vertical slats with small spaces between the slats. Disputes among animals often end when
the "chasee" gets out of the sight of the chaser. Unexpected noises are common in animal facilities
and may be startling to animals. The natural tendency for the prey species is to hide while they try to
determine the source of the noise. Large human figures peering into cages may also be frightening,
causing the animals to seek refuge. If there is nowhere safe to hide, the animals will be stressed.
Opportunities and Structure for Exercise
Space for exercise is important, particularly in young animals. Animals like to run or hop and this is
important for bone and muscle development. Space alone is usually not enough as can be seen in the
pacing of some large cats in zoos. There should be structures within the space to allow climbing,
stretching, swinging, etc. Even relatively small structures within a cage will be used for exploration.
Mice are often seen clinging upside-down to the food hopper.
Environmental Enrichment and Research Results
Improving the environment is not just a nicety for research animals. There is a considerable body of
literature now that demonstrates the influence of an animal's physical and social environment on
research results. One of the earlier demonstrations showed that social and physical stimulation of rats
resulted in a thicker cerebral cortex with more dendritic connections. Tumours in isolated mice grow
faster than the same tumours in mice housed at appropriate densities. Isolation of mice has been
shown to increase the toxic effects of some drugs.
It has also been shown that environmental enrichment is beneficial at any stage of an animal's life.
The effects may be different between young and old animals but the old will also benefit. For this
reason it is important to consider environmental enrichment as a variable in an experiment and to
account for it. It is not an option, however, to omit environmental enrichment to reduce the variables
in a study unless the investigator is prepared to include all the deleterious effects of an impoverished
environment on the study. Even then, it would be difficult to say that the results represent the normal
state of the animal. However, if an investigator feels that attempts at environmental enrichment will
jeopardise the results of a study, then this should be justified to the Animal Care Committee.
Environmental enrichment encompasses more than just the physical and social environment of a
group of animals. Because we interact with them at various levels, we may have a profound effect on
their life. We should treat animals in a manner that minimizes any discomfort or stress they may
experience at our hands. All the environmental enrichment in the world will not be of any value if an
animal fears the arrival of a human being at its cage. It may not be just the presence of a person but
it may also include the sounds and smells associated with an experimental procedure, for example.
Our activities in the animal facility may be disturbing, even if they do not directly involve the animals.
Noise is disturbing to animals and we should minimise extraneous noises as much as possible. Some
people bustle and have an air of urgency about them that is unsettling to animals. Doors are allowed
to slam shut or objects fall on the floor. Equipment like cage washers, vacuums etc. may be
upsetting, particularly to pregnant animals.
If we try to see things as the animals might see them, we will probably be able to improve their living
conditions. In return, people working with the animals, particularly the animal care technicians, feel
better about their jobs when they see the animals responding to their enriched environment.
It must be emphasised that changing an animal's environment, whether it be giving it a clean cage
devoid of familiar "homey" smells or adding toys or other objects for enrichment, will be a variable
that should be accounted for. It is important, then, not to make changes to the environment without
the agreement of the principal investigator and if changes are made, they should be applied
consistently to all animals in the study. It should be remembered that there may be effects of
withdrawing enrichment, for example, if the animals move from a facility with very complex
environments to one where there is minimal complexity.
Examples of Environmental Enrichment (General)
There follows a series of examples of enriched environments for animals. The form of enrichment
provided for a species should take into account the normal environment for that species and how the
species interacts with its environment. It is not necessary, or even generally feasible, to duplicate
exactly the natural environment but the substitutes should allow the animal to perform as many of its
natural behaviours as possible.
Further examples of environmental enrichment may be found in the literature. Comfortable Quarters
for Laboratory Animals. Eds. Viktor and Annie Reinhardt may be accessed via the Internet at
http://www.awionline.org/pubs/cq02/cqindex.html.
The CCAC publication "Guide to the Care and Use of Experimental Animals Vol.1 2nd edition, has a
section on the Social and Behavioural Requirements of Experimental Animals (Chapter VI) and it can
be found on the CCAC website.
An extended list of references may be found on the CCAC website at www.ccac.ca
Examples of Environmental Enrichment (Mice)
Even a simple glass bottle will provide variation in the environment for a mouse in a small cage. In
this case, the mouse and her offspring are using the bottle as a nesting spot. In some cases, the mice
use the bottle as a urinal, thereby keeping the rest of the cage dry and clean. If this happens, the
bottles must be changed frequently. Mice will not use it once there is a significant amount of urine in
the bottle and will block up the entrance.
A divider in the cage like the one shown allows the mouse to use different "rooms" for different
purposes. In this case one of the small rooms has been selected for the nest. It is important to
provide mice with nesting materials so that they can construct a suitable nest. This does not just
apply to breeding mice as male mice will also build nests. The nesting material is given in the large
"room" and the mice select where they will build the nest. Most select one of the smaller "rooms".
Examples of Environmental Enrichment (Rats)
This blue Plexiglas structure was introduced into a large bin approximately 100cm X 160 cm, to
provide the rats with something to play on and to exercise in. There were 20 rats each weighing
about 200g in the bin.
This picture was taken one morning when most of the rats were resting. There are twenty rats in the
picture and the space where most are resting was about 1000 cm2. (The CCAC recommended
minimum space for a single rat greater than 150 g is 250 cm2.) Rats do not mind lying on top of each
other but they need space to do other things. Crowding occurs when the space is so limited that the
animals cannot get away from each other and this is stressful.
This is the same structure as in the previous picture. The picture was taken within a few minutes of
the structure being turned upright. The rats demonstrated two behaviours that we should try to
accommodate. They wanted to hide under something and they wanted to explore. Rats, particularly
young rats will make use of any structure in their cage to play, explore, hide etc. It is not necessary
to provide something that would be present in their natural environment.
Examples of Environmental Enrichment (Guinea Pigs)
These guinea pigs are housed in large bins with shavings in the bottom. They have places to hide
when they are startled and they are given hay as roughage.
Examples of Environmental Enrichment (Rabbits)
These rabbits are group housed as befits a social species. They have places to hide and shelves to
climb on. The sawdust allows them to "dig" although they cannot dig burrows. They groom each
other and indulge in other amicable interactions. Female rabbits may be kept this way at any age but
male rabbits will start to fight when they reach puberty.
Examples of Environmental Enrichment (Ferrets)
Ferrets are playful animals that like to run, tumble and hide. Even simple structures can be fun. The
empty feed bag shown here allows them to run inside, tumble around and run out again. Three
ferrets that escaped from their pen and set off the security alarms were recaptured by placing a bag
on the floor. The report from security described the capture of the three masked bandits.
Examples of Environmental Enrichment (Cats)
These two cats have a large indoor pen with access to the outside. They have individual resting areas
but they prefer to lie together. It is important to provide animals with as much choice as possible, the
choice to be together or to be apart in this case. They also have scratching posts and other toys in
the pen. Animals do not necessarily get along together all the time and so it is important to provide
group housed animals with space to be apart when they want to be.
Examples of Environmental Enrichment (Sheep)
Sheep are frequently used in laboratories and these flock animals become extremely distressed if they
are held in isolation from other sheep. If isolation is necessary for some reason, an effort must be
made to allow them to see and smell other sheep otherwise the stress of the isolation will have
profound effects on the experimental results.
Examples of Environmental Enrichment (Swine)
Environmental enrichment includes the human. Some animals evoke a stronger reaction in people
than others but all animals should be treated with respect and kindness. This Yucatan pig enjoys a
back scratch and will lie down to have his stomach scratched. The pen has sawdust on the floor and
the pigs spend a large proportion of their active time rooting and "ploughing" the sawdust. These pigs
have an outdoor run also and urination and defecation occur there, keeping the inside sawdust dry
and clean.
Examples of Environmental Enrichment (Monkeys)
Environmental enrichment can include the provision of a variety of foodstuffs. While specially
prepared diets are nutritionally complete they are usually not very interesting. This monkey has been
given a popsicle but Jello and various fruits and vegetables may also be given without risking an
imbalance in his diet. Some of these extra treats may be used to carry medications if this is required.
An extension of the use of feedstuffs to carry medication is the training of animals to take their own
medication. This monkey has been given a syringe with juice carrying the medication and he will take
the juice voluntarily. Although we can force most of our experimental animals to do what we want, it
is always better to have their cooperation. The level of stress on the animals, technicians, and
investigators is less when force is not needed. Monkeys and dogs have been trained to present arms
or legs for blood sampling. The training requires patience but the rewards are great for both the
animals and the investigators.
"Old Bill" lives in a large pen with logs for climbing and sitting. He receives a variety of foodstuffs that
he has selected as his favourites. The food is placed so that he climbs each day and gets exercise.
The floor of the pen is covered in sawdust that contains sunflower seeds, raisins or peanuts so that Bill
spends much of his time foraging as he would in the wild. He is approximately 20 years old and his
hair is thinning, but he still has an interest in the female monkey in the next pen.
The examples of environmental enrichment shown above give an idea of the range of improvements
that can be made in the way animals are housed and handled. Many research animals have not been
covered but species should not be held in environments that fail to meet their basic social and physical
needs. This applies to all animals but it is especially important for wild animals being held in
captivity. However, even those animals that have been bred in captivity for many generations retain
many of the traits seen in their wild counterparts. Laboratory mice and rats are still prey species and
still behave to protect themselves from predation. They need safe places within their cages, places to
hide. We must remember that mice are not just small rats. Rats prey on mice in the wild so we
cannot keep mice and rats together within the same airspace as the pheromones from the rats will be
distressing to the mice.
Examples of Environmental Enrichment (Birds)
Chickens, quail, ducks, and turkeys, and other birds are often used in research. There is sometimes a
conflict between the commercial housing of birds, particularly chickens, quail, ducks and turkeys, and
the research housing of the same birds. It is common for investigators to invoke the need to do
research using the same housing conditions that are used in the industry so that the results of the
research will be applicable. Many of the industry standards for the housing of these birds are under
review, both from within the industry and among the public that purchase the product. For example,
many members of the public are concerned by the battery system of housing hens to produce eggs
and there have been many attempts in recent years to move away to more bird-friendly housing (e.g.,
free range systems and aviaries). There is a need, too, in research institutions to provide modified,
enriched environments for birds to avoid the demonstrably stressful housing used by the industry.
Not all birds need space to fly but the provision of aviaries or flight cages for research pigeons helps
maintain their health. Most birds like to peck and scratch in dirt, looking for food to eat. It is said
that chickens will spend up to 50% of the day looking for food in this manner. In addition, they
consume a much wider variety of foods than those kept in small cages with a processed diet brought
to the cage.
Most birds engage in dust or water bathing and this is an important component of their maintenance
behaviour. Most birds perch and so they should be given the opportunity to perch.
Other Animals
Fish, amphibians, and reptiles are common research animals. Although there has been little research
done on these animals in terms of environmental enrichment, there is considerable information on
their husbandry in captivity and on their behaviour in the wild. It is reasonable to try to provide these
animals with some form of environmental enrichment. This will vary widely among the species. Small
fish, for example, may benefit from having places to hide, even if predation is not a threat in an
aquarium with only one species. Hiding places may be "caves" on the bottom or plants growing in the
water. The unpredictable movements of plants, even artificial plants, may provide stimulation and
novelty to the fish. It may be necessary to cover some sides of aquaria so that the fish are not being
continuously startled by the sight or shadow of large humans passing by.
Reptiles and amphibians have specific requirements depending on the species. As cold-blooded
animals they are somewhat more dependent on their physical environment to meet their needs. It is
important to design the environments to meet the requirements of the animal, based on observation
of wild animals. If group housing is used, then issues of territoriality, hierarchy, etc. must be
considered. Many reptiles require different temperatures depending on their activity so cooler refuges
should be available within the environment. Amphibians and reptiles need places to hide. Climbing
animals should have things to climb on. Animals that spend time in the water in the wild should be
able to do so in the laboratory. The guide is to try to provide the right conditions for the animals to
fulfill most of their major behavioural activities.
Summary
In the past, animals were kept in cages or pens that provided very little substrate for them to engage
in many of their natural behaviours. Environmental enrichment is a phrase used to cover a wide
range of additions or modifications of the environment to allow animals a more varied life. Although
many of the changes are indeed to the physical environment, changes in social opportunities for the
animals are also important; and social opportunities include interactions with people. A major benefit
of environmental enrichment is the reduction in stress in the animals with beneficial influences on the
research projects they are involved in.
Sample Questions
Which one of the following statements regarding environmental enrichment is incorrect?
a.
b.
c.
d.
e.
It
It
It
It
It
benefits the animal
benefits the animal technicians
is not required by the CCAC
can affect experimental results
can be simple and inexpensive
Which one of the following is likely to provide the most effective enrichment for an animal?
a.
b.
c.
d.
e.
Toys
Treats
Nesting material
Another animal
A larger cage
Module 08 - Basic Diseases and the Animal Facility
Module Objectives
At the end of this module, the reader should:
1.
2.
3.
4.
5.
Understand how animals become infected
Understand how diseases spread
Understand how diseases may be introduced to an animal facility and what steps should be
taken to exclude disease
Understand how diseases may be contained if they do gain access to a facility
Understand the basics of health monitoring programs
Introduction
The spectre of disease in the animal facility sends shivers down the backs of facility managers,
veterinarians and investigators who have experienced outbreaks previously. This is partly because
they understand the devastating effects some diseases have on the research program, and the huge
amount of work and cost involved in cleaning up after a disease outbreak.
Facility managers and veterinarians are not alone in their efforts to prevent infectious organisms from
gaining entry to the animal facility. All people who regularly work in the animal facility (e.g.,
technicians, researchers, research staff, graduate students) must understand how diseases may be
introduced and spread. Facility guidelines and standard operating procedures (SOPs) designed to limit
the risk of introducing or spreading disease must be followed by everyone. Mechanical systems must
work as expected (e.g., to sterilize cages, to maintain air pressure gradients) so even the facility
maintenance staff is involved.
Diseases may be broadly classified as infectious or non-infectious. This module concentrates on
infectious disease. Infectious diseases are caused by a variety of organisms, such as viruses, bacteria,
yeast, fungi, and parasites.
Laboratory animals, like people, are regularly exposed to potentially infectious microorganisms;
however, not all such exposures result in infection. Whether a laboratory animal becomes infected
depends on a number of factors related to the infectious organism, and the animal host. For example,
microorganisms vary in virulence, or the animal may be exposed to only a small number of infective
particles. The animal, species, or strain may be partially or entirely resistant to infection, or more
susceptible because it has a deficient immune system, is stressed, or poorly nourished.
If an organism does infect an animal, there are several possible outcomes. The infection may be silent
or latent, in which case the animal displays no outward evidence of infection; or the infection may
cause overt disease with the animal showing a variety of clinical signs depending upon the organs or
systems affected. The disease may run its course with complete recovery with or without treatment,
leave some damage (residual pathology from the disease), or even lead to the death of the animal.
Any animal that recovers from the disease and those animals that have had a latent infection may
become carriers of the infectious organism.
Infectious diseases pose a threat to animal colonies through a wide variety of mechanisms. Clinically
ill animals are poor research animals because of the disruption to their normal physiology and
biochemistry. Recovery may be prolonged and recovered animals often continue to carry and shed the
organisms that caused the disease, acting as potential sources of infection for healthy animals. Latent
or silent infections may also adversely affect the results of an experiment due to changes in the
animals' biochemistry or immune system.
In thinking of means of controlling infectious diseases, it is important to know how diseases spread,
the routes of infection and the routes of excretion of the organism from an infected animal.
Routes of Infection
There are just a few portals of entry into an animal for infectious agents. The two most common are
by inhalation into the lungs and by ingestion through the mouth into the gastrointestinal tract.
Inoculation through the skin represents a special case (e.g., insects or needles). In these cases, the
disease may spread beyond the skin. Some diseases are sexually transmitted. Occasionally, infection
of the skin, eyes or ears may result in disease spreading beyond these organs.
Routes of Shedding
Organisms are excreted by a variety of routes. For example, organisms may be excreted from the
lungs by coughing and sneezing or from the gastrointestinal tract through faeces. Organisms may also
be excreted in urine, saliva, milk, pus, from the reproductive tract, or through vectors such as
mosquitoes. Skin-based diseases (e.g., fungal infections) may cause shedding of organisms from the
skin.
Disease Spread
There are only a few ways for disease to spread between animals (or between people and animals).
Diseases spread: (a) by direct contact between animals, (b) via the environment, or (c) by means of
fomites.
a.
b.
c.
Direct contact. Animals must be in direct contact with each other for the disease to spread.
This applies particularly to skin diseases (e.g., ringworm) but could also include sexually
transmitted diseases.
Indirect contact through the environment. The environment is important for disease
transmission. Respiratory disease is a prime example of this form of transport where the
infectious organisms are in the air before they are inhaled. Water and bedding may also be
considered part of the environment and so contamination of these by one animal may result in
transmission to others.
Fomites. Fomites are inanimate objects that have inadvertently become carriers of infectious
organisms. Contaminated cages or food, or the utensils for delivering them, are examples but
there are a variety of objects which animals come in contact with which may help transmit the
infectious organisms.
Intermediate hosts or vectors are special cases for disease transmission that should not occur in
animal facilities. These include biting insects (e.g., mosquitoes) in which infectious organisms may just
be transported between animals (vector) or the infectious organism may undergo some development
in the intermediate host.
Excluding Disease
Many facility design features, facility equipment, and standard operating procedures have as their
main objective the exclusion of undesirable organisms and the containment of disease if it should
occur.
There are three potential sources of infection: animals, people and experimental procedures. For each
of these, the following points will be discussed in terms of excluding disease or preventing it from
entering an animal facility.
Animals
•
•
•
•
•
Sources of animals
Arrival procedures
Quarantine
Interlopers
Other animals
People
•
•
•
•
Restricted access
Entry requirements
Clothing requirements
Pets
Animal and Facility Related Procedures
•
•
•
•
•
Cells, tissues, fluids, etc.
Sources of food, bedding, water
Equipment used for housing, changing, feeding, watering
Surgical facilities
Facility functions (e.g., ventilation, cagewashing, sterilizing facilities, caging systems, barriers,
airlocks etc.)
Animals
Sources of animals. Animals should normally be obtained from reputable suppliers where regular
testing of the animals is carried out and where the results of these tests are available. Animals,
especially genetically modified animals, obtained from institutions which cannot supply clean bills of
health should be regarded with a high index of suspicion if they are to be introduced into a diseasefree colony. Ensuring the disease-free status of immunocompromised animals from such sources is a
particular problem, as they may not develop antibodies to pathogens which is the most common
method used for health monitoring.
Arrival procedures. Research animals arriving at institutions have passed through a number of areas
where their health status may have been compromised (trucks, airport freight areas, airplane
compartments). Shipping crates are usually not entirely impervious to micro-organisms and airports
may be infested with wild rodents. Thus, there is a real possibility that the disease-free animals that
left the supplier are no longer disease-free when they arrive. In some cases, when the delivery can be
controlled either by the vendor or the purchaser, the risk may be less (e.g., when the vendor is close
to the purchaser and purpose specific vehicles are used for transport).
Quarantine. The conditioning/quarantine of all incoming animals serves two purposes: it allows the
animals to be acclimated after a long trip, and it provides the opportunity to determine if the health
status of the animals meets the requirements for entry into the disease-free colony. The quarantine
period should be long enough to demonstrate that contamination did not occur during transport.
Remember immunocompromised animals may not develop antibodies, and sentinel animals will be
required. When rodents come from sources where their health status cannot be assured, a longer
period of quarantine may be required to determine their health status.
Interlopers. The entry of wild rodents into an animal facility is cause for concern. Some of these
animals may carry organisms that facilities want to exclude. Wild rodents may come into close contact
with colony animals, but may also contaminate feed, bedding and other materials destined for colony
use. An active pest control programme is important.
Other animals. It is common for an institution to house both disease-free animals and animals of
unknown status in the same general areas. There may be SOPs in place to reduce the likelihood of
transfer of a pathogen from one colony to the other. The use of air pressure gradients or airflow
patterns assist in separating the colonies. It should be remembered that procedures and facilities are
only as good as the people who work with them and that human or mechanical failures do occur.
The main focus of this module is the laboratory animal facility in which animals known to be free of
certain specified micro-organisms are housed - primarily rodents and rabbits. However, many other
research animals do not have highly defined health profiles (e.g., dogs, cats, farm animals, nonhuman primates, fish, etc.). While the precautions regarding entry of these animals into the facility
may be less stringent, the requirement to prevent disease spread remains.
People
Restricted access. One of the risk factors for infections in disease-free colonies is the number of
people who have access to the animals. It is important to limit the people traffic to those that must
have access. Access may be restricted for people who have visited another animal facility during the
same day. Bioexclusion SOPs may require that a specific time period occur from the previous animal
facility contact.
Entry requirements. In addition to the restrictions mentioned above, entry requirements for all
other people (e.g., physical plant workers, visitors, accreditation teams, etc.) should be enforced.
Equipment may need to be brought in that cannot be sterilized. It may be desirable to have all visitors
put on fresh clothing or shower before entering the unit.
Clothing requirements. Protective clothing should be worn when working with animals. A complete
change of clothing into facility clothing may be desirable for entry into some areas such as SPF
colonies. Showering in may be necessary in some areas. Protective wear could include bonnets,
masks, gloves and foot wear depending on the level of protection required both for the animals and
the people.
Pets. It has been documented that people who have rodents at home, for whatever reason, may
spread rodent viruses. This is really a special case of transferring unwanted organisms from an
infected colony to a disease-free colony. It is recommended that people working with disease-free
rodents should not keep rodents as pets.
Normal microorganisms carried by people may be a source of animal disease in some cases.
Immunocompromised animals are often susceptible to organisms that do not cause disease in
immunocompetent animals. Staphylococcus aureus and Klebsiella pneumoniae are two organisms that
some people carry which may cause disease in immunocompromised mice.
Animal and Facility Related Procedures
Cells, tissues, fluids, etc. Animal cell lines may be contaminated with rodent viruses or Mycoplasma.
An outbreak of mouse pox in 1998 was traced to contaminated mouse serum. These materials should
be tested for contamination before they are used on animals. Human cell lines that have been
passaged through or maintained in animals may also be contaminated.
Sources of food, bedding, water. There is the possibility of food and bedding becoming
contaminated before it reaches the animal facility. Animal facility SOPs should ensure that any
damaged bags are rejected and the outsides of the other bags are disinfected. Unwanted organisms
can be introduced in the drinking water, which is a particular concern for immunocompromised
animals. Automatic watering systems must be thoroughly cleaned and disinfected on a regular basis.
Equipment used for housing, changing, feeding, watering. The equipment used in an animal
facility must be kept clean to prevent disease spread within the colony. In some cases, routine
autoclaving of the equipment is used to back up the cagewashing facilities. Food, bedding and water
may be sterilized at the same time. Irradiated food is often used rather than autoclaved food.
Surgical Facilities. These are usually shared facilities and may represent a crossover point for
disease-free and possibly infected animals. Although it is unlikely that the two groups of animals will
be in the surgery at the same time, it is important to ensure that there is proper sanitation between
uses. This applies to all other shared facilities (e.g., radiology units, procedure rooms.).
Facility functions (e.g., ventilation, cagewashing, sterilizing facilities, caging systems,
barriers, airlocks, etc.). There are a number of facility functions that serve to minimize the risk of
bringing unwanted organisms into the facility. It is important that the ventilation system does what it
is supposed to in terms of the movement of air through the various areas. As an electro-mechanical
system, it is subject to failure (e.g., a damper may fail to open or close when requested, or a fan may
not operate as required). Add to this human foibles such as propping open a door and ventilation
patterns can change. Airlocks are employed in modern facilities to limit the movement of air between
discreet areas within the facilities and between the facility and the outside.
Facility SOPs should be in place to ensure that cage washing facilities and autoclaves are checked
routinely to ensure that they are performing as required. The use of Standard Operating Procedures
will also help ensure that the correct procedures are used each time.
Containment of Disease
Although it is most desirable to keep diseases out of a colony, sometimes it is necessary to take steps
to contain a disease and limit its spread. For example, animals may be isolated from each other,
thereby eliminating direct spread of an agent. This may not be practical in some cases and may be
detrimental to the well-being of the animals. Microisolator cages may be used to limit airborne
transmission. Cage changing should be conducted in a ventilated change station so that airborne
particles from one open cage do not land in the next open cage. The same procedure should be used
for cages on ventilated racks.
Microisolators represent containment at the cage level. Flexible film isolators provide containment at a
higher level. These units may contain a number of cages and the air entering and exiting is HEPA
filtered. Clean cages are introduced and dirty cages are removed through airlocks with appropriate
controls to prevent pathogens from escaping. A number of these units may be placed in the same
room, each isolating the animals from the others.
Containment barriers may also occur at the room or facility level.
Specific procedures should be in place to complement physical barriers. These should be in the form of
SOPs for all tasks carried out in the barriers, all aimed at maintaining the containment. Perhaps
equally important, people should be aware of what they should not do (e.g., open a microisolator cage
lid to look at the mice without using a change station). Animals that jump onto the floor should be
considered contaminated.
Summary
Do not:
•
•
Prop open doors; it interferes with the ventilation system
Lift the lids of microisolators for any reason unless they are in a properly ventilated changing
station
•
•
•
•
•
•
•
•
Refill water bottles (replace with a new bottle instead)
Put rodents that have jumped onto the floor back in their cages
Move from contaminated areas to uncontaminated areas
Use the same needle to inject two different animals
Use the same instruments for surgery on two different animals without sterilizing them
Swap enrichment devices between cages
Save food from the hoppers when the whole cage is being changed
Keep rodents at home if you work in a disease-free rodent facility
•
•
Follow all facility SOPs
Clean and disinfect common equipment and areas after each use (e.g., procedure tables,
anaesthetic machines)
Wear protective clothing, gloves, masks, caps, gowns, shoe covers, etc. as required by facility
guidelines
Change protective clothing between individual or groups of animals as required
Ensure that equipment for cleaning and sterilizing is working up to standard
Ensure that a health monitoring program is in place (the design of a health monitoring
program is specific to a facility, and is beyond the scope of this module; however readers
should be aware that such a program is required).
Do:
•
•
•
•
Health Monitoring
There are a number of diseases that should be excluded from animal facilities. Some organisms cause
severe disease in the animals and so render them unsuitable for research purposes. Some organisms
may be present in the animal without overt signs of disease appearing until the animal is stressed, for
example after a surgical procedure. Some organisms may be present in animals without causing any
disease but still represent a threat to research because of the changes they cause to the immune
system, for example. These latent or silent diseases usually do not provide any clinical signs of their
presence. Some are transmitted to humans and may pose a risk to people, while in other cases the
diseases do not cause overt disease in the animals but may interfere with their use in research. For
example, Q fever, caused by Coxiella burnetii, seldom causes overt disease in sheep but is usually
highly infectious for people and may cause a variety of symptoms ranging from mild, influenza-like
symptoms to severe pneumonia and infection of heart valves.
A health monitoring program is required for any of the reasons given above and it has become more
of a necessity with the increase in transfer of animals, particularly mice, between institutions. The
health monitoring program should seek evidence for the presence of specific organisms that have may
represent a threat to the animals, the research or people. The list will be different for different species
and perhaps even within species. (Note: Link to this in a separate window: See list of unacceptable
organisms for rats and mice) The organisms include viruses, mycoplasmae, bacteria, internal and
external parasites, fungi, etc. The program will generally use a number of techniques (e.g., serology,
microbial culture, PCR technology, microscopy, histopathology) to evaluate the health status of the
animals.
The frequency of testing of a colony depends on a number of factors, some of which increase the need
for increased testing (e.g., frequent delivery of animals from many sources), and some of which
decrease the need (e.g., good quarantine and management procedures).
The testing process may simply involve taking samples from existing animals for serology, culture,
etc. Sheep or cattle are usually tested in this manner. For colonies of small rodents, sentinel animals
are frequently employed so that the main colony is not disrupted. If colony animals are used, then
there are several caveats to be recognized.
Since the whole colony will not be tested, enough animals must be tested to be reasonably sure that
an infection does not exist in the colony. As an example, if the incidence of infection is 30% in a
colony, 10 animals per 100 must be tested to be 95% confident that at least one infected animal will
be identified.
Often older animals are picked when colony animals are used for surveillance. This may result in some
interesting reports, especially from the histopathology examinations as some of the animals may be
showing age-related changes.
If sentinel animals are used in the health monitoring program, they must be free of any of the
unwanted organisms. The major consideration for sentinel animals is that they should be
given every opportunity to become infected if an organism is present. Genetically modified
animals should not be used as sentinels, because they may not mount a measurable antibody
response.
Dirty bedding from colony animals may be placed in the sentinel cages since a number of diseases are
spread by the fecal-oral route. The sentinel animals should be exposed to the air from colony animals
(i.e., they should not have microisolator tops on their cages) and they should be close to the outflow
for air in the room. (One design of ventilated rack routes all the air from the rack through the cage
containing the sentinels.)
When the animals are tested, the hope is that all results will come back negative. However, it should
be remembered that the tests are not infallible and so while the results of a single testing may be
encouraging, repeated negative tests are more conclusive.
Sometimes there are positive results, particularly from serology. False positives do occur and if there
is just one positive from an appropriate sample, then retesting may show that the colony is disease
free.
If a test comes back with a positive result, repeat samples are often taken. Repeat positive results are
handled in a number of ways depending on the organism, its ability to spread, its effect on research,
etc. The course of action may also depend on the type of animals in the facility. Immunocompromised
animals are at a greater risk than immunocompetent animals. If the infected animals are unique, this
will influence the course of action
Among the options a facility may consider would be: live with the organism if it does not pose a threat
to the research, other animals or people; isolate and contain the infected colony; depopulate the
infected colony, do a comprehensive decontamination and restock with known disease free animals.
The source of disease introduction should be sought as part of the recovery process from a disease
problem in an animal facility. Animal sources, transportation, people, cell lines, etc., should all be
checked to avoid a repeat of the problem
Monitoring for Non-infectious Disease
The previous discussion dealt primarily with the detection of infectious diseases since they may spread
and threaten a whole colony. However, non-infectious diseases are becoming more important with the
large numbers of genetically modified animals being produced. The "secondary" diseases being
identified in these animals may affect both the well-being of the animals and the research being
conducted on them. A broad health monitoring program for genetically modified animals should also
be in place.
Observing and Reporting Disease
Research scientists, research technicians, graduate students, animal care staff all have a role to play
in minimizing the impact of disease in an animal facility. Important elements in health monitoring are
the identification of sick animals and the implementation of procedures for dealing with the animals.
Animals may be ill because of something unrelated to the experimental protocol. It is important that
sick animals are promptly reported to the veterinary staff for diagnosis. It may be appropriate to
isolate the animal during the diagnostic period or it may be necessary to euthanize it to secure a
definitive diagnosis. The health monitoring program outlined above should be seen as a snapshot of
colony health at the time the samples were taken. Very low levels of infection or diseases still in the
incubation phase may be missed so it is important to obtain a diagnosis on sick animals between the
routine tests.
Standard Operating Procedures should be established for the reporting of sick animals and for
subsequent procedures (e.g., isolation, euthanasia, necropsy, diagnosis).
The following list of unwanted microorganisms is given for interest and is not part of the examinable
material.
LIST OF UNDESIRABLE ORGANISMS IN ANIMAL FACILITIES
Immunocompetent Mice
Bacteriology (Culture of the oropharynx and of the cecal/colonic content)
•
•
•
•
•
•
•
Bordetella bronchiseptica
Citrobacter rodentium
Corynebacterium kutscheri
Pasteurella pneumotropica
Salmonella sp.
Streptobacillus moniliformis
Streptococcus pneumonia
Serology (ELISA)
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Cilia-associated respiratory bacillus (CARB)
Ectromelia virus
Encephalitozoon cuniculi (ECUN)
Epizootic diarrhea of infant mice (EDIM)
Hantaan virus
K virus
Lymphocytic choriomeningitis (LCMV)
Minute virus of mice (MVM)
Mouse adenovirus (MAD)
Mouse cytomegalovirus (MCMV)
Mouse hepatitis virus (MHV)
Mouse parvovirus (MPV)
Mouse thymic virus (MTV)
Murine encephalomyelitis virus (GDVII)
Murine mammary tumor virus (MTLV)
Mycoplasma pulmonis
Pneumonia virus of mice (PVM)
Polyoma virus
•
•
Reovirus 3
Sendai virus
Polymerase chain reaction (PCR)
•
•
•
Helicobacter bilis
Helicobacter hepaticus
Pneumocystis carinii
Parasitology
•
•
Endoparasites (Direct examination of cecal and colonic contents, and pooled flotation)
Ectoparasites (Direct microscopic examination of hair samples)
Immunodeficient Mice
Bacteriology (Culture of the lower respiratory tract and of the colonic content)
•
•
•
•
•
•
•
•
•
•
•
Bordetella bronchiseptica
Citrobacter rodentium
Corynebacterium kutscheri
Salmonella sp.
Streptobacillus moniliformis
Streptococcus pneumonia
Pseudomonas aeruginosa
Pasteurella sp.
B-hemol Streptococcus
Klebsiella pneumonia
Klebsiella oxytoca
Serology (ELISA)
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Cilia-associated respiratory bacillus (CARB)
Ectromelia virus
Encephalitozoon cuniculi (ECUN)
Epizootic diarrhea of infant mice (EDIM)
Hantaan virus
K virus
Lymphocytic choriomeningitis (LCMV)
Minute virus of mice (MVM)
Mouse adenovirus (MAD)
Mouse cytomegalovirus (MCMV)
Mouse hepatitis virus (MHV)
Mouse parvovirus (MPV)
Mouse thymic virus (MTV)
Murine Encephalomyelitis virus (GDVII)
Murine mammary tumor virus (MTLV)
Mycoplasma pulmonis
Pneumonia virus of mice (PVM)
Murine Encephalomyelitis virus (GDVII)
Reovirus 3
Sendai virus
Polymerase chain reaction (PCR)
•
•
•
•
Helicobacter bilis
Helicobacter hepaticus
Helicobacter sp.
Pneumocystis carinii
Parasitology
•
•
Endoparasites (Direct examination of cecal and colonic contents, and pooled flotation)
Ectoparasites (Direct microscopic examination of hair samples)
Rats
•
•
•
•
•
•
•
•
Bacteriology (Culture of the lower respiratory tract and of the colonic content)
Bordetella bronchiseptica
Citrobacter rodentium
Corynebacterium kutscheri
Pasteurella pneumotropica
Salmonella sp.
Streptobacillus moniliformis
Streptococcus pneumonia
Serology (ELISA)
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Cilia-associated respiratory bacillus (CARB)
Encephalitozoon cuniculi (ECUN)
Hantaan virus
Killham Rat Virus (KRV)
Lymphocytic choriomeningitis (LCMV)
Rat parvovirus (RPV)
Mouse adenovirus (MAD)
Mycoplasma pulmonis
Pneumonia virus of mice (PVM)
Polio virus (GDVII)
Reovirus Type 3 (REO-3)
Sendai virus
Sialodacroadenitis virus (SDA)
Toolan's H-1 Virus (H-1)
Polymerase chain reaction (PCR)
•
•
•
Helicobacter bilis
Helicobacter hepaticus
Pneumocystis carinii
Parasitology
•
•
Endoparasites (Direct examination of cecal and colonic contents, and pooled flotation)
Ectoparasites (Direct microscopic examination of hair samples)
Sample Questions
Which one of the following best describes a static microisolator rodent cage?
a.
b.
c.
d.
e.
It
It
It
It
It
is a disease barrier at the cage level
requires a complex ventilation system
should only be used for nude mice
must be sterilized before use
does not require a ventilated change station
Which one of the following best represents a fomite?
a.
b.
c.
d.
e.
An insect in the mange mite group
A contaminated weigh scale for mice
A disinfectant that kills viruses
A sterilized forceps
Another animal
Which of the following may be a source of contamination of a disease-free colony?
a.
b.
c.
d.
e.
University veterinarian
Visitors
Physical plant workers
Animal care technicians
All of the above
Module 09 - Pain, Distress and Endpoints
Module Objectives
This module is devoted to the experience of pain and distress, and the approaches and methods we
can use to evaluate that experience in animals. A subsection is devoted to the issue of setting more
humane endpoints so that the pain and distress in invasive experiments is minimized. The anatomy
and physiology of nociception is covered in the following module on analgesia. Nociception is the
scientific term used to describe the neuro-physiology of pain perception, including the nerve pathways
involved with transmitting messages to the central nervous system that are interpreted as "pain".
The objectives of this section are to:
•
•
•
•
•
•
describe the experience of pain in animals
outline the sources of pain
outline how we can obtain evidence of the pain experience in animals
describe stress and distress in animals
outline how we can obtain evidence of the distress experienced by an animal
present some signs of pain and distress in various animal species
Consequences to Animals
As an introduction to this module, the student is asked to think about the consequences of the
following normal activities within an animal facility. Consider each scenario and try to identify why
such common procedures should be stressful, before looking at the explanations.
Cleaning the animal's cage.
Holding the animal.
Turning on the lights in a darkened animal room.
The isolation of social animals will be stressful to them and this may turn to distress with the animals
showing behaviours that are contrary to their best interests. Often these are stereotypies, i.e.,
behaviours without any obvious purpose being repeated over and over again. What might you see in
the following animals?
Rabbit
Mouse
Non-human primate
Pain and Distress in Animals
Introduction
Pain, stress, distress, fear and anxiety are sensory states that people are able to describe. Pain is the
word we use when something hurts e.g., after an injury of some kind, an infection, a headache or
sometimes an emotional event. We feel stressed when we are harried, when there seems to be too
little time to do everything and so many demands are being made on us, or perhaps when you have to
take a course before you can work with animals in your research program!! Usually, we are able to
deal with the situations. However, when the level of stress reaches a point where we are unable to
deal effectively with it, our responses may become counterproductive to our own well-being and we
become distressed. Fear and anxiety are important modulators of the pain experience. Freedom from
fear can reduce the pain experienced in humans and animals, which is why tranquilizers are useful
adjuncts to analgesia or anesthesia.
Definitions and Terminology
The following definitions of distress, discomfort and pain were developed by CCAC based on FELASA
(Federation of European Laboratory Animal Science Associations) definitions.
Distress: Distress is a state associated with invasive procedures conducted on an animal, or with
restrictive or other conditions which significantly compromise the welfare of an animal, which may or
may not be associated with pain, and where the animal must devote substantial effort or resources to
the adaptive response to challenges emanating from the environmental situation.
Discomfort: Discomfort is viewed as a mild form of distress.
Pain: Pain is an unpleasant sensory and emotional experience associated with actual or potential
damage or described in terms of such damage.
Suffering: The term "suffering", as in "pain and suffering" is not used in this module in relation to
animal experiences, because for some animals (the lower vertebrates) that capacity may not be
present in the way that humans perceive it. Instead, the word "distress" is used.
Pain and distress in a research animal may be very difficult to assess. We cannot ask the animal how
it feels, and so must rely on other means of determining the level of pain being experienced by an
animal. Although animals cannot communicate with us verbally and describe their sensations, they
respond to pain, stress, fear and anxiety in ways similar to humans. They may show fear and anxiety
when confronted with a predator, including us, and they show signs of pain when they hurt (e.g., not
using a limb when it has been injured). Novel or strange situations may be stressful, as is isolation for
a sociable animal. A stressful situation that is not resolved may progress to a state of distress. For
example, inappropriate housing conditions that do not permit a normal range of behaviours may result
in the development of purposeless behaviours like pacing or weaving.
Even if there are no obvious signs of pain or distress, we should not assume that the animals are free
of pain or distress. We may simply not be recognising the signs. We are trying to make an
assessment on a totally different species when we often cannot make such an assessment on other
humans.
The challenge for us is to identify the signs that suggest an animal is in pain or distress. And even if
we cannot identify the signs, we should not assume that all is well. Animals, particularly species that
have evolved as prey species, will successfully hide signs of pain and stress if it is not in their best
interests and if they are able. For example, it is difficult for an animal in the wild to disguise the fact
that it has a broken leg but it may disguise other signs of injury or disease to mislead a potential
predator. In the lab, mice that have had surgery to implant ova in the fallopian tubes will behave
normally if observed soon after they recover from the anaesthetic. This is seen even when a gaseous
anaesthetic is used and the animals are mobile within minutes of turning off the gas. However, if the
animals are observed without them knowing, they may be sitting apart from the group, licking or
scratching at the wound, stretching frequently, etc. The signs may indicate that the animals are
having some pain or discomfort.
Pain, stress and distress produce some similar behavioural, physiological and biochemical changes which we know as the "fight or flight" mechanism. These changes are the result of a stimulation of
the hypothalamic-pituitary-adrenal (HPA) axis, and include alterations in blood pressure, heart rate, or
a change in behaviour.
Fear in a cat. Notice the snarl and the flattened ears. This cat is indicating that
it does not want to be touched. Its next action may be to strike out or to flee.
There may be a wide variation in response to a painful or stressful situation in different species and
between animals of the same species. Some animals will vocalize if they are hurt and the sounds will
be quite different from those usually associated with the species. A guinea pig that is hurt will squeal
and try to get away. It will rarely ever bite. A rat, on the other hand, will squeal and try to run away,
but it will also bite. There is evidence that different strains of mice respond differently to painful
stimuli. Furthermore, a strain that reacts at a low intensity for one stimulus may resist a second
stimulus until it reaches a high intensity.
It is also important to emphasize the temporal nature of pain. Pain is not constant or consistent. It
changes all the time both in intensity and form, so the appropriate treatment must accommodate
these changes.
Birds may show acute pain by vocalising and wing flapping. Open-mouthed breathing may occur and
if the pain persists, feathers become ruffled through lack of preening. A decrease in activity and
protection of an injured part may also be seen. In some birds, a state of immobility and
unresponsiveness may develop. Chronic pain is also seen in birds (e.g., heavy broilers with arthritis;
these birds will respond to analgesics by being more mobile and limping less).
Pain in amphibians, reptiles and fish is still a poorly studied subject. However, since they have similar
anatomical, physiological and neurological features to mammals and since they respond to aversive
stimuli, it may be concluded that they feel pain. Some published guidelines on pain and analgesia in
birds and fish are available. The effectiveness of various analgesic drugs in these animals has
received limited study.
Some animals cope with stress better than others. Some coping mechanisms are internal (e.g.,
hormonal responses to stress), while some are external (e.g., somewhere to hide may be the coping
solution when confronted by a predator, including a person). If there is nowhere to hide in a small
barren cage, this coping mechanism may fail to relieve the stress and the animal will move quickly
from being temporarily stressed to being distressed.
The term frustration is often used to describe the reaction of an animal to stressful situations and
conflicts that they cannot resolve. Frustration may develop in a laboratory animal when an
impoverished environment curtails the normal behavioural repertoire, for example. Increased
aggression in pigs and poultry has been ascribed to frustration when these animals are kept in an
impoverished environment. Hungry chickens that were presented with food they could not reach
began a preening procedure that differed from normal preening in its "frantic" appearance. This was
considered to be a sign of frustration in the chickens.
In this module, we will look for evidence of pain, stress and distress from four angles.
1.
2.
3.
4.
Is there any situational evidence that pain, stress or distress could or should exist? Has there
been an injury that makes us believe that the animal must be in pain?
Are there any signs that the animal is behaving in an abnormal manner? Has it stopped
grooming and looking after itself?
Are there physiological changes that suggest the animal is in pain or distress? Has the
respiration rate increased?
Are there biochemical changes that indicate that the animal is stressed, distressed or in
pain? Have cortisol levels increased beyond normal levels?
We should be able to gather evidence from the situation and the animal's behaviour without
interfering with the animal. However, physiological and particularly biochemical evidence may require
restraining the animal and taking blood or other samples. These actions may exacerbate any pain or
distress the animal is experiencing and so the findings must be interpreted with care.
Although pain, stress and distress will be discussed separately, it will soon be noted that many of the
signs overlap. This is to be expected since stress is a common factor in pain and distress. The object
is to provide the student with a framework within which it will be possible to identify factors that could
have a profound effect on experimental results. Even some of the simplest and common procedures
in the animal facility may cause the animal some stress (e.g., changing cages, turning on lights).
Looking for Evidence of Pain in Animals
Situational Evidence
Our own experience with pain and distress can tell us something about an animal's experience,
because there is an evolutionary continuum from animals to man, not only with respect to the
physical, but also with respect to the behavioural and psychological systems. Situations that cause us
pain probably cause pain in animals as well. However we must approach interpretation of animal
thinking and behaviour with caution. This is sometimes called "critical anthropomorphism". Animal
models have been used extensively to evaluate and test drugs with antinociceptive properties, and to
determine the mechanisms whereby pain relief is obtained. This clearly shows that we believe that
animals have the same, or a closely similar, neural substrate subserving pain processes, as do
humans.
Pain is usually associated with physical damage to tissue. Pain is the feeling we experience when pain
receptors are stimulated in the tissues. Most tissues have pain receptors and the density varies from
tissue to tissue. The skin is well supplied with pain receptors while deeper organs tend to have fewer
receptors. Skin incisions are painful, as we know from experience. As a result of tissue damage,
chemicals are released that either stimulate pain receptors directly (e.g., histamine, bradykinin) or
sensitize them to stimulation (e.g., prostaglandin). The brain is believed to be devoid of pain
receptors, so injuries to the brain itself are not painful. However, other tissues within the skull do
have pain receptors (e.g., arteries) and so may cause "headaches".
Traumatic damage to tissues from a surgical procedure is usually easily recognized. It is important to
note that pain and stress can be significantly reduced by good surgical technique and planning so this
should be addressed before rather than after the event. A major factor in perioperative pain is
surgical skill. We should not substitute analgesics for incompetent surgical technique. Trauma may
occur as a result of a slip or a fall and the external evidence may not be so obvious.
Infectious diseases cause tissue damage and the resulting inflammation is part of the body's attempt
to repair the damage. Conjunctivitis, cystitis and mastitis are examples of infectious diseases that are
associated with pain but not all infectious diseases have a pain component. Some immune diseases
may result in tissue damage (e.g., some forms of arthritis).
Compounds injected into animals may cause tissue damage and pain. Some drugs are known to
cause pain when injected. For example, the pH of the solution may not be appropriate. There may be
two components to the pain, one associated with the drug itself and one associated with the injection
of a relatively large volume into a closed compartment for example a muscle bundle. The latter
should not be a factor if the proper volumes for injection are followed. (See www.eslav.org/efpia.htm)
All of these examples provide us with evidence that pain could be expected. However, they do not
give any idea of how much pain is occurring. That depends on a number of factors that vary the
response to pain in different species, different strains, different temperaments, etc.
Pain in Animals: Behavioural Evidence
The most easily acquired evidence of pain in an experimental animal comes from noting changes in
behaviour. Animals change their behaviour in response to pain, particularly pain that persists. The
changes depend on the level of pain, the tolerance of the animal, the species and even strain of the
animal, the situation under which the pain occurs and a variety of other factors. Short-term pain
(e.g., an injection) may be very well tolerated, depending on the site of injection and the substance
injected. There may be no behavioural changes as a result of a subcutaneous injection of a nonirritating material. On the other hand, a subcutaneous injection of an irritating material may result in
the animal scratching at the site of injection. This repeated scratching at a specific site constitutes a
change in behaviour. The same reaction may be seen in animals infected with mites that bite and
irritate the skin.
Severe short-term pain may produce an aversive reaction in an animal. A normally compliant, friendly
animal may attempt to bite someone causing it pain. Alternatively, the animal may attempt to flee
from the cause of the pain.
Chronic or long term pain (e.g., arthritis, orthopedic procedure) is likely to produce more subtle
behavioural changes. Animals and people react to pain through coping mechanisms that are both
internal and external. There are descending pathways from the brain to the spinal cord that will
inhibit neuronal activity and reduce the sensation of pain. In addition, compounds are released that
have opioid effects (e.g., endorphins and enkephalins). These internal reactions to pain will reduce
the external behavioural changes. Animals in pain will often withdraw from their social group,
choosing instead to remain alone, to be less active and less responsive to external stimuli.
Sometimes, persistent pain will cause an animal to traumatize further the area that is hurting. An
extreme form of this is seen in animals that have undergone the severing of nerves to the foot. A
neuroma frequently develops and this causes pain that seems to come from the denervated foot. The
animal's response may include chewing the toes as it attempts to relieve the pain. More often,
persistent pain will result in the animal scratching at the site. In our own experience, a persistent itch
will cause us to scratch repeatedly at the site.
A second complicating factor is that many laboratory animals are nocturnal and so are asleep during
the part of the day when we are working and the lights are on. Again it may be difficult to discern
signs of pain. However, switching off the lights and using a red light to observe them can overcome
this difficulty. Most of these nocturnal animals will become active within a few minutes of the lights
being switched off. Animals that are inactive or those that remain separated from the group may be
in distress or pain. It is possible to see abnormal postures such as hunched back or tiptoe gait that
may also point to pain.
Food and water intake is often altered when an animal is in pain, regardless of the source of the pain.
Because many animals used in science are housed in groups it is difficult to detect these changes.
Loss of body weight can be used as an indirect measure of a decrease in food and water intake.
Animals expected to develop pain can be closely monitored to allow accurate measurement of body
weight.
Grooming is an important activity for animals and a failure to groom is an early sign of pain. The hair
may be standing up (piloerection or staring) rather than lying down smooth. It may be dull rather
than shiny and it may be matted or clumped, particularly around the face and mouth and the anal and
genital openings.
Pain in Animals: Physiological Evidence
Painful experiences stimulate the hypothalamic-pituitary-adrenal (HPA) axis. Many of the physiological
changes seen in pain are a reflection of the release of adrenalin and noradrenalin. Some of these
changes may be observed without handling the animal. Dilation of the pupils and an increase in
respiration rate may be directly observed. Measuring the body temperature, heart rate or blood
pressure in small laboratory animals usually requires some interventions such as restraint or previous
surgery to implant recording devices. In larger animals (e.g., cattle) it is possible to see the pulse in
the jugular vein with each heart beat and so the heart rate may be determined. Palpation of the heart
or a peripheral artery will also provide a measure of the heart rate, which will be elevated in an animal
in pain (and also in a nervous animal). The blood pressure will also be elevated and this may be
measured using a cuff in some animals (e.g., around the tail in small rodents or over a peripheral
artery using Doppler equipment).
The Doppler monitor shown on
the left is of a style commonly
used for larger animals, larger
than a rabbit. An appropriately
sized cuff is placed over a
peripheral artery according to
the instructions from the maker.
The machine may be set to
measure the pressures
automatically at given time
intervals or it may be used
manually.
Physiological evidence of pain is best observed in the early stages after a painful stimulus. As the pain
persists, the changes tend to lessen so that the changes in heart rate, respiration rate etc. are not so
dramatic and may be within the normal range.
Pain in Animals: Biochemical Evidence
Several biochemical changes accompany painful stimuli. However unless the animal has been
previously catheterized so that blood can be collected during the painful stimulus, these changes are
not likely to be observed. Some of the compounds or their metabolites may appear in the urine.
Corticosteroids (e.g., cortisol) and catecholamines (e.g., adrenaline) will be elevated, from stimulation
of the HPA axis. The metabolic rate will be increased and there will be elevation of endorphins and
enkephalins. There are also changes in the immune system associated with pain (and distress), some
of which can be measured. The widespread effects of corticosteroids may be seen in a range of
biochemical parameters, electrolyte changes, metabolic changes, immune factors, etc. This presents
the investigator with the difficulty of separating the normal from the pain-induced abnormal
biochemical changes.
In addition to the four sources of evidence for pain already discussed, other evidence may be useful in
special circumstances. For example if an animal in pain is treated with pain relieving drugs
(analgesics) and its behaviour and physiology return to normal, we feel confident that it was in pain
before.
Summary comment
In coming to the conclusion that an animal is in pain, we need to examine evidence from each of the
areas discussed. One sign alone may be sufficient to identify an animal in pain, but more often we are
trying to identify more subtle signs of pain. In these cases, several pieces of evidence will add up to a
correct diagnosis.
Evidence of Stress and Distress
We will look for evidence of stress using the same four areas we looked at for pain.
1.
Is there any situational evidence that stress could or should exist?
2.
Are there any signs that the animal is behaving in an abnormal manner?
3.
Are there physiological changes that suggest the animal is stressed?
4.
Are there biochemical changes that indicate that the animal is stressed?
Pain is also a stressor, so many of the same signs will be found. However, since there are other forms
of stress than just pain, we need to be able to recognize these also.
Stress and Distress: Situational Evidence
Are there any reasons to expect that the animal may be under stress? Is a social animal (and most of
the animals commonly used in biomedical research are social animals that are most comfortable living
in small groups) being housed alone without contact with its own kind? Often animals are housed
alone as part of an experimental protocol. However, the effect of this must be recognized within the
context of the experiment. It is known, for example that in mice, the growth of some tumours is
increased if the mice are housed singly when compared with housing in groups.
The rabbit is alone in a metal cage, with a wire floor that will
allow feces and urine to pass through to the tray below. The
water bottle will provide the rabbit with one of the few means of
diversion in this otherwise barren cage. It is common for single
rabbits in a cage like this to play with the water bottle, allowing
the contents to dribble through its mouth to the tray below. Pigs
in stalls may also develop this habit.
Is the environment stimulating for the animals or does it provide little opportunity for the animals to
indulge in normal behaviours? A physically impoverished environment, like a socially impoverished
environment is known to be stressful to many species. This is especially so if it is combined with a
restriction in activity.
Are the animals new to their surroundings or have they just been transported? Animals in these
situations often show signs of stress, particularly in their behaviour. And transportation may be as
little as being moved from one room to the next, even within their home cage.
Some experimental manipulations that are not associated with pain may be stressful. Even picking up
an animal in a careless manner or by an unfamiliar person may trigger signs of stress.
Food and water deprivation of even short duration may be stressful to animals used to good supplies
of these nutrients.
Stress and Distress: Behavioural Evidence
Again the stressed animal will show some of the behavioural changes noted in association with pain
although not the pain specific signs (e.g., lameness, protecting an injured part). Stressed animals
may become quiet and unresponsive or they may become hyperactive (e.g., observe the frantic
activity of mice when they are placed in a nice clean cage, free from any odours of their marking). In
some cases, the animals may return to apparently normal behaviour, even though the stressful
situation still persists. This is described as coping and is a response to stress, i.e., the animal tries to
return all systems to normal.
Stress and distress: Physiological Evidence
The physiological changes described under pain are seen in animals stressed by non-painful situations
although the level of change may be less. Again these changes result from stimulation of the HPA axis
with the release of corticosteroids and catecholamines. Changes in heart and respiratory rates, blood
pressure and metabolic rate are all seen. Studies of the immune system have shown changes
including suppression of some elements of this system. This ties in well with the well-recognized
development of disease in stressed animals and people. These changes in the immune system are
being investigated as a subtle indicator of a stress problem when other signs have returned to normal
values.
Stress and Distress: Biochemical Evidence
As indicated above, there are raised levels of corticosteroids and catecholamines in stress. These may
return to normal, even in the continued presence of the stimulus, and so their absence cannot be
considered evidence of no stress.
Distress may be considered to be the state of an animal that has been unable to cope with stress and
where its responses not only fail to alleviate the stress but also are detrimental to the animal's wellbeing.
The behavioural signs noted in stressed animals in the early stages persist. In addition, the animal
develops stereotypies or maladaptive behaviours. A stereotypy is a repeated behaviour that has no
obvious purpose (e.g., the pacing of a tiger in a small enclosure, excessive licking or grooming,
feather pecking in battery chickens, bar chewing by pigs in stalls). Stereotypies should be seen as
indicators of stressful conditions that are causing the animals to be distressed. They are the animals'
attempts to cope with the stress and in most cases they do not cause harm to the animal. In a few
cases, the stereotypical activity may result in physical harm to the animal. For example, excessive
grooming seen in a number of species may result in the formation of hairballs in the animal's stomach
or actual trauma to the skin. This type of stereotypy is often seen when social animals are held in
isolation (e.g., rabbits or non-human primates).
The eating and drinking habits of animals in distress are usually altered. Frequently they refuse to eat
and drink and remain passive and unresponsive to their environment.
The physiological signs of distress are quite variable. They include the signs of stress given above but
modified depending on the success of coping mechanisms. In addition, the changes in behaviour,
particularly in eating and drinking will result in weight and hydration changes, depending on the
duration of the distress. There may be changes noted in some of the formed elements of the blood,
particularly eosinophils, neutrophils and lymphocytes. Distressed animals may be more susceptible to
infectious diseases.
As with the physiological signs, the biochemical signs may be quite variable but are similar to those
seen in stress. Since the HPA axis is intimately involved, there may be changes in adrenal gland
secretions and even exhaustion of adrenal function may occur. Other hormonal changes may occur
(e.g., the estrous cycle may be disrupted in non-human primates).
Signs of Pain and Stress: Summary
The following table summarizes the signs of pain and stress and gives an expectation of their
occurrence. Since pain and stress are seen in different intensities, the animal's response depends on
the species, age, situation and a number of factors. For example, some animals vocalize often as part
of their normal behaviour (e.g., cats) while others seldom vocalize except in extreme situations (e.g.,
rabbits). This table is presented as a checklist of things to look for rather than a definitive or
complete description. Additional information on the species specific signs of pain is available in the
CCAC Guide: (click here)
Sign
Pain
Stress
Protecting injured part e.g., limb, Commonly seen; may be
abdomen
reluctant to move
Seldom seen
Vocalizing
Especially if forced to move
May occur in isolated stressed
animal
Respiration
Rate increased and may be
shallow
Rate may be increased if animal
is also fearful
Attitude
May be depressed and
unresponsive to stimuli
Usually alert and responsive,
sometimes depressed
Food and water intake
Usually decreased
Often decreased; some stressed
animals may overeat
Urination and defecation
Reduced volume and frequency
Both may be increased with
diarrhea sometimes
Appearance
Unkempt, piloerection, reduced
self care
Unkempt, piloerection and
reduced self care
Eyes
May be sunken, occasional
discharge
Discharge especially in rats and
mice
It is our responsibility, when animals are used for research, teaching and testing to ensure that pain is
minimized, including for those studies in which pain may accompany the research being done. We
should seek evidence for pain, stress and distress in animals from several points of view. Situational
and behavioural evidence may be found by observation while physiological and biochemical evidence
requires the collection of information by handling or otherwise manipulating the animal. Since there is
no single parameter that defines any of the states, we must synthesize our opinion based on all
available evidence.
Assessment of Pain and Distress in Animals with a View to
Setting More Humane Endpoints in Invasive Studies
Objectives
The objectives of this section are to:
•
•
•
•
•
define the term "endpoint" in relation to minimising the potential for an animal to experience
pain or distress in invasive studies
present the ethical and scientific concerns important in choosing the appropriate endpoint in a
given study
describe the observations upon which endpoints can be based
make reference to recommended endpoints in the published literature, and provide research
examples
list some questions that can help the principal investigator and the animal care committee
choose the appropriate endpoint in a given invasive experiment, and help ensure that no
animals will go past the endpoint and thus experience unnecessary pain or distress
Introduction
When some level of animal pain or distress may be inherent or unavoidable in a research program in
relation to a condition being studied (like cancer or infectious disease), or as an undesirable side effect
of the procedures, it is our responsibility to minimize that pain or distress. Anticipating the expected
pain and formulating a pain control plan are important. One way of limiting the potential pain and
distress is to decide ahead of time at what point such an experiment will stop, to avoid pain and
distress that would occur if the experiment continued.
When an invasive research project is being planned, the principal investigator should consult the CCAC
guidelines on: choosing an appropriate endpoint in experiments using animals for research, teaching
and testing
http://www.ccac.ca/en/CCAC_Programs/Guidelines_Policies/GDLINES/ENDPTS/APPOPEN.HTM to
ensure that choosing the appropriate endpoint is done in compliance with the CCAC guidelines.
Defining "Endpoint"
For our purposes, the term "endpoint" can be defined as the point at which an experimental animal's
pain and/or distress is terminated, minimized or reduced by taking actions such as humanely killing
the animal, terminating a painful procedure, or giving treatment to relieve pain and/or distress.
The general guideline in the CCAC endpoints guidelines CCAC guidelines on choosing an appropriate
endpoint in experiments using animals for research, teaching and testing states: "In experiments
involving animals, any actual or potential pain, distress, or discomfort should be minimized or
alleviated by choosing the earliest endpoint that is compatible with the scientific objectives of the
research. Selection of this endpoint by the investigator should involve consultation with the laboratory
animal veterinarian and the animal care committee." This statement acknowledges that there are
both ethical and scientific considerations that go into finding an appropriate endpoint. Arriving at that
decision involves the researcher and the people at the institution given the responsibility for acting on
behalf of the animals used in research teaching and testing.
The various stages of an animal's condition in an invasive experiment can be depicted on a scale from
"normal" through "moribund" to death at the other end of the scale.
When a procedure affects an experimental animal, its condition starts to change from being a
"normal" healthy animal. In studies that involve infection, cancer, or arthritis, for example, as the
condition progresses there may be increasing pain and distress. Eventually the condition of the
animal may reach a point where it becomes obvious that unless action is taken to terminate the
condition, the animal will go on to die. This point on the scale is called the "limiting clinical signs".
One example of this comes from the regulatory safety testing literature involving the rabies vaccine
challenge test in mice (used to test the efficacy of a batch of rabies vaccine). The traditional endpoint
for this test has been death in the control animals (and perhaps in some dilutions of vaccinated
animals). Using a 4 stage clinical scoring system:
Score 1: ruffled fur, hunched back; Score 2: slow movements, circling plus >15% weight loss; Score
3: trembling, shaky, convulsions; Score 4: lameness, paralysis, permanent recumbency;
The researchers found that all mice progressing to score 2 went on to die. These observations were
the most significant predictors of further deterioration in the animal's condition, and a score of 2 was
the earliest point at which those signs appeared. Therefore the experimental endpoint could be set at
a score of 2 rather than waiting until the mice died, without affecting the outcome of the test.
Cussler, Morton, Hendriksen 1998. Humane endpoints in vaccine research and quality control, Proc
Int Conf, Nov 1998. (click here for: cussler.pdf )
When the cardinal signs of the condition are unknown, a pilot study using a few animals under close
observation may provide the information to allow the earliest endpoint to be found.
Ethical and Scientific Concerns in Choosing an Appropriate
Endpoint
The above example also raises the issue of the balance between scientific concerns and ethical ones in
choosing an endpoint. Selection of an endpoint by the investigator is important because he/she has
defined the scientific objectives, and if those are not met because an experiment was terminated too
early, then the study and the animals' lives are wasted. The endpoint should not change the outcome
or invalidate the results. The objective should be to have a scientifically valid experiment, while at the
same time holding any pain and distress to a minimum.
Which Observations of Behaviour and Physiology are Best for Selecting the Endpoint?
There is no single answer for this question. Each research project where an endpoint is defined to
minimise animal pain and distress will probably have a distinct set of observations needed to
accurately identify the animal whose condition has progressed to the endpoint. A study of the pain of
castration in lambs will use different observations than a study of bacterial infection in mice.
Nevertheless the approach to making (and recording) the observations will be much the same.
For most studies, there are five areas in which observations of the animal should be made:
•
•
•
•
•
external physical appearance
changes in behaviour (both when the animal is at rest and when it is stimulated)
changes in body weight (and related changes in food and water intake)
body temperature
changes in clinical signs (e.g., heart rate, respiratory rate, etc.).
Of these, measuring and recording body weight and body temperature should be considered for
almost every endpoints assessment.
A scale can be set up for each observation (for parametric signs) with increasing changes from normal
identified, and tracked by using a checklist to record the observations. That way the changing
condition of the animal can be followed from one observation time to the next. This approach also
helps ensure that the observations are as objective as possible. An endpoint can be then be pre-set;
that point when the scoring of the animal's condition has reached the endpoint.
There are two types of observations that can be made:
1.
Changes from normal (also called parametric signs) on a continuous scale from normal. These
observations include: body weight; body temperature; heart rate; respiratory rate; level of
activity or other behaviour. For example a scale could be set up for body weight as normal,
10% weight loss, 20% weight loss; 30% weight loss.
Some of the technologies that assist in the recording of these important observations include
infrared thermometers, implantable physiological recording and telemetry microchips,
computerised activity monitors, etc.
2.
Presence or absence of signs (also called non-parametric signs). Signs such as ruffled coat,
closed eyelids, nasal discharge, limping, hunched, recumbent, circling, vocalisation, self-
trauma, diarrhea, dyspnea, seizures could be recorded as being present or not. Viewing the
recording sheet over several observations would reveal if an increasing number of signs were
present.
To be effective a checklist should be specific for each experimental protocol, for each species, and
strain, should list all the common and the cardinal signs in order of observation, and each sign scored
as present or absent, or degree change from normal. The CCAC endpoints guidelines provide
additional information on the process for developing such checklists. (CCAC guidelines on: choosing
an appropriate endpoint in experiments using animals for research, teaching and testing - click here )
Using observational checklists to track the condition of patients has been used in human medicine for
some time. The APACHE II scoring system (Acute Physiology and Chronic Health Evaluation) for
evaluating seriously ill people in critical care has a scale from "0" being normal to "+4" indicating an
observation that is significantly changed from normal. The evaluation is made for more than ten
parameters. (Click here for: APACHEII.pdf
)
A Sample Clinical Scoring Checklist
The checklist for the clinical and physiological observations made in an acute pneumonia model in
cattle used to develop vaccines, included:
•
•
•
•
a rhinitis or nasal score (0 normal to 4 very severe rhinitis),
a respiratory distress score (0 normal to 4 very severe respiratory distress),
a depression score (0 normal to 4 moribund),
and a strength score (0 normal to 4 recumbent, unable to rise).
In addition temperature and body weight were recorded. When an animal reached an overall sickness
score of 3, that was set as the endpoint and the animal euthanised. This choice of endpoint was
based on early experience with the model.
Some Endpoints Recommendations from Published
Guidelines
Body Weight Changes
The rate, duration and extent of weight loss are all important. A weight loss of 20% is considered an
endpoint, in the CCAC endpoints guidelines. Similarly the UK Coordinating Committee on Cancer
Research (UKCCCR) Guidelines for the Welfare of Animals in Experimental Neoplasia (click here
)
recommend that weight loss exceeding 20% should be one of the endpoints in cancer research. A
number of other endpoints for animal models of cancer research are also presented in this
publication. For example, the total mass of the tumour should not exceed 10% of the normal
bodyweight for that animal. (CCAC guidelines on: choosing an appropriate endpoint in experiments
using animals for research, teaching and testing - click here )
Body condition scoring may be necessary to identify loss of body condition in cancer studies where the
general weight loss may be cancelled out by the growth of the tumour.
Body Temperature Changes
A number of studies have shown that in mouse models of infectious disease (bacterial and viral),
animals whose body temperature dropped more than 6°C went on to die, and so this would be one of
the recommended endpoints in studies of this nature.
Change in Activity Level
Lethargy, depression, and sleepiness accompany many disease conditions (produced in part by the
actions of cytokines released during the acute phase response). In rodents observing this decrease in
activity may require observing them during the dark phase of the room's lighting system. This can be
mimicked by turning the room lights off and observing the activity level under red light (the red light
test).
How Often Should the Observations be Made?
The CCAC endpoints guidelines recommend a minimum of two or three observations each day during
critical periods, and more frequently if necessary to ensure that no animal's condition progresses past
the set endpoint. In pilot studies, continuous monitoring using video equipment can be helpful in
identifying critical times.
Assisting the Principal Investigator, the Animal Care Committee and the Veterinary and
Animal Care Staff in ensuring that CCAC Guidelines on Endpoints are satisfied.
The challenges for principal investigators include: setting the earliest scientific endpoint possible;
defining limiting clinical signs; using best technologies for obtaining necessary observations. The
challenge for animal care committees is to balance the requirements for good science, with the
responsibility to minimise pain and distress. This would include pressing for earlier, data driven
endpoints whenever possible. The challenges for veterinary, animal care and research staff include:
ensuring careful, objective monitoring of all animals; documenting observations made; identifying
animals nearing pre-determined endpoints.
To help meet these challenges, asking the following questions will hopefully generate the discussion
needed to meet our collective responsibilities in ensuring that experimental animal pain and distress
are minimised in invasive studies.
What are the scientific justifications for using the proposed endpoint?
Scientific justification for a proposed endpoint is related to the goals of the project. The work must
have scientific merit, and the endpoint must fulfill the scientific requirements. Scientific justification
should not rest wholly on comparison with published data, as this does not permit refinement of
endpoints. A pilot study might be used to compare a new scientifically justifiable endpoint with data
from a previous study using an older, later endpoint.
There may be some studies in which going beyond normally accepted endpoints could be scientifically
justified, for example, cancer treatments or treatments for other serious diseases. For these to be
acceptable from a welfare point of view, the animals would need to be treated as if they were in
intensive care, and provided with all possible measures to alleviate their pain and distress while
allowing the study to proceed.
What is the expected time course for the animals, from the initial treatment to first signs of
pain/distress to the death of the animal, based on previous information with the specific
model under study?
When this knowledge is not available from previous studies, a pilot study with a few animals might
provide a means of assessing the time-course of events during a study, to predict the time at which
the effects on the animals are the most severe, and the times at which the animals need the most
careful monitoring. This information is needed to decide when the most intensive monitoring of the
animals should take place so that the endpoint is reached when the relevant personnel are present
and can terminate the experiment
When are the effects to the animal expected to be the most severe?
Knowledge of the time course of the development of a tumour, or infectious disease can also assist in
determining when the animals require the most attention. Conditions that are acute, i.e., that
progress to severe dysfunction or death in a short period of time, are of particular concern.
Providing special care for animals whose condition is severely compromised would help ease the pain
and distress they experience.
If the course of the disease and expected signs of the adverse effects are unknown, could
an initial pilot study, under close observation by the investigator and/or laboratory animal
veterinary staff answer these questions?
Observation by the investigator should ensure that the necessary scientific objectives are being
reached, while the laboratory animal veterinary staff can provide the expertise with regard to clinical
signs of pain and/or distress.
Has a checklist of observations, on which the endpoint will be based, been established?
If not, the pilot study affords the opportunity to compare observations of control animals with treated
animals and to identify indicators that can be used to establish the earliest possible endpoint.
Who will monitor the animals (identify all responsible) and keep records?
All the persons involved in the care and monitoring of the animals should be identified at the outset,
and must be skilled at recognizing signs of pain and distress.
Has a clear chain for reporting observations been established?
It is crucial that the individual(s) responsible for monitoring the animals have a clear reporting line so
that the individual with responsibility for deciding on the termination of the experiment is informed
promptly of changes in the animal that indicate the selected endpoint is imminent.
What will be the frequency of animal observations: a) during the course of the study; and
b) during the critical times for the animals?
The animal care committee must be assured that the animals are going to be monitored with sufficient
frequency to enable the staff responsible to identify any animals approaching the endpoint.
Do the investigator(s), animal care and technical staff have the training and expertise to
monitor the animals adequately?
The animal care committee must also be assured that the individuals responsible for monitoring the
animals have the training and experience to do the monitoring.
What provisions have been made to deal with any animals that show unexpectedly severe
signs and symptoms?
Provision should always be made to deal with unanticipated pain and/or distress.
For toxicological studies, have existing toxicological data been evaluated?
Data mining: It may be possible to predict clinical signs from background data or databases for similar
chemicals or substances. Information from human or veterinary clinical practice with similar
substances may also be useful.
Summary
As noted in the CCAC endpoints guidelines, in experiments involving animals, any actual or potential
pain, distress, or discomfort should be prevented, minimized or alleviated by choosing the earliest
endpoint that is compatible with the scientific objectives of the research. Any distress or pain
experienced by the animals in the course of biomedical research, teaching and testing, that is not
necessary to achieve the scientific objectives of the research should be avoided. That is our ethical
responsibility.
Sample Questions
Which one of the following best describes the typical physiological response to stress or pain?
a.
b.
c.
d.
Decrease in body temperature;
Increase in heart rate;
Decrease in respiratory rate;
Immune system stimulation.
Repetitive non-productive behaviors called stereotypies may occur in which one of the following cases?
a.
b.
c.
d.
e.
The animal has just been moved to a procedures room;
The animal is bored or frustrated;
The animal has been restrained for injection;
The animal has just been weaned;
After the animal has been anesthetized.
Which one of the following statements is not correct?
a.
b.
c.
d.
e.
Scientific endpoints for a study should be reached before humane endpoints.
Appropriate endpoints may be exceeded if another method of reducing pain and distress is
employed, so that useful scientific data may be collected and animals will not be wasted.
Pilot studies, using small numbers of animals, should be used to define suitable humane
endpoints if the outcome of a new procedure is unpredictable.
Pilot studies should be used to identify technical problems that may develop in a novel
procedure.
Pilot studies need not be reviewed by the institutional Animal Care Committee because very
few animals will be used.
Module 10 – Analgesia
Objectives
The objectives of this module are:
•
•
•
•
•
•
to review pain in animals
to define analgesia
to define nociception
to outline the anatomy and physiology of nociception
to discuss the major groups of pain relieving drugs; their properties, limitations and side
effects
to discuss the legal requirements in using controlled drugs for analgesia
What is not in this module?
Drug Doses: Drug doses will not be discussed within this module, because it would have required the
consideration of every possible circumstance, every species and strain. However, the investigator
should be aware of the mode of action of the drug to ensure unacceptable complications are not added
to the study. The investigator is advised to consult a veterinarian about specific drugs for pain relief.
As a prelude to this module and as a reminder of some of the material covered in Module 5 on pain in
animals, four questions are posed here to introduce some of the important themes in the module.
The student should consider the questions before clicking the link to the commentary.
Which of the following conditions may be accompanied by pain?
a.
b.
c.
d.
e.
Surgical incision site
Arthritis
Eye infections
Abscesses
Gastrointestinal upsets
Which of the following signs are often associated with pain in animals?
a.
b.
c.
d.
e.
f.
Reluctance to move
Reduced food intake
Failure to groom adequately
Ocular discharge
Lameness
Isolation from the group
We should try to relieve pain in animals because:
a.
b.
c.
We expect to have our own pain relieved
Animals feel pain
Pain has an effect on many body systems and so may distort research
d.
e.
results
We have an obligation to avoid causing unnecessary pain and suffering to
animals
The Canadian Council on Animal Care Guidelines require the use of
analgesics
Which factors influence the choice of drug to relieve pain?
a.
b.
c.
d.
e.
The
The
The
The
The
severity and duration of the pain
duration and effectiveness of the drug
most effective drug with the fewest side effects
least stressful effective route of administration for the animal
most effective drug that does not interfere with the study.
The four questions above represent different aspects of pain control in animals. Pain may originate
from a variety of sources and while the pain of surgical wounds may be the most common source, we
should not forget other causes. The signs of pain will vary from species to species and with the source
of the pain. Highly localized pain may cause signs restricted to that locality e.g., the signs of pain
from a sore foot may be restricted to the same leg while a generalized infection will provide more
widespread evidence of pain.
Introduction to Analgesia in Experimental Animals
We have an obligation to reduce or abolish pain in animals whenever it occurs and particularly if it
occurs in research, teaching or testing.
Many investigations that use animals for research and testing have the potential to cause pain or
suffering that should be alleviated. When studies may result in pain, the principal investigator should
include a plan of action to use analgesic agents, including contingency plans for unexpected events.
Veterinary expertise should be consulted.
Some experimental situations are obvious candidates for pain therapy. Pain occurs following most
surgical procedures, but the severity depends on the invasiveness of the surgery, the organs involved,
post surgery complications, etc. In some cases, the pain is of relatively short duration in the
immediate post-operative period, while in other cases pain may last considerably longer.
For example, animal models of arthritis should be considered painful as the human diseases that they
model are known to be painful. Similarly, some forms of cancer in humans are painful, particularly
those that are found in bone. However, even solitary tumours may induce pain, depending on their
site. Those that ultimately ulcerate through the skin may have been causing pain for a considerable
time.
Infectious diseases result in a range of pain and distress symptoms. Fever and pain in muscles and
joints, "flu-like" symptoms, may be experienced in a variety of viral infections. Gastrointestinal
diseases, urinary tract diseases, hepatitis, peritonitis, respiratory diseases, abscesses, conjunctivitis,
may all be painful. Antibody-antigen interactions, as in allergies or following immunization, may be
itchy or painful.
The examples given above provide concrete indications for pain therapy. In very painful states, this
may include the use of the most potent analgesic agents, the opioids. However, if we consider our
own situations, there are also less serious conditions that have us reaching for the medicine cabinet.
We are used to thinking of pain relief in terms of a single analgesic. However, as we learn more about
the nature of pain, it is clear that effective pain control may depend on using a number of drugs. Pain
can also vary with time, and the pain treatment should match the situation. This may involve using
different drugs at different times in the course of treating pain in an animal. The usefulness of local
anesthetics in providing short-term pain control should not be forgotten.
In addition, we should examine the timing of analgesic administration. The use of analgesics before
surgery has been reported to lessen the need for post-surgical pain control although there is still some
controversy over the effectiveness of this approach.
For the most part we are the cause of pain and distress in experimental animals and we should
alleviate that pain and distress as completely as possible using all the tools at our disposal.
Concern Over The Impact Of Analgesics On Research Results
Although some investigators may worry about the effects of pain relieving drugs on their experimental
model, the effects of the pain and distress that the animal may suffer are of greater concern. The
physiological and neuroendocrine effects of unrelieved pain and distress may be extensive. More
consistent results will probably be obtained from an experimental model when the animal is not
experiencing pain and distress. It is a moot point then, whether the risk of side effects from drugs is
a greater threat to the experimental protocol than the problems associated with extensive
pathophysiology. Further, the side effects of most drugs are quite well known and with careful
selection acceptable pain relief can be achieved with a minimum of interference with the investigation.
Analgesia in Laboratory Animal Models
Analgesia Means "Without a Sensation of Pain"
Analgesic drugs, with the exception of local anesthetics, usually do not completely abolish painful
sensations but reduce the intensity of the pain and make it tolerable. Nearly everyone has taken an
analgesic for a painful condition (e.g., severe headache; after a tooth extraction) and has found the
pain diminished and more tolerable. The same effect probably occurs in animals although it is difficult
to be certain of this.
The approach to pain therapy should be a balance between providing the animal with adequate pain
relief without causing the animal further problems. Pain relieving drugs have side effects that must be
considered. For example, under certain conditions, many non-steroidal anti-inflammatory drugs
(NSAIDs) may cause gastric ulcers and renal damage. Many people are unable to take pain-relieving
doses of aspirin for this reason and this has fuelled the search for new NSAIDs.
Pre-emptive Analgesia
There is increasing evidence that the administration of analgesics prior to surgery reduces the need
for analgesics in the post-surgical phase. This information has come primarily from the human field
where subjective information on pain is easily obtained. (Woolf and Wall 1986, Lascelles et al. 1995
References from Pain Management book chapter 5) Lascelles et al (1997) and Gonzalez et al. (2000
Pain 88: 79-88) found a similar effect in animals. There is some dispute about the effectiveness of
pre-emptive analgesia in man and several reports have been unable to demonstrate any significant
reduction in the post-surgical need for analgesics. (Katz, J. Eur. J. Anaesthesiol Suppl 1995 10:8-13
Pre-emptive analgesia: evidence, current status and future directions)
Administering Analgesics to Laboratory Animals
Oral administration of drugs in food or water may seem attractive as there will be minimal
interference with the animal. However, it does depend on the animal eating or drinking a required
amount to obtain sufficient drug and this does not always happen. The taste of the drug may
discourage the animal from eating it. Water consumption in normal rodents is quite variable and so
accurate dosing of any compound in drinking water is difficult. It should be remembered that some
analgesics, particularly the opioids, suppress appetite.
Although each class of analgesic drug is considered separately, this does not mean that only one
should be used at a time. However, care must be taken to ensure that there are few or no reactions
between the drugs that would either increase the side effects or diminish the analgesic effects.
There are many factors to be considered in choosing an analgesic drug including those listed below.
Many of these factors will be discussed in greater detail in the ensuing text. However, each situation
should be evaluated separately and a decision made to minimize the pain experienced by the animal.
•
•
•
•
•
•
•
•
•
Species
Cause of the pain
Severity of pain
Route of administration
Volume required
Duration of effect
Potential side effects of drug, good or bad
Effects on study
Effects of administration on animal
Pain: Sites of Origin, Pathways, and Neurotransmitters
The precise mechanisms of pain appreciation in the central nervous system have not been fully
elucidated although much is known of the pathways involved. Some of the transmitters and receptors
that play an important role in pain have been identified. This has led to the development of drugs that
are designed to interrupt pain pathways at specific sites, usually by occupying receptors and blocking
the signals. Interruption or modification of pain transmission can occur at several sites and employ
different classes of drugs. This opens up the possibility that optimal pain control may require more
than one analgesic. This approach has been adopted to control pain in some human patients, and
should be discussed with the veterinarians in cases of pain in laboratory animals too.
A detailed description of the pain pathways of the central nervous system is beyond the scope of this
module. It would suffice to say that there are three primary sites at which modification of pain
transmission can occur: the periphery; the spinal cord; the cerebrum. Most drugs have actions at
more than one site.
At the periphery, the responsiveness of pain receptors is enhanced by the presence of prostaglandins.
These prostaglandins are formed in response to tissue trauma. This means that the receptors will
respond to a lesser stimulus than before they were sensitised. A number of endogenous compounds
(e.g., histamine, serotonin) may be responsible for the actual pain sensation.
In the spinal cord, information on pain is received by cells in the dorsal horn and is passed on to
higher centres in the brain along tracts in the spinal cord.
Pain fibres coursing into the cerebrum may end in a number of sites, particularly the reticular
formation, the thalamus and the cerebral cortex. In the reticular formation, the pain stimuli may
evoke arousal, changes in heart rate, blood pressure, respiration and other activities. It is in the
thalamus and cerebral cortex where the appreciation or conscious awareness of pain is to be found.
Neural Pain Pathways
The diagram above also shows pathways coming from the brain down to the spinal cord. Stimulation
of these descending pathways can reduce and even abolish some forms of pain. The body also
produces chemicals including endorphins that act on the same receptors as externally administered
opioids, to provide pain relief. The significance of descending inhibitory pathways and chemicals in
the control and modification of pain sensations is unclear, particularly in animals. Many animals (and
humans) appear to tolerate pain and show very few behavioural alterations following a painful insult.
This may be due in part to the central inhibitory effects and in part to other biological factors. We
must accept that if pain relief is required for a human an animal should have pain relief for the same
problem.
Post-traumatic Hypersensitivity (Windup)
It has long been recognised that trauma to an area is often accompanied by an area of
hypersensitivity surrounding the trauma. This is sometimes called secondary hyperalgesia to
distinguish it from the increased sensitivity to pain within the trauma area called primary
hyperalgesia. Hyperalgesia is recognised by an increase in pain produced by stimuli at the threshold
for pain or by a decrease in the pain threshold in that area.
Windup
Following an injury, dorsal horn cells are bombarded by stimuli originating from pain receptors. Over
a period of time, the receptive field of these cells increases. While it is likely that many chemical
processes are involved in central sensitisation, N-methyl D-aspartate (NMDA) receptors are thought to
be key in the process. This process of increasing central sensitisation of dorsal horn cells has been
called windup.
Analgesic Drugs
There are three major groups of analgesic drugs:
•
•
•
Opioids
Non-steroidal anti-inflammatory drugs (NSAIDs)
Local anesthetics
There are also several useful drugs that do not fit into these groups:
•
•
Alpha-2 adrenergic receptor agonists
N-methyl-D-aspartate (NMDA) receptor antagonists
Opioids
Drugs of this classification act on one or more of the principal receptors and their subtypes in the
brain. The activity is either stimulation (agonist), partial stimulation (partial agonist) or blocking
(antagonist) of the receptor. Stimulation of some receptor types may produce unwanted side effects
(e.g., respiratory depression) and so the goal of manufacturers is to produce opioids that give good
analgesia without the side effects. Some of these drugs are described as agonist/antagonists,
indicating that they stimulate some receptors while inhibiting others.
One group of opioids, the opioid antagonists, is important for its ability to reverse most of the effects
of opioids.
Effects of Opioids
The effect of opioids on any particular system varies markedly depending on the drug and the species
of animal. The following effects are seen to a greater or lesser degree with all opioids.
Sedation: Opioids produce sedation in some species but in others may cause increased activity,
especially with higher doses (e.g., cats may become excited if given high doses of morphine and rats
may show increased activity including pica with higher doses of buprenorphine).
Cardiovascular effects: Usually there is slowing of the heart (bradycardia) and a fall in blood
pressure (hypotension). Dilation of peripheral blood vessels, as well as changes to the heat regulation
centre in the hypothalamus, may contribute to heat loss and result in hypothermia.
Respiratory effects: Respiration is usually depressed through reductions in respiration rate and tidal
volume with a decrease in the respiratory centre sensitivity to carbon dioxide. Some opioids inhibit
ciliary activity in the trachea and this may be significant for an animal recovering from anesthesia.
Gastrointestinal effects: Gastrointestinal propulsive movements (peristalsis) are reduced. Appetite
is suppressed and animals may be unwilling to eat.
Immune system effects: Opioids have been shown to have subtle effects on the immune system
(e.g., suppression of cytotoxic activities of natural killer cells).
Analgesia and pain tolerance: This is due primarily to stimulation of µ receptors although other
receptors are known to have analgesic effects. All of the commonly used opioid analgesics are µ
receptor agonists or partial agonists.
The effects of opioids described above do not represent any single drug or animal species. There is
considerable variation between species and among the different opioids. For this reason, it is
important to consult with a veterinarian.
As with other drugs, the actions described are for normal healthy animals. Pre-existing conditions
may increase the level of the responses and may result in severe reactions in the animal. Care should
be taken to identify any potential side effects in compromised animals.
Summary of Opioid Effects
Analgesia
For moderate to severe pain
Nervous
system
May cause deep sedation in some species,
hyperactivity in others
Cardiovascular
Slowing of heart; vasodilatation with increased heat
loss
Respiratory
Reduced rate and tidal volume; depressed ciliary
activity in trachea
Gastrointestinal Decreased activity in gastrointestinal tract; appetite
suppression
Immune
system
May affect natural killer cells
Other
Abnormal behaviours; metabolic and endocrine effects
Duration of Action of Opioids
The effects of opioids are quite short in animals following a single injection (one to three hours for
most drugs, with species variation). Buprenorphine may provide effective analgesia for a longer
period of time.
Routes of Administration of Opioids
Opioids are usually given by subcutaneous injection but may be given by other routes, including
intravenously. Patches impregnated with the opioid fentanyl are available to provide continuous
absorption of the drug through the skin over a prolonged period. Epidural use of opioids provides
prolonged analgesia in some species, including man, without the usual side effects. Epidural
administration of drugs is difficult in small animals. (Link to diagram of epidural injection). Opioids
may also be given orally, but their effectiveness is lessened by the degree of metabolism associated
with the first pass through the liver. Buprenorphine has been incorporated into instant jelly powder
such as Jell-O® for administration to rats.
Opioids in Combination with Other Drugs
Opioids are sometimes administered in combination with other drugs and the combined effects may or
may not be beneficial. For example, fentanyl may be combined with the tranquilising drug droperidol
or fluanisone to provide short-term anesthesia. However, the effect of the fentanyl is of much shorter
duration than the tranquilliser and so the animal may remain sedated for quite a long time but not
have the benefit of the analgesia. Care should be taken when giving an opioid with any other drug
which depresses respiration or encourages heat loss because the effect of the two will be more
profound than for either alone.
Hypnorm© combines the potent opioid analgesic fentanyl with the tranquillizer fluanisone.
This combination will permit short surgical procedures in some laboratory animals.
Adverse Reactions Caused by Opioids
The most life-threatening effects of opioids involve the depression of the respiratory and
cardiovascular systems. These are particularly important if the opioids are being used in conjunction
with other drugs known to have similar effects (e.g., anesthetic agents). The loss of body heat that
occurs when opioids are used may further compromise the respiratory and cardiovascular systems and
for this reason, animals should be kept warm when they are recovering from anesthesia, with or
without opioids. Some animals respond to opioids with excitation, rather than sedation. In these
species (e.g., swine, sheep) the occurrence of convulsions would also represent a life-threatening
situation.
Specific antagonists to the opioids may be used to counteract the respiratory and cardiovascular
depression. Naloxone, for example, may be used but the analgesic effects of the opioids will also be
reversed.
Examples of Opioids
Click on drug for more details
Butorphanol
Buprenorphine
Partial agonist
Partial agonist
Buprenorphine (brand names include Buprenex©; Temgesic©) is a partial agonist that
provides longer pain relief in animals than most opioids.
Butorphanol (brand names include Torbugesic ©) is a partial opioid agonist.
Non-Steroidal Anti-inflammatory Drugs (NSAIDs)
NSAIDs reduce pain by interfering with the production of prostaglandins from arachidonic acid.
Prostaglandins, produced at a site of inflammation, sensitize pain receptors in the area.
NSAIDs block prostaglandin production from arachidonic acid through the inhibition of the enzymes
cyclo-oxygenase 1 and 2 (COX-1 and COX-2). COX-1 occurs normally in the body e.g., in the
stomach and kidney, while COX-2 is produced at sites of inflammation. It is desirable to inhibit COX-2
only but most of today's NSAIDs block both enzymes, leading to some undesirable side effects. There
are now some selective COX-2 inhibitors on the market.
Effects of NSAIDs
NSAIDs provide analgesia for mild to moderately painful conditions, although their effect on visceral
pain is considered to be poor. As with the opioids, different animals react differently to NSAIDs.
NSAIDs have two other desirable effects, namely they reduce fever and inflammation. These effects,
along with fewer side effects than the opioids, have led to the widespread use of NSAIDs for treating
acute or chronic pain (e.g., headaches or arthritis).
There are few effects on the cardiovascular or respiratory systems.
Gastric ulceration is possible with most NSAIDs especially when given orally. This effect is least with
p-aminophenol derivatives of which acetaminophen (Tylenol©) is the best known. Acetaminophen has
the least anti-inflammatory activity of the NSAIDs.
Interference with platelet aggregation occurs with all NSAIDs. It persists for the life span of affected
platelets following acetylsalicylic acid (aspirin) administration. This effect can result in prolonged
bleeding but it is useful in reducing intravascular clotting in some studies.
Interference with prostaglandin mediated renal function occurs because the NSAIDs block
prostaglandin production in the kidneys (and in the gastric mucosa) where prostaglandins are
important for normal function. Animals with already impaired renal function may be pushed to renal
failure following the administration of NSAIDs in the event of fluid or blood loss.
Summary of Actions of NSAIDs
Analgesia
Effective for mild to moderate pain
Nervous
system
Should not cause sedation in normal doses
Cardiovascular Few effects on heart and blood vessels; affects
platelet aggregation
Respiratory
Few effects; overdoses of some may lead to acidosis
Gastrointestinal
All have potential to cause ulceration
Immune
system
Anti-inflammatory effects may inhibit antibody
production
Other
May be nephrotoxic; hypersensitivity may occur;
metabolic effects
As with other drugs, the actions described are for normal healthy animals. Pre-existing conditions
may increase the level of the responses and may result in severe reactions in the animal. Care should
be taken to identify any potential side effects in compromised animals.
Duration of Action of NSAIDs
The duration of action depends on the effect being considered and the specific NSAID. For the more
commonly used NSAIDs (e.g., flunixin, ketoprofen, meloxicam) the analgesic effect may last for about
24 hrs. There are species differences also in the excretion of NSAIDs and so veterinary advice should
be obtained before long-term therapy is initiated.
Routes of Administration
NSAIDs may be given by oral, intramuscular and subcutaneous routes. It is often convenient to give
these drugs to small animals in their drinking water. However, in some cases, the taste may
discourage the animals from drinking normally and so the required dose may not be ingested.
NSAIDs in Combination with Other Drugs
NSAIDs are seldom given in combination with other drugs in animal care although there are some
products available for human use (e.g., acetaminophen plus codeine for more severe pain or
acetaminophen plus pseudoephedrine for cold symptoms). However, care should be taken when
giving an NSAID with some other drugs that have complementary effects. For example, NSAIDs may
cause gastric ulceration and this will be made worse if the animal is receiving a corticosteroid at the
same time. Similarly, the use of an NSAID at the same time as other anticoagulation therapy may
result in unexpected bleeding.
Adverse Reactions to NSAIDs
The major adverse reactions to NSAIDs given at therapeutic doses come from their ability to cause
gastric ulceration and kidney toxicity. This varies from drug to drug and from animal to animal.
Animals with gastric ulceration may show signs of abdominal pain and have blood in their feces,
however these signs may not be obvious so appropriate monitoring is necessary. These problems are
more frequently seen with prolonged administration of NSAIDs. Overdoses of NSAIDs may also
include respiratory signs and persistent bleeding due to platelet inhibition.
Examples of NSAIDs
Click on drug for more details
Ketoprofen
Flunixin
Banamine© contains flunixin as its active ingredient.
Local Anesthetics
Local anesthetics provide pain relief by blocking pain stimuli from reaching the central nervous system
(brain and spinal cord). They differ from the previously discussed opioids and NSAIDs in that they
abolish pain rather than diminish it and make it more tolerable. However, they are limited in their
usefulness by the need to reach the activated pain receptors or the nerves leading from these
receptors to the brain or spinal cord. An advantage of local anesthetics is their local activity with few
systemic effects. This allows for local pain relief without distorting other physiological systems that
could interfere with the experiment.
The primary use of local anesthetics in analgesia therapy is in the relief of pain in the skin. Wound
edges infiltrated with local anesthetics provide relief for up to about six hours after surgery, the time
over which the acute pain of surgical wounds is at its greatest and when there is the greatest need for
pain relief. Local anesthetics may also be infiltrated around nerves if the opportunity arises.
Intercostal nerves, for example, may be blocked to relieve pain following a thoracotomy and to permit
better respiration.
Summary of Actions of Local Anesthetics
Analgesia
Block pain transmission at receptors and along nerves
Nervous
system
High doses may cause convulsions
Cardiovascular High doses may affect the heart and lower blood
pressure
Respiratory
No effects if respiratory nerves are not blocked
Gastrointestinal
No effects at normal doses
Immune
May impede phagocytosis
Duration of Effects
Local anesthetics last for up to six hours, depending on the agent. The effects will be prolonged if the
preparation included epinephrine. Topically applied local anesthetics may last for less than one hour.
Routes of Administration
Local anesthetics may be given by several different routes depending on the objectives. Local
infiltration may be used to block receptors in the skin and underlying tissues, or to block nerves
coursing through the area. In the latter case, anesthesia (and paralysis) is provided at a site remote
from the injection site. Topical application on mucous membranes or subcuticular structures will
effectively block receptors in these areas. For example, local anesthetic sprays are used to block
receptors on the vocal cords to prevent spasm during endotracheal intubation. When given into the
spinal epidural or subarachnoid spaces, all activity in the nerves will be blocked, so that there will be a
loss of sensation and a loss of motor function.
Local anesthetics are poorly absorbed through intact skin. One preparation that is absorbed through
skin is a mixture of prilocaine and lidocaine, however absorption is relatively slow and anesthesia is
limited to about five millimeters under the skin. This is enough to anesthetise the cutaneous
receptors. The preparation requires a minimum of 30 minutes to penetrate the skin and will require
longer to achieve maximum penetration. This is effective when applied to the surface of the rabbit's
ear over the artery or vein and wrapping with a occlusive bandage for 30 minutes before injecting or
taking a blood sample.
EMLA© cream is a mixture of prilocaine and lidocaine
Local Anesthetics in Combination with Other Drugs
Frequently epinephrine is added to local anesthetics to cause local vasoconstriction and to slow the
uptake of the local anaesthetic and increase the effective duration. Although the amounts of
epinephrine are small, they may be sufficient to cause an elevation of blood pressure and heart rate.
This effect is well recognized by dentists who use this type of preparation.
Adverse Reactions to Local Anesthetics
Overdoses of local anesthetics may cause convulsions in people and animals. In general, the more
potent the anaesthetic, the lower the toxic dose. Procain, prilocaine, lidocaine have low relative
anesthetic potencies and high toxic doses while tetracaine and bupivacaine have a high relative
anesthetic potency and low toxic doses. Toxic doses vary between animal species and range from 1-5
mg/kg to 15-25 mg/kg. These figures usually refer to local anesthetics injected intravenously,
however, toxic levels may be achieved following administration by other routes.
Special care must be taken when giving local anesthetics to small laboratory animals, as it is very
easy to exceed the toxic dose. Injectable local anesthetics come in solutions of 0.5-5.0%. Even the
lowest concentration has 5mg/ml and for the least toxic local anesthetic, 0.1 ml could be toxic for a
20g mouse. Inadvertent intravenous injection of local anesthetic may cause a decrease in cardiac
function with a fall in blood pressure.
Examples of Local Anesthetics - Click on drug for more details
Lidocaine and Prilocaine (EMLA)
Summary of Major Effects of the Above Three Groups of
Analgesic Drugs
The following table summarizes the major effects of the three main groups of pain relieving drugs.
These are general effects and are applicable, more or less, to all of the drugs in each group. However,
they should be considered guidelines only. More detailed descriptions will be found in pharmacology
texts or in texts dealing with pain relief. A veterinarian should be consulted to assist in the selection
of the most appropriate analgesia regimen.
Table: Summary of Major Effects of Analgesic Drugs
Opioids
NSAID
Local Anesthetics
For severe pain
For mild to
moderate level pain
and for chronic
pain
Block all painful
stimuli for
duration of
anesthesia
Nervous
System
Sedation
depending on
drug, dosage
and species;
may cause
excitation
No effects at
normal doses
No sedation;
excitation and
convulsions with
overdose
Cardiovascular
Fall in blood
pressure and
heart rate;
dilation of
peripheral blood
vessels
No general effect;
some may cause
platelet changes
and a possibility of
bleeding
May cause some
vascular changes
due to added
epinephrine
Respiratory
Depressed
No general effect;
respiration rate overdoses may
and depth; cilia affect respiration
activity in
trachea inhibited
Gastrointestinal Appetite
suppression;
gastrointestinal
movements
reduced
No effect unless
nerves associated
with respiration
are involved
May cause ulcers in No effects
stomach
Immune
system
Subtle effects on May inhibit the
immune system
some cells of
the immune
system
Other
Behavioural
changes
No effects
demonstrated
Hypersensitivity
Alpha 2-Adrenergic Receptor Agonists
The alpha2-adrenergic receptor agonists have been used extensively in animals to supplement other
anesthetic agents and to provide increased analgesia. While they provide sedation in most species,
the level of analgesia is quite variable, depending on the species, and the dose of the drug. Alpha2adrenoreceptors are found throughout the body, particularly in the vascular system. Stimulation of
these receptors results in an initial increase in blood pressure followed by a fall in blood pressure and
heart rate. This lowering effect on blood pressure and heart rate is one of the important side effects
limiting the use of these drugs to the healthiest animals. Respiration rates are reduced and this
results in an elevation of carbon dioxide and a lowering of oxygen in the blood.
Rompun© is an example of an alpha-2 receptor agonist. The active drug is xylazine.
The most commonly used drugs of this group are xylazine and medetomidine. They are sometimes
used in combination with other drugs (e.g., ketamine) to produce general anesthesia (although it is
preferable to use inhalation anesthetics in most cases). Specific antagonists are available (e.g.,
atipamezole) so the depressant effects on the cardiovascular systems and the respiratory systems can
be reversed if clinical problems arise.
Example of Alpha 2-Adrenergic Receptor Agonists - Click on drug for more details
Xylazine
Ketamine
NMDA Receptor Antagonists
N-methyl-D-aspartate (NMDA) receptors are important for the transmission of some aspects of pain in
the central nervous system. In particular, they appear to be involved in the development of
hypersensitivity that accompanies injuries or inflammation.
The main NMDA receptor antagonist in use at present is Ketamine. It is used as an anesthetic agent
in conjunction with other analgesics, particularly the alpha-2 adrenergic agonists. Less than
anesthetic doses are required to block central sensitisation but the duration of action is short and it is
not yet a practical means of controlling pain. Dextromethorphan is also an NMDA receptor antagonist
but its use has been primarily as a cough suppressant.
The NMDA receptor antagonists prevent the development of wind-up and may also prevent the direct
transmission of pain from the viscera.
Ketamine is the active drug in Ketalean(TM)
Ketamine is the only commonly used member of this group of drugs. It is an anesthetic with relatively
poor analgesic qualities. It has not been used extensively to provide pain relief, for example, after
surgery because of its short duration of action. Dextromethorphan, commonly used as a cough
suppressant, also blocks NMDA receptors and may have a role in pre-emptive analgesia. {Articles on
pre-emptive analgesia may be found in a number of journals, particularly those that deal with pain
and anesthesia. This is a website to one such journal where this material may be read. (click here)}.
Administration of Analgesic Drugs
These drugs may be given by a variety of routes. The route of administration and the frequency
should not be stressful to the animal. They no more enjoy getting injections than we do. The volume
of injections should be considered. Very small volumes to very small animals increase the risk of
inaccuracies and the chance of an accidental overdose, thus a dilution of the drug may be needed
before it is given to the animal. Large volumes injected into muscles may be painful and so cause the
animal more stress. The following website has some useful information on recommended volumes to
be given by a variety of routes to research animals. These volumes are considered to be the
maximum that should be given in one site. (click here)
Legal Requirements Associated with Use of Analgesics
Opioids: Many of the opioids are very potent compounds that have the potential to be addictive.
They are controlled drugs. Only qualified persons may prescribe them. Careful record must be kept
of each dose given. The records are subject to examination by the inspectors from the Office of
Controlled Substances of Health Canada.
NSAIDs: Many NSAIDs are available as over-the-counter drugs (e.g., aspirin™, acetaminophen).
Some of the newer compounds may require a prescription for acquisition. Although there is not a
legal requirement to keep a record of administration, this should be standard practice.
Local anesthetics: Local anesthetics are available on prescription. Although there is not a legal
requirement to keep a record of administration, this should be standard practice.
The alpha2-adrenergic receptor agonists: the alpha2-adrenergic receptor agonists are available
on prescription. Although there is not a legal requirement to keep a record of administration, this
should be standard practice.
NMDA receptor antagonists: The NMDA receptor antagonists are available on prescription.
Although there is not a legal requirement to keep a record of administration, this should be standard
practice.
Summary Statement
The relief of pain both in people and animals is an inexact science. Evidence of pain is sometimes
difficult to demonstrate but the relief of that pain may be even more difficult to confirm. There are
many analgesic drugs and their effects differ from species to species and even within a species. The
side effects of the drugs may make it difficult to be certain that pain has been relieved. Nevertheless,
we must try to relieve pain, particularly that which we have caused.
Sample Questions
You discover that, due to an error in calculation, a group of rats has been receiving 2 times the
therapeutic dose of a NSAID. Which one of the following side effects might be expected ?
a.
b.
c.
d.
e.
Hyperactivity
Vomiting
Increased clotting time
Sedation
Fall in blood pressure
A rat has undergone a research procedure that requires open chest surgery. Which one of the
following is the most appropriate analgesic ?
a.
b.
c.
d.
e.
Morphine
Acetylsalicylic Acid
Bupivicaine
Ketamine
Xylazine
Which of the following signs may indicate that an animal is in pain ?
a.
b.
c.
d.
e.
Lameness
Isolation from the group
Disinterest in surroundings
Decreased food intake
All of the above
Module 11 – Anesthesia
Objectives
The objectives of the section on anaesthesia are:
•
•
•
•
•
To introduce the student to the administration of anaesthetics to laboratory animals
To discuss anaesthesia under the following broad headings:
o Preanaesthesia
o Effects of anaesthetic agents
o Anaesthetic administration
o Anaesthetic emergencies
o Recovery from anaesthesia
To provide information on the effects of drugs used during anaesthesia
To consider the consequences of anaesthesia and the surgical procedures on recovery
To discuss anaesthetic emergencies and their treatment
Introduction
"Animals must not be subjected to unnecessary pain or distress. The experimental
design must offer them every practical safeguard whether in research, in teaching or
in testing procedures…" (Ethics of Animal Investigation, CCAC 1989)
In this module we will discuss the alleviation of pain during painful procedures such as surgery,
through the use of anaesthetic drugs. Anaesthesia is also used for producing muscle relaxation,
suppressing reflexes, and producing loss of consciousness for purposes other than prevention of pain
perception. For example, anesthesia is required for MRI, CT scans and in some cases for radiology.
Because of the wide variability of laboratory animal species, strains, and strains, as well as
anaesthetic agents, an appropriate anaesthetic regimen should be developed in consultation with a
veterinarian prior to the commencement of any study.
Related Information Covered in Other Modules
The relief of pain after a surgical procedure, or at other times, is covered in the module "Analgesia".
The signs of pain and distress in animals are covered in the module "Pain and Distress and Endpoints".
Readers can refer to these modules for further information.
The need to use an anaesthetic to perform a procedure implies that the procedure would be painful for
an awake animal. In addition there may be some residual pain after the animal recovers from the
anaesthetic and analgesics should be used. Some drugs described here appear in both the anaesthesia
and analgesia modules.
Preanaesthetic Treatments
There are several reasons why the use of preanaesthetic agents should be considered:
•
•
•
•
to
to
to
to
reduce apprehension in the animal
allow a reduction in the dose of anaesthetic required
reduce some of the side effects of the anaesthetic agent
provide some analgesia after the anaesthetic has worn off
The preanaesthetic agent should allow a reduction in the dose of anaesthetic required. This applies
particularly to injectable anaesthetics where control of the depth and duration of anaesthesia is often
more challenging than with inhaled anaesthetics. The combination of preanaesthetic and anaesthetic
drugs must provide sufficient pain blocking so that the animal does not feel pain during the surgical
procedure.
Most anaesthetic agents do more than produce unconsciousness and pain relief. They are potent drugs
that may affect every system in the body. The effects on the respiratory and cardiovascular systems
may be particularly significant during the anaesthetic period, however effects on other organ systems
may be more significant depending on the goals of the research. While some preanaesthetic agents
may reduce anaesthetic side effects, more often it is the reduction in anaesthetic dose that provides
the greatest relief from the side effects.
An ideal preanaesthetic agent should provide some analgesia after the surgery. Some analgesics are
quite short acting in animals and depending on the length of the surgery may not last into the
recovery period. It is important to ensure that analgesia continues through the surgical period, into
and beyond recovery.
The effects of the ideal preanaesthetic as described above are not to be found in a single agent.
Sometimes more than one drug is used but with care the undesirable side effects do not outweigh the
potential beneficial effects.
Summary
The ideal preanaesthetic agent should:
•
•
•
•
Reduce apprehension
Allow a reduction in anaesthetic doses
Reduce or eliminate some of the undesirable side effects of anaesthetics
Provide some analgesia after the anaesthetic has worn off
Types of Preanaesthetic Drugs
The major groups of preanaesthetic drugs are analgesics (particularly the opioids), tranquilizers and
anticholinergic drugs. Paralytic or neuromuscular blocking drugs are also used as adjuncts to
anaesthesia, particularly in human surgery.
Analgesics
Some analgesics, as well as providing pain relief, also have a desirable sedative effect. They calm the
animals and allow a reduction in the amount of anaesthetic required along with a smoother recovery
from anaesthesia.
Moreover, a pre-emptive strike against pain is thought to result in a reduced need for analgesics after
the surgery. For these reasons, some analgesics may be given as preanaesthetic agents. Of the
analgesics, the opioids are commonly used as preanaesthetic agents.
Opioids
Among the effects of opioids that make them useful as preanaesthetic agents are analgesia (they
make pain more tolerable without abolishing it completely) and CNS depression (there is usually
depression although some opioids may produce excitation and convulsions in some species). As a
general rule, the suitability of any drug for a given species should be checked before using it.
The combination of analgesia and CNS depression are both desirable for anaesthesia. This allows for a
reduction in the dose of anesthetic agent and provides pre-emptive analgesia.
Tranquilizers
The principal drugs in this group are the phenothiazine derivatives (e.g., acepromazine,
chlorpromazine), the benzodiazepines (e.g., diazepam, midazolam), and the butyrophenones (e.g.,
droperidol, fluanisone). The alpha 2 adrenergic receptor agonists (e.g., xylazine, medetomidine) could
be considered as tranquilizers as well as analgesics but they are seldom used for their tranquilizing
effect alone because of their profound cardiovascular effects.
The principal effect of tranquilizer drugs administered prior to anaesthesia is the reduction of anxiety
in animals. This effect may be achieved with low doses and may not be accompanied by CNS
depression. At higher doses, CNS depression may be profound, depending on the drug, and the
species. Most of the tranquilizers do not have any analgesic effects and so a lack of response or a
diminished response to painful stimuli should not be interpreted as a sign of analgesia. The alpha 2
adrenergic receptor agonists provide some analgesia as well as tranquilization. Tranquilizers generally
cause minimal cardiovascular and respiratory depression and some have an effect in reducing the
occurrence of cardiac arrhythmias during anaesthesia. There are some exceptions. The alpha 2
adrenergic receptor agonists have profound effects on both cardiovascular and respiratory systems.
Some tranquilizers (e.g., chlorpromazine) can cause moderate to severe hypotension, and some
phenothiazine derivatives decrease the seizure threshold in certain species.
Summary of effects of tranquilizers
•
•
•
•
Minimal or no analgesia
Minimal CNS depression
Anxiolytic (calming)
Minimal cardiovascular and respiratory depression
Anticholinergic Drugs
Anticholinergic drugs block the muscarinic actions of the neurotransmitter, acetylcholine. Acetylcholine
is a neurotransmitter at many sites throughout the body and so the effects of anticholenergic drugs
are widespread.
Anticholinergic drugs are used to block two effects in particular during anaesthesia, secretions in the
respiratory tract in response to the irritating nature of some inhalant anesthetics, and bradycardia
(slowing of the heart) which accompanies most anaesthetics. Respiratory secretions are a complicating
factor especially in animals with small airways where even a low level of secretion may compromise
respiration. With the advent of less irritating volatile anaesthetics, concerns about respiratory
secretions have been reduced, and the primary use is to minimize the bradycardia that follows
stimulation of the vagus nerve. Salivation is reduced in many animals although not in ruminants. The
muscarinic receptors in the eye are also affected resulting in dilated pupils (mydriasis). There may be
a decrease in gastro-intestinal motility.
The most commonly used anticholinergics are atropine and glycopyrrolate. Some rabbits possess
atropinase, an enzyme which rapidly breaks down atropine and reduces its effectiveness in this
species. Glycopyrrolate may be used in place of atropine in cases where the anticholinergic effect is
required for a longer period of time. The effects are similar to atropine although the increase in heart
rate may be less.
Types of Anaesthetic Agents
Anaesthetic agents should produce a loss of sensation with a minimum of side effects and they should
have a calming effect on the animal during the recovery phase. While there is not a requirement for a
loss of consciousness during anaesthesia, that is the case with general anaesthetics. Local
anaesthetics for example will produce quite localized or even regionalized anaesthesia without any loss
of consciousness. As well, it is advantageous for an anaesthetic agent to provide some level of
analgesia during the recovery phase.
There are three broad groups of anaesthetic agents namely volatile anaesthetics like isoflurane and
halothane, injectable anaesthetics like ketamine, propofol and barbiturates, and local anaesthetics like
lidocaine, procaine and bupivacaine. For general anaesthesia inhalant anaesthetics are highly
preferred as the anaesthesia is much easier to control and the agent quickly cleared from the body.
Volatile Anaesthetic Agents
The common effects of anaesthetic agents described above apply to the volatile anaesthetics. These
drugs are usually supplied as liquids and require a vaporizer and a carrier gas such as oxygen to
deliver them to the patient. Altering the concentration of the anaesthetic agent in the inspired gases
easily controls the depth of anaesthesia. In the event that the animal becomes too deeply
anaesthetised, the anaesthetic agent is quickly removed from the animal through the lungs. It is
important to scavenge waste anaesthetic gases to minimize exposure of people to these agents.
Isoflurane
•
•
•
•
•
Highly volatile and must be administered using a calibrated vaporizer to prevent exposure to
high concentrations of gas
Must be scavenged to avoid occupational exposure
Respiratory depression greater than with halothane and may necessitate external ventilation
Little hepatic metabolism and a lessened risk of hepatitis
Rapid recovery (1-3 minutes)
Halothane
•
•
•
•
•
•
Highly volatile and must be administered using a calibrated vaporizer to prevent exposure to
high concentrations of gas
Must be scavenged to avoid occupational exposure
May cause cardiac arrhythmias
May cause hepatitis in humans but rare in other species
Will cause malignant hyperthermia in genetically susceptible pigs
Rapid recovery (1-3 minutes) except from very long and deep anaesthesia
Nitrous Oxide
•
•
•
•
•
Comes as a gas in cylinders
Low anaesthetic potency and cannot produce anaesthesia in animals by itself
Causes minimal cardiovascular and respiratory depression
May be used to reduce the concentration of other anaesthetic gases although this effect is less
than that seen in humans
Use with caution in ruminants
Injectable Anaesthetics
The general effects of anaesthetics apply to the injectable anaesthetics, with some exceptions.
Ketamine, for example, does not cause significant cardiovascular depression at the usual anaesthetic
doses. Injectable anaesthetics are easily administered requiring little more than a needle and syringe,
but once they have been injected it is very difficult to control their effects. There are no specific
antidotes for many of these drugs and recovery from anaesthesia depends on redistribution of the
drug from the blood to the tissues or its metabolism or a combination of both processes.
There are many injectable anaesthetic drugs in use, ketamine, propofol, pentobarbital, methohexital,
thiopental. The following notes on a few injectable anaesthetics highlight some important features or
exceptions from expected effects. Full details on the activities of the drugs in particular species should
be obtained from the veterinarian.
Ketamine
•
•
•
•
•
Poor analgesia in most laboratory species and should not be used alone
Increased muscle tone
Many reflexes remain although animal is unresponsive to pain (e.g., swallowing and blink
reflexes)
Usually used in combination with another drug (e.g., xylazine, diazepam)
Duration of anaesthesia depends on dose.
Sodium Pentobarbital
•
•
•
•
•
Narrow safety margin
Poor analgesia until animal is completely unconscious
Excitation during the recovery phase
Gives up to 60 minutes of anaesthesia
Controlled drug status
Urethane
•
•
•
Provides long periods of surgical anaesthesia with little respiratory depression
Urethane is carcinogenic
Animals should not be allowed to recover from urethane anaesthesia
Local Anaesthetic Agents
Local anaesthetics are dealt with in more detail in the Analgesia module. They are used particularly for
pain relief following surgical procedures in small animals. They are also employed for regional
anaesthesia in larger animals (e.g., sheep and cattle for procedures such as dehorning, castrations
and caesarean sections). Frequently they are supplemented with tranquilizing drugs to help provide
restraint.
Animal Factors in Anaesthesia
There are a number of factors related to the animal that impact on the quality of anaesthesia. These
factors should be considered when the type of anaesthetic agent is being chosen.
Species. Different species require different doses of anaesthetic agents. This applies particularly to
the injectable anaesthetics. In general, the smaller animals require a higher dose in mg/kg of a given
anaesthetic than larger animals. Familiarity with the effects of an anaesthetic agent in one species
should not be assumed in another species. The volatile anaesthetics are more consistent in their
application between species. The mean alveolar concentration of the anaesthetic agent required for
anaesthesia is similar among species and this is controlled by the concentration of the agent in the
inspired gases. Differences in the respiratory tract in birds (fixed lungs, air sacs) and other nonmammalian species must be considered when administering inhalation anaesthetics.
Strain. Strain differences have been noted even within the same species. Some strains of pigs are
more susceptible to malignant hyperthermia during halothane anaesthesia than others (a genetic
trait).
Age. Young animals and old animals may have an increased risk for anaesthetic complications. In
older animals, pathological changes if present in the respiratory system, may result in complications.
Young animals may not have developed all the processes required to metabolize the drugs and so may
have longer than expected recovery from anaesthesia. Volatile anaesthetics allow more refined control
of the anaesthesia in both groups.
Weight. Very fat animals may not breathe as effectively during anaesthesia as thinner animals,
leading to the problems associated with hypoventilation. In addition, if an agent is given on a mg/kg
basis, there may be a relative overdose because the fat does not participate to a great degree in the
circulation and distribution of the drug. If part of the recovery from an anaesthetic depends on its
removal from the blood into tissues including fat (e.g., the short acting barbiturates) then animals
with very little fat (e.g., greyhounds, calves) may experience longer than usual recovery from
anaesthesia.
Sex. There is some evidence for a difference between the sexes for some anaesthetics.
Health of an Animal. Pre-existing disease or pathology may complicate an otherwise smooth
aneasthesia. Any disease in the lungs will further compromise respiration during anaesthesia. Liver
disease may interfere with the metabolism of anaesthetic agents and kidney disease may limit their
excretion. Surgically altered animals (e.g., hypophysectomy, adrenalectomy, thyroidectomy) may be
at increased risk at subsequent anaesthesias
Demeanor. An exited animal with high levels of circulating adrenalin, elevated heart rate and blood
pressure is at an increased risk when undergoing anaesthesia.
Previous Anaesthesia. Some of the injectable anaesthetics are not completely cleared from the body
for several days (e.g., pentobarbital), even if the animal has recovered consciousness and is behaving
normally. Care must be taken if a second anaesthetic quickly follows the first. For those anaesthetics
that are extensively metabolized as part of the excretory process, a second anaesthetic may result in
more rapid metabolism of the drug than the first, with a shorter period of anaesthesia.
Other Factors. Some non-anaesthetic drugs have effects on anaesthetic agents. Chloramphenicol
may lengthen the duration of pentobarbital anaesthesia and some antibiotics potentiate the actions of
muscle relaxants.
Side Effects of Anaesthetic Drugs
Like many drugs, anaesthetics also have other effects that may not be desirable. It may be necessary
to take account of these side effects whether the animal is anaesthetized for a surgical procedure or
for a physiological study of an organ system. The side effects described below occur to a greater or
lesser degree with all general anaesthetics.
Central Nervous System (CNS) Depression. The commonly used anaesthetics provide CNS
depression to the point of loss of consciousness. This does not mean that all neuronal activity has
been abolished. Many of the reflexes that are used to assess anaesthetic depth are retained after
unconsciousness. However if anaesthetic depth increases, these are gradually lost and even automatic
functions like respiration may be lost.
Cardiovascular Depression. Anaesthetics usually cause a decrease in cardiac output and a fall in
blood pressure. These effects are a combination of a direct influence of the anaesthetic on the heart,
reducing its contractility and an effect on the heart and blood vessels by way of the nerve supply to
these tissues.
Respiratory Depression. One of the effects of anaesthetic agents is to cause a loss of muscle tone
and a decrease in contractility. In the respiratory system, this results in smaller breaths i.e., the tidal
volume is decreased. At the same time the respiratory rate is decreased. There is also a decreased
sensitivity in the receptors that detect the level of oxygen and carbon dioxide in the blood. The overall
effect is to reduce the respiratory capability of the animal.
Loss of Temperature Control. Anaesthetic agents inhibit the mechanisms responsible for
maintaining a steady body temperature. These include the temperature regulating centres in the brain
and processes like shivering. The result is a tendency for the animal's temperature to drift downwards
towards the environmental temperature. Hypothermia is a major consideration in anaesthesia
especially for small animals such as rodents, and controlled supplemental heat must be provided to
maintain body temperature.
Hormone Release Depressed. Generally, the release of hormones is depressed. Prolactin release
may be increased by some general anaesthetics.
Depression of Other Functions. Gastro-intestinal motility is depressed by general anaesthetics as is
liver function. Urinary excretion is decreased.
Anaesthetic Techniques
Inhalation Anaesthesia
This type of anaesthesia requires the animals to breathe in the anaesthetic. Initially the anaesthetic
concentration is highest in the alveoli and as the gas passes into the bloodstream, the animal becomes
anaesthetised. At the end of the surgery, the concentration in the inspired gas is reduced to zero and
the agent passes from the blood to the alveoli and the animal recovers. The balance between blood
and alveolar levels controls the depth of anaesthesia.
There are several techniques for anaesthetizing animals with volatile anaesthetics. The animal may be
placed in a chamber and the chamber flooded with the anaesthetic gas in the carrier gas at the
required concentration. Once anaesthetized, the chamber should be opened in a fume hood and the
animal may be removed and placed on a nose cone or some other apparatus for delivering the
anaesthetic to prolong the anaesthesia, if that is necessary. This technique is most suitable for small
animals. Assisted ventilation may be required in some species (e.g., sheep).
Courtesy of Dr Paul Flecknell, University of Newcastle
Animals may be masked down by placing a mask over the nose and mouth and allowing them to
breathe the anaesthetic gases. Even fairly large animals may be anaesthetized in this manner but it
usually results in some accidental exposure to the gases. Some species hold their breath when the
mask is placed on their face.
Courtesy of Dr Paul Flecknell,
University of Newcastle
Animals may be anaesthetised initially with a short acting anaesthetic, injected intravenously, an
endotracheal tube placed in the trachea and the tube connected to an anaesthetic circuit delivering the
volatile anaesthetic. This system has several advantages. It makes it possible to ventilate the animal
using a respirator and so maintain normal breathing and respiratory values. It makes it easier to deal
with anaesthetic emergencies should they occur and it allows for better control of stray anaesthetic
gases in the room. However, endotracheal intubation is difficult in some small animals (e.g., rodents)
and the reduction in diameter of the airway in the trachea may significantly affect respiration. Care
must also be taken not to significantly increase the dead space i.e., the part of the respiratory system
where exchange of gases with the blood does not occur. The ventilation rate and the tidal volume
should be set to maintain normal blood gases and this should be discussed with a veterinarian.
Courtesy of Dr Paul Flecknell, University of Newcastle
Occupational Health and Safety Concerns
Human exposure to the inhalation anaesthetic gases should be avoided. Hepatic toxicity may occur
with exposure to some volatile anaesthetics. Others are known to be carcinogenic (e.g., urethane).
Procedures should be in place to collect or remove all waste anaesthetic gases that leak out or are
expired by the anaesthetized animal.
Injectable Anaesthesias
Anaesthetics may be injected by a number of routes, intravenously, intraperitoneally, intramuscularly
and subcutaneously. Injectable anaesthetic drugs may be used to produce general, regional or local
anaesthesia. For regional anaesthesia, anaesthetics may be injected into the subarachnoid or epidural
spaces to block both the sensory and motor nerves entering or emerging from the spinal cord at that
level.
Anaesthetics such as the thiobarbiturates should be given intravenously because their high pH
(alkalinity) causes tissue damage if injected subcutaneously or intramuscularly.
Anaesthesia through Drugs Placed in the Animal's Environment
This refers particularly to anaesthetics administered in the water of fish and amphibians. There are
several anaesthetics that can be added to the water and the animals allowed to swim until they
become anaesthetised. Tricaine methanesulfonate (TMS; MS-222®) is commonly used to anaesthetise
frogs and fish. In water, it is very acidic and must be buffered with sodium bicarbonate.
Cold-induced "anaesthesia" - Hypothermia
Induction of hypothermia has been used for immobilizing neonatal rodents since they do not yet have
well-developed thermoregulatory mechanisms, and for immobilising amphibians and reptiles, for
surgical procedures with an apparent wide safety margin. It is known that a neural tissue temperature
less than about 9°C (5°C is sometimes cited as the desired core body temperature) results in blockage
of transmission in the brain and central nervous system to produce unconsciousness. The lack of
response to surgery trauma during such levels of hypothermia has been accepted as an indication of
insensitivity to pain. However, there are important welfare concerns about the chilling down and
warming up periods, the methods of doing so, and the absence of post-operative analgesia with this
technique. Definitive studies on the anaesthetic and analgesic effects of hypothermia as the sole agent
have not been reported, and since safe and effective alternatives are available, these should be used.
Assessment of Anaesthesia
Anaesthesia has been described as a series of four Stages.
•
•
•
•
Stage 1: the period between administration of an anaesthetic and loss of consciousness.
Stage 2: the period after loss of consciousness, which may include actions such as
uncontrolled movement, delirium, vocalization.
Stage 3: the level at which surgery can be performed. Stage 3 anaesthesia is divided into four
planes.
o Plane 1: "light" anaesthesia - the animal still has blink and swallowing reflexes, and
regular respiration.
o Plane 2: "surgical" anaesthesia - the animal has lost blink reflexes, pupils become
fixed and respiration is regular.
o Plane 3: "deep" anaesthesia - the animal starts losing the ability to use the respiratory
muscles and breathing becomes shallow; may require assisted ventilation.
o Plane 4: the animal loses all respiratory effort, and breathing may stop entirely.
Stage 4: anaesthetic crisis! Respiratory arrest and death from circulatory collapse imminent.
Reflexes are used to estimate the depth of anaesthesia and while there is some variation in the
activity of these reflexes with different anaesthetics, they are consistent enough to be useful as a
group. A single reflex should not be used as the sole determinant of anaesthetic depth.
•
•
•
•
•
Pupillary Reflex. Shine a light in the eye and the pupil constricts. This reflex is present at the
start of Stage 3 and starts to decrease and will be absent by about the middle of Stage 3.
Palpebral Reflex. Touch the corner of the eye and the animal blinks. This disappears early in
Stage 3.
Corneal Reflex. Touch the cornea and the animal blinks. This disappears early in Stage 3. Be
careful not to damage the cornea if this reflex is tested.
Withdrawal Reflex. Pull a limb gently, pinch the toe and the animal will pull back the limb.
This reflex indicates whether the animal feels pain or not and should be absent before surgery
starts. This will occur early in Stage 3.
Laryngeal (Swallowing) Reflex. Stimulation of the larynx will cause the animal to swallow.
The stimulation may be from outside, for example, an attempt to pass an endotracheal tube or
may be internal for example the presence of secretions at the larynx. This is a mechanism to
prevent accidental aspiration of fluids into the lungs. This reflex will disappear early in Stage
3.
There are some other signs that will help judge the depth of anaesthesia.
Respiratory efforts change as anaesthesia deepens. In Stage 1, the animal is awake and respiration
may be quite rapid due to the excitation of being handled. It is evenly apportioned between the chest
and abdomen and is quite regular. In Stage 2, breathing is still evenly apportioned between chest and
abdomen but is less regular and breath holding may occur. Early in Stage 3, breathing is regular with
equal contributions from chest and abdomen. As anaesthesia deepens, breathing becomes shallower
and more predominantly abdominal. Late in Stage 3 it becomes irregular and in Stage 4 will stop.
Muscle tone decreases from a maximum during Stage 2 all the way through Stage 3. Jaw tone is a
good indication of muscle tone.
Response to Surgical Stimuli. Care should be taken to note any responses to surgical stimuli. Once
it has been determined by the reflexes that an appropriate depth of anaesthesia has been reached,
surgery will commence. The first incision should be observed to determine if the animal makes any
response. A response could include a movement, a pause in respiration or a deeper breath if the
animal is breathing spontaneously or an increase in heart rate or blood pressure. If there is surgery in
the abdomen, traction on the abdominal viscera is known to be painful and this may provide another
indication of whether the anaesthesia is adequate or not.
DEPTH OF ANAESTHESIA
Stages
Reflexes/Signs
Stage 1: the period between administration of an
anaesthetic and loss of consciousness.
Respiratory efforts change as anaesthesia
deepens. In Stage 1, the animal is awake and
respiration may be quite rapid due to the
excitation of being handled. It is evenly
apportioned between the chest and abdomen and
is quite regular.
Stage 2: the period after loss of consciousness,
which may include actions such as uncontrolled
movement, delirium, vocalization.
Muscle tone decreases from a maximum during
Stage 2 all the way through Stage 3. Jaw tone is
a good indication of muscle tone.
Respiratory efforts. In Stage 2, breathing is still
evenly apportioned between chest and abdomen
but is less regular and breath holding may occur.
Stage 3: the level at which surgery can be
performed. Stage 3 anaesthesia is divided into
four planes.
•
•
•
•
Plane 1: "light" anaesthesia - the animal
still has blink and swallowing reflexes, and
regular respiration.
Plane 2: "surgical" anaesthesia - the
animal has lost blink reflexes, pupils
become fixed and respiration is regular.
Plane 3: "deep" anaesthesia - the animal
starts losing the ability to use the
respiratory muscles and breathing
becomes shallow; may require assisted
ventilation.
Plane 4: the animal loses all respiratory
effort, and breathing may stop entirely.
Pupillary Reflex. Shine a light in the eye and the
pupil constricts. This reflex is present at the start
of Stage 3 and starts to decrease and will be
absent by about the middle of Stage 3.
Palpebral Reflex. Touch the corner of the eye
and the animal blinks. This disappears early in
Stage 3.
Corneal Reflex. Touch the cornea and the animal
blinks. This disappears early in Stage 3. Be
careful not to damage the cornea if this reflex is
tested.
Withdrawal Reflex. Pull a limb gently, pinch the
toe and the animal will pull back the limb. This
reflex indicates whether the animal feels pain or
not and should be absent before surgery starts.
This will occur early in Stage 3.
Laryngeal (Swallowing) Reflex. Simulation of
the larynx will cause the animal to swallow. The
stimulation may be from outside, for example, an
attempt to pass an endotracheal tube or may be
internal for example the presence of secretions at
the larynx. This is a mechanism to prevent
accidental aspiration of fluids into the lungs. This
reflex will disappear early in Stage 3.
Respiratory efforts. Early in Stage 3, breathing
is regular with equal contributions from chest and
abdomen. As anaesthesia deepens, breathing
becomes shallower and more predominantly
abdominal. Late in Stage 3 it becomes irregular
and in Stage 4 will stop.
Stage 4: anaesthetic crisis! Respiratory arrest
and death from circulatory collapse imminent.
Body temperature should be measured and steps taken to prevent the fall in body temperature that
usually accompanies anaesthesia, particularly in small animals.
Capillary refill time may give an indication of the adequacy of cardiovascular function. If a pink paw
is squeezed, it will go white but the pink will return within about two seconds. If the time is
significantly longer than this, then blood flow through the capillaries is compromised, usually because
anaesthesia is too deep and the blood pressure is too low.
If a pulse can be felt and if the anaesthetist is experienced with respect to the feel of the normal
pulse, it is possible to get an indication of the cardiac output. In addition, the pulse rate can be
counted and compared with the heart rate as heard through a stethoscope or counted from an
electrocardiogram. There should not be a difference between the two.
Blood pressure gives a good indication of the effectiveness of the heart's contractions and the
resistance to flow in the peripheral vessels. It may be measured either indirectly (e.g., a Doppler
system or directly with a catheter in an artery). A falling mean arterial pressure is a sure indicator of
deepening anaesthesia, if there are no other possible causes like severe haemorrhage.
Additional Monitoring. If an arterial blood sample and the capability to measure blood gases are
available, then this information provides a very accurate picture of the effectiveness of the ventilation.
This information can be used to fine tune ventilation to maintain normal physiological parameters. End
tidal CO2 and pulse oximetry are non-invasive methods of obtaining information on the blood gas
status.
Anaesthetic Management
Whenever possible, and particularly for more complex surgeries, there should be an "anaesthetist"
involved. Anaesthetic management accounts for all the processes and events during a period of
anaesthesia that will result in freedom from pain during the surgical procedure and a return to a
normal physiological state as soon as possible after recovery. The assessment of the depth of
anaesthesia is only one part of anaesthetic management. An important component is ensuring that all
equipment is functioning properly.
During the course of the surgical procedure, there will have been changes in the fluid balance of the
animal that should be corrected. Some of these are normal: continued production of urine, and losses
from the respiratory tract. Continuous saliva production, especially in ruminants, will deplete body
water and electrolytes. An anaesthetized sheep may produce 800 ml/hr of saliva high in bicarbonate,
which is lost to the animal because it cannot swallow while under anaesthesia. This secretion is not
reduced by anticholinergic drugs like atropine and will soon lead to fluid depletion and an acid/base
imbalance.
Blood loss may account for a serious loss of fluids and this is particularly important in small animals
that have a very small blood volume. (It is convenient to estimate the blood volume of an animal at
70 ml/kg. This is only an estimate for the purpose of rapid simple calculation of blood loss or the
volume of blood that might be taken from an animal. A 20g mouse has about 1.4 ml of blood and a
loss of 0.2 ml represents 14% of its blood volume.)
The loss of fluids through evaporation may be significant if deep body cavities are open during the
surgery. It has been estimated that the fluid loss through evaporation from an open human abdomen
is about 500 ml/hr.
Fluid losses should be replenished throughout the surgery, rather than waiting to the end.
Unexpectedly large losses should be replaced as soon as possible. It is not always possible or desirable
to replace blood with blood but the consequences of a reduced circulating blood volume on
cardiovascular performance must be considered.
If vascular access has been established, it is important to ensure that this access is patent. This is
usually accomplished by slowly running fluids through the needle or catheter. Occasionally, the needle
becomes displaced or there is a kink in the tubing and the vascular access is lost through clotting of
the needle.
Paralytic Agents (Neuromuscular Blocking Drugs)
Neuromuscular blocking drugs are used as an adjunct to anaesthetics to provide greater muscle
relaxation during a surgical procedure or when control of respiration is necessary. These compounds
paralyze skeletal muscle so that voluntary control of the muscles is lost. Most significantly, there is
loss of activity in the muscles of respiration and in muscles that are responsible for some reflexes used
to judge the depth of anaesthesia (see above). Loss of the muscles responsible for respiration means
that an artificial method of respiration must be used. Loss of reflexes means that it is difficult to
ensure how deeply the animal is anaesthetized.
The major concerns about the use of paralyzing agents are that they produce paralysis, but not loss of
consciousness or pain relief, and that their effects may last beyond the anesthesia. Thus an animal
may appear to be anaesthetized (i.e., unresponsive to any painful stimuli) while in reality, it is unable
to respond because of the muscle paralysis.
Emergencies
The three major emergencies while an animal is anaesthetized result from anaesthetic overdose, blood
loss and equipment failure. Any of these could result in the death of the animal.
Anaesthetic overdoses with injectable anaesthetics are the most difficult to deal with because there
are no specific reversal agents. Respiratory depression is the most easily observed effect of the
overdose and takes the form of a decreasing ventilatory effort. If this occurs artificial ventilation
should be initiated to try to maintain normal blood gases until the animal metabolizes the anaesthetic
or otherwise reduces the concentration in blood. It is advisable to have respiratory support equipment
always available when animals are anaesthetized.
Courtesy of Dr Paul Flecknell, University of Newcastle
Blood loss will be recognized if a large vessel is accidentally cut. However, the continual loss of small
amounts of blood may add up to a serious problem. This may occur if the animal has been given anticoagulating drugs like heparin and there is no spontaneous clotting. If the level of anaesthesia has
been deep at the start of the surgery, there may have been little bleeding due to the low blood
pressure. The surgeon may not have had to deal with the many small bleeding points that would have
been obvious if the blood pressure had been higher. Many of these will start to bleed as the animal
recovers or if the anaesthesia is lighter. If blood loss is anticipated then appropriate replacement fluids
should be available as well as a venous catheter placed at the start of anaesthesia so that fluids,
drugs, etc., may be administered if necessary.
The anaesthetist should be aware of the functioning of the equipment at all times and have backup
plans to protect the animal if there should be a failure. Such failures could include a massive electrical
shutdown that halts all electrically driven devices.
Recovery from Anaesthesia
Recovery from anaesthesia more or less mirrors the induction pathway although the timing differs.
Thus while rapid induction pushed the animal through Stage 2 and avoided the excitement stage,
recovery is slower and some excitation may be seen particularly following barbiturate anaesthesia.
Very close monitoring of the animal must continue into the recovery phase, and particular care must
be taken to ensure that the airways are patent and that breathing is unimpeded.
Any anaesthesia-induced abnormalities present at the end of the anaesthesia, such as hypothermia or
dehydration, should be corrected. One dangerous effect of hypothermia is that the anaesthetic drugs
will not be metabolized as quickly and the duration of the anaesthesia unnecessarily prolonged.
There are a number of factors that will influence the rate and quality of the recovery.
If a preanaesthetic agent was used, this may increase the time to full recovery because of the
tranquillizing effects of some of these drugs. On the other hand they will make recovery calmer with
less of the excitement phase.
The anaesthetic agent will determine the rate of recovery. Volatile anaesthetics are quickly blown off
and the animal regains consciousness. Injectable anaesthetics are slower since they depend on
metabolism and excretion for their inactivation.
The duration of anaesthesia may govern the rate of recovery, especially for those agents where rapid
recovery is due to clearing into tissues from the blood with metabolism later (e.g., thiobarbiturates).
Long anaesthesia with one of these drugs will result in saturation of the tissues and prolonged rather
than rapid recovery. The longer the period of anaesthesia, the more difficult it is to keep everything
normal, even with very extensive monitoring. It is likely then that recovery will be different compared
to a short anaesthesia.
The quality of the anaesthesia may be important in the recovery. If anaesthesia was turbulent with
periods of very deep anaesthesia, the likelihood of abnormal physiology and biochemistry is greater,
with delayed recovery.
The surgical procedure, especially if it was accompanied by blood loss may affect recovery. The
duration of the surgery and the insult on the organs may be important. For example, a thoracic
procedure may result in incomplete expansion of the lungs and the compromised ventilation may need
longer to return the respiratory parameters to normal.
Some of the animal factors described as influencing anaesthesia may also affect recovery. Older and
younger animals, for example, may not metabolize the drugs as quickly.
Sample Questions
When comparing volatile anesthetics with injectable anesthetics, which one of the following
statements is correct?
a.
b.
c.
d.
e.
Volatile
Volatile
Volatile
Volatile
Volatile
anesthetics
anesthetics
anesthetics
anesthetics
anesthetics
allow for greater depth of anesthesia.
enable more precise control of anesthetic depth.
are more easily administered.
are safer for the operator to use.
cause fewer cardiovascular changes.
An animal's response to an anesthetic agent may depend on:
a.
b.
c.
d.
e.
Age
Weight
Medical condition
Species
All of the above
Module 12 - Euthanasia of Experimental Animals
Objectives
•
•
•
•
•
To discuss the principles of euthanasia - the humane killing of animals
To discuss the emotional impact on people of killing animals
To outline the criteria for a humane killing technique
To describe the advantages and disadvantages of a number of methods of euthanasia
To discuss the importance of choosing the correct method of euthanasia based upon the
tissues to be collected for analysis
Introduction
Experimental animals are killed for various reasons. The reasons include: to provide cells or tissues for
in vitro research; to collect blood, tissues or other samples at the end of a study; to do veterinary
pathology or diagnostics; to prevent unnecessary pain and suffering when the approved endpoint is
reached and when they are no longer needed or are culled from a breeding program.
Whenever an animal is killed in the course of research, teaching and testing, it must be done with
respect and in a way that ensures the death is as painless and distress-free as possible. The CCAC
Guide to the Care and Use of Experimental Animals states: "In the use of animals in research,
teaching, and testing it is essential that the scientific community take on the mantle of responsibility
for applying scientific judgement and new knowledge to ensure that, when the life of an animal is
taken, it is assured of a "good death"."
Terminology
The word euthanasia means a gentle death. The derivation of the word is from the Greek: eu - well or
good; thanatos - death. Euthanasia is defined as a quiet or easy death, or means of producing one.
We tend to use many different words in an apparent effort to avoid stating plainly that an animal is
being humanely killed: "put down"; "put away"; "put to sleep"; "put out (of its misery)"; "sacrifice".
The term "sacrifice" is still used in scientific publications and presentations.
Principles for Humane Killing
The main welfare principles for a humane method of killing an animal are:
•
•
there should be very rapid (immediate) unconsciousness and subsequent death
there should be no pain or distress accompanying the procedure
These points are emphasised in the CCAC guidelines and in the Canadian Veterinary Medical
Association position statement on euthanasia. The CCAC guidelines state: "The most important
criterion of acceptance of a euthanasia method as humane is that it have an initial depressive action
on the Central Nervous System (CNS) to ensure immediate insensitivity to pain." (click here)
The CVMA position statement on euthanasia reads: "The Canadian Veterinary Medical Association
believes that when an animal has to be killed, its death must be quick, and cause the least possible
pain." (click here)
In addition, the circumstances leading to the humane killing of the animal should not produce fear or
psychological stress on the animal. Other criteria used to evaluate methods of euthanasia are
discussed below.
The application of these principles requires professional judgement, well-maintained equipment, and
technical competence, coupled with an understanding of the animal, its behaviour and its physiology,
and an understanding of the environmental and ecological impact, the sensitivities of other personnel,
and the concerns of the general public.
Training and Expertise of Personnel
Personnel must be adequately trained to ensure that euthanasia is carried out in the most humane
manner, and that it is done with professionalism and respect. Training should include: recognizing
pain and distress in the behaviour of an animal, proper methods of handling and restraining the
animal, proper application of the method and use of equipment, recognizing and assessing
unconsciousness, methods of ensuring the death of the animal, and recognizing and confirming death.
This training is beyond the scope of this module and should be provided by the institution separately.
Handling of the Animal Prior to Euthanasia
Any restraint of the animal necessary for humanely killing it should be done in a gentle, careful
manner to minimise fear, distress and/or pain. Where the restraint may cause fear, distress or pain,
the use of tranquillizers or sedatives should be considered.
Equipment Used to Perform Euthanasia
Any instruments or devices used for euthanasia of animals should allow for easy observation of the
animals and be professionally designed and kept in good repair so that their use effectively produces
rapid unconsciousness and death. They should be cleaned of all animal tissue, blood, or excreta after
each use.
Evaluating the Humane-ness of a Method for Killing an
Animal
When expert panels evaluate methods of killing animals, they use a number of criteria to determine
whether a given method is humane and therefore acceptable. The following criteria were used by the
AVMA Panel on Euthanasia, in their 2000 report:
http://www.avma.org/resources/euthanasia.pdf
•
•
•
•
•
•
•
•
•
•
•
ability to induce loss of consciousness and death without causing pain, distress, anxiety or
apprehension
time required to induce loss of consciousness
reliability
safety of personnel
irreversibility
compatibility with requirement or purpose
emotional effect on observers or operators
compatibility with subsequent evaluation, examination or use of tissue
drug availability and human abuse potential
compatibility with species, age, and health status
ability to maintain equipment in proper working order
•
safety for predators/scavengers should the carcass be consumed
Most of these criteria are self-explanatory. However, some will be discussed briefly: a) irreversibility;
b) scientific concerns relating to compatibility with requirement or purpose, and with subsequent
evaluation, examination or use of tissue; c) emotional effects on observers or operators.
Irreversibility - Ensuring the Death of the Animal
While any acceptable method of euthanasia rapidly renders the animal unconscious and insensitive to
pain, there must also be assurance of the death of the animal. Only when there is assurance that
blood is no longer being delivered to the brain because the heart has stopped and all other
movements such as respiration or reflex activity have ceased, should the animal be considered dead.
For some methods this involves two steps, the application of the method producing initial
unconsciousness, and secondly ensuring that the animal cannot regain consciousness or recover (for
example, exsanguination, opening the chest, severing major blood vessels, cervical dislocation after
CO2 euthanasia).
Scientific Concerns Relating to Choice of Euthanasia Method
Euthanasia methods may affect tissues and thus impact on subsequent biochemical, histological or
electron microscopic analyses. The direct effects are generally subtle or absent. Methods which cause
anoxia may produce pulmonary congestion and edema, depending on the rapidity of death. Tissue
hypoxia may produce alterations (e.g., metabolic acidosis). Alterations in the central nervous system
tissues probably occur more rapidly than to other tissues or organs, so it is important that tissues be
prepared as rapidly as possible following unconsciousness and death of the animal. Proper handling of
the animal prior to death to avoid stress or fear is also important.
Where there are scientific concerns about the impact on research results of a chemical method of
euthanasia and a physical method of euthanasia is proposed, it must be justified and approved by the
animal care committee based on scientific evidence. There should be an evaluation of the skills of the
personnel, and of the location and equipment to be used, before any physical method is approved.
Emotional and Psychological Impact on Humans
There may be emotional and psychological effects on the people performing the euthanasia, and on
observers, that must be acknowledged. In research laboratories staff may become attached to the
animals and experience uneasiness at having to euthanize them at the end of a study. Regular
exposure to the task may raise defence mechanisms that may result in callousness or rough handling
of the animals. A number of steps can be taken to minimize the negative impact of having to perform
euthanasia. Euthanizing animals can be made less distressful by ensuring that people are skilled in the
techniques, that they have a good understanding of the physiological events associated with dying
(assurance of unconsciousness, reasons for body movements), and are using the most esthetically
acceptable techniques. A forum for open discussion of an individual's concerns about euthanasia, and
support, should be available. Any person who feels uncomfortable with euthanizing an animal should
discuss it with his/her supervisor or the veterinarian.
Modes of Action of Euthanasia Agents
Euthanasia agents cause death by: a) brain hypoxia; b) direct depression of neurons necessary for life
to function, and; c) physical disruption of brain activity and destruction of neurons necessary for life to
function.
Choosing an Acceptable Method of Euthanasia
There are many methods available for humanely killing an experimental animal. Before any method is
used, it must be considered by the animal care committee during protocol review. The approval of the
proposed euthanasia method should always include consultation with a veterinarian. Appropriate
records should be kept of euthanasias, method/drug and personnel involved.
A number of the commonly accepted methods of euthanasia, with a few key points related to those
methods (advantages, disadvantages, comments), are presented here. Full discussions of a given
agent or method are available in the listed references. In particular, Appendix XIV of the CCAC Guide Methods For Euthanasia By Species (Methods In Order Of Acceptability) - should be consulted. The
reader is encouraged to bookmark that section of the CCAC Guide for future reference.
Unacceptable methods or agents are merely listed in this module without comment. The reader is also
referred to the institutional veterinarian for additional information on any euthanasia method.
Classification of Euthanasia Agents
Methods of humanely killing animals are usually grouped as chemical (inhalation, injectable, or other)
or physical methods.
Chemical Agents - Inhalation
Inhaled euthanasia agents are delivered either as vapours (from a liquid) or gases to the animal,
usually in a closed chamber to avoid human exposure.
Volatile Inhalant Anesthetic Vapours
Any of the commonly used inhalation anesthetics (e.g., halothane, isoflurane, and others) can be used
to overdose and kill an animal. Rodent preference tests have indicated halothane is the least aversive
to inhale. Thus halothane is considered the most acceptable, and is the only one discussed here. All
these agents must be used in closed chambers (for small animals) and the vapours must be
scavenged to avoid human exposure.
Halothane Anaesthetic
Advantages:
•
•
•
easily achieve high vapour levels in closed containers
quick acting
relatively non-irritating to inhale
Disadvantages:
•
•
human health risk on exposure to vapours
must be used in circumstances that ensure no human exposure to vapours
Inhalant Gases
There are several gases that can be used to kill an animal, carbon dioxide, carbon monoxide, and inert
gases such as argon and nitrogen. Of these, carbon dioxide is considered acceptable for euthanasia in
small laboratory animal species, and is the only one discussed here.
Carbon Dioxide
Carbon dioxide (CO2) is heavier than air and is nearly odourless. Compressed gas cylinders of CO2 are
readily available. At concentrations above 70%, unconsciousness usually occurs in less than a minute.
Advantages:
•
•
•
inexpensive and readily available
rapid onset of unconsciousness
minimal risk for human exposure
Disadvantages:
•
•
•
•
•
•
difficult to ensure optimal concentration in chamber
irritating to inhale and to mucous membranes
aversion in rodents
longer to unconsciousness than inhalant anesthetics
neonatal mammals, amphibians and reptiles have higher CO2 tolerance and should not be
euthanized by CO2 exposure
should not be used in any animal with the ability of prolonged breath-holding
Comments
Welfare concerns regarding the use of CO2 for euthanasia have been raised. Among the issues are
questions of what concentration of CO2in the chamber is optimal for rapid unconsciousness with a
minimum of distress, avoiding the reflex respiratory reactions to anoxia, optimal procedure for placing
the animal(s) in the euthanasia chamber, and the irritant properties of CO2 when inhaled. When CO2
euthanasia is used it should be done according to an approved SOP (Standard Operating Procedure).
Chemical Agents - Injectable
Acceptable Injectable Euthanasia Agents
A number of commercially produced injectable euthanasia agents are available. These are discussed
here.
Barbiturates
All barbituric acid derivatives are generally excellent euthanasia agents if given intravenously at high
doses. Intraperitoneal administration can also be acceptable when the intravenous route would cause
distress. They act by depressing the central nervous system (anesthetic properties). Concentrated
solutions of sodium pentobarbital are the most widely used. The Canadian Veterinary Medical
Association (CVMA) considers intravenous injection of concentrated barbiturates to be the most
humane method of euthanizing companion animals. From the CVMA Position on Euthanasia:
"Intravenous injection of a concentrated barbiturate is widely considered the most humane method of
euthanising companion animals. It is rapid acting, reliable, and effective and is therefore superior to
all other forms of euthanasia." Records must be kept of all barbiturate use.
Advantages:
•
•
•
speed of action to unconsciousness
smooth induction
relatively inexpensive
Disadvantages:
•
•
•
controlled drugs available only to licensed veterinarians
intravenous injection requires skill in smaller animals
drugs persist in the animals and carcasses must be disposed of in a manner that prevents
scavenging
Comments
Intraperitoneal injection may be acceptable in some circumstances where intravenous injection would
be distressful, or where intravenous access is not available.
T-61
T-61 is an injectable euthanasia agent comprised of three drugs: a local anaesthetic, a strong
hypnotic, and a paralytic. It must be administered intravenously at the dose and rate recommended
by the manufacturer.
Advantages:
•
it is not a controlled drug and so availability is less restricted, however it needs to be ordered,
stored and used like a controlled drug because of the potential for abuse
Disadvantages:
•
•
must be given as recommended; intravenously, slowly
in dogs there may be vocalisation and muscle contractions upon injection
Comments
In most circumstances the disadvantages outweigh the advantages and barbiturates are the preferred
injectable agents.
Chloral Hydrate
Chloral hydrate is a sedative that has a slower action in depressing the central nervous system. Death
occurs due to hypoxemia as respiratory centres become depressed. It may be conditionally acceptable
in some species in high doses when given intravenously. (click here)
Chemical Agents - Other Routes
Tricaine Methane Sulfonate (TMS or MS 222)
Tricaine methane sulfonate is a benzoic acid derivative that is used for anesthesia of fish and
amphibians, and can also be used for euthanasia in these species. Stock solutions are dissolved in
water (concentration greater than 250 mg/litre) and buffered, and the animal is immersed in the
solution until dead.
Immersion in benzocaine hydrochloride solution may be an alternative to TMS for euthanasia of fish or
amphibians.
Unacceptable Chemical Euthanasia Agents
•
•
Strychnine (rodent pesticide)
Nicotine
•
•
•
•
Magnesium sulfate
Potassium chloride
Any neuromuscular blocker (paralytic)
Other toxic chemicals
Some of these chemicals may be acceptable for killing the animal if it is already deeply anesthetized,
for example at the end of a study when blood sampling is taking place and these chemicals are
injected to ensure death. Potassium chloride may be useful in the field to reduce the dose of
euthanasia drug needed, where the carcass may be accessible to scavengers.
Physical Methods of Euthanasia
Whether any physical method of killing an animal is humane depends very much on the skill and
training of the operator, and on properly functioning equipment. The skill of the operator must be
monitored. Properly applied, most physical methods can produce a humane death of the animal. The
use of any physical method requires justification to the animal care committee.
Cervical Dislocation
Cervical dislocation may be acceptable in small rodents and some species of poultry. Training and skill
of operator is essential.
Advantages:
•
•
•
done properly it produces rapid unconsciousness
accomplished without equipment
avoids tissue contamination with chemicals
Disadvantages:
•
•
•
skill and experience required
the procedure may be aesthetically unpleasant
limited to small animals
Decapitation
Decapitation involves severing the neck and head. Guillotines specifically designed for the procedure
should be used. The devices should be kept in good repair to ensure that decapitation is humanely
done.
Advantages:
•
•
rapid loss of consciousness
avoids contamination of brain with chemicals
Disadvantages:
•
•
•
animal must be carefully restrained
the procedure may be aesthetically unpleasant
personal injury may occur
Stunning
Stunning the animal by a blow to the head may be acceptable in small or young animals with a soft
skull. Death of the animal must be ensured secondarily. Training and skill of operator is essential.
Penetrating Captive Bolt
A penetrating captive bolt "pistol" causes concussion by trauma to the brain. The bolt, spring loaded in
the front of the device, is driven out by a gunpowder charge, penetrating the skull and brain. With
accurate placement of the device against the skull of the animal, there is sudden unconsciousness
progressing to death of the animal due to brain trauma.
Advantages:
•
effective use causes immediate unconsciousness
Disadvantages:
•
•
•
•
the procedure may be esthetically disturbing
animal must be adequately restrained
training and experience of personnel essential
device must be properly maintained
Shooting
Shooting an animal appropriately can produce immediate unconsciousness proceeding to death, and is
a humane way to kill an animal. Using a firearm to kill an animal should only be done by highly skilled
operator, and in accordance with legal use regulations. Guidelines for aiming the firearm to the head
to penetrate the brain are available.
Shooting as a means of collecting wild animal specimens is acceptable under certain conditions,
depending on the skill of the personnel and use of appropriate firearms. The term "collection" is
appropriate for this method of killing wild animals.
Advantages:
•
•
immediate loss of consciousness if bullet destroys much of the brain
may be best option for killing wild or free-ranging animals
Disadvantages:
•
•
dangerous to personnel and to other animals in the area
may not directly hit the brain in free ranging animals
Kill Traps
Kill traps are occasionally used to collect field specimens of small rodents, but they do not always
result in a quick humane death of the animal.
Other Physical Methods of Killing Experimental Animals
The following method may be acceptable under certain conditions, if approved by an animal care
committee.
Thoracic (cardio-pulmonary) compression involves forceful pressure on the chest of small birds in the
field when alternative methods are not available. This is not an appropriate technique in laboratory
settings.
Adjunctive Methods
Some additional methods of physically killing animals are acceptable when the animals are already
deeply anaesthetized for other reasons. These adjunctive methods include exsanguination (bleeding
out the body) quick-freezing the body in liquid nitrogen, pithing (rendering the animal brain dead by
destroying the cerebral hemispheres using a sharp probe introduced into the brain cavity through the
foramen magnum).
Carcass Disposal
A Reminder: All experimental animal tissues/carcasses must be disposed of according to institutional
policy.
Summary Statement
The humane killing of animals requires knowledge, skill, respect for the animal, and an understanding
of the many factors that are part of the choosing a humane method. The primary welfare principles for
a humane method of killing an animal require that there should be very rapid (immediate)
unconsciousness and subsequent death, and there should be no pain or distress accompanying the
procedure.
Web-based References on Euthanasia
CCAC. 1993. Guide to the Care and Use of Experimental Animals, Volume 1, Chapter 12 Euthanasia.
2000 Report of the AVMA Panel on Euthanasia.
http://www.avma.org/resources/euthanasia.pdf
European Commission, Recommendations for Euthanasia of Experimental Animals, Part 1. 1996.
Laboratory Animals 30: 293-316.
http://www.lal.org.uk/pdffiles/LA1.pdf
European Commission, Recommendations for Euthanasia of Experimental Animals, Part 2. 1997.
Laboratory Animals 31: 1-32.
http://www.lal.org.uk/pdffiles/LA2.pdf
Additional Reference
Seaman WJ. 1987. Postmortem Change in the Rat: A Histologic Characterization. Iowa State
University Press.
Sample Questions
Which one of the following should be the primary criteria for choosing a method of euthanasia?
a.
b.
c.
d.
e.
Safety for personnel
Minimal cost and regulation
Rapid unconsciousness and pain free application
Aesthetic aspects of the procedure
Ease of administration
Which one of the following methods of killing an animal is unacceptable?
a.
b.
c.
d.
e.
Exposure to carbon dioxide
Administration of strychnine
Immersion in MS 222
Penetrating captive bolt
Shooting with a firearm
Appendix A - Timeline
Back to Module 02
Galen of Pergamum,
a Greek physician, catalogued
experiments performed by Alexandrian
physicians from 300BC, on differences
between sensory and motor nerves and
tendons.
129 - 199
0
125
Landmarks in Animal Based Research
150
175
Key Moral Statements
200
1250
St Thomas Aquinas
1260
declared in his Summa Theologiae that
humans were unique and opposed the
use of data based on vivisection on the
grounds that all other animals were
incapable of rationality because they
possessed no mind.
Andreas Vesalius
Illustrated public lectures of
anatomy with systematic
non-human vivisection.
1514 - 1564
1250
1275
1300
1325
1500
1525
1550
Engl
this o
Ro
162
The first people to r
subjects were profe
Rene Descartes
1595
exaggerated the Christian centered
prevalent humanist attitude into a
mechanistic philosophy, the concept of
beast-machine, which provided
a convenient ideology for early
vivisectionists.
us
William Harvey
lectures of
stematic
ection.
Demonstrated the circulation of blood using
animals, extrapolated the results to humans and
showed the value of vivisection for comparative
physiological investigation.
1628
1550
1575
1600
1625
1650
1675
Alexander Pope
1688-1744
Samuel Johnson
1709-1784
Marshall Hall
pioneered welfare issu
science by proposing t
cal procedures be reg
that took into consider
suffering of animals.
English essayists and poets who argued that animals may feel pain and that
this ought to be taken into consideration.
Robert Boyle
1627-1690
Robert Hooke
1635-1703
1790
Richard Lower
1631-1691
rst people to record their genuine concern for the welfare of some of their experimental
cts were professional physiologists based on a moral objection to perceived cruelty
Humphrey Primatt 1776
1824
extended the principle of justice beyond the
sphere of humans, to include all animals. The
anthropocentric world view was being challenged
by the notion that animals ought to be protected
for their own sake. Whether an animal had a soul
or not was no longer an issue.
UK Roya
for the pre
of Cruelty
Animals fo
Jeremy Bentham
August Comte
The beginnings of the theory
of utilitarianism. Shift from an
anthropocentric world view
towards animals’ capacity to
suffer.
Development of Positivi
differentiation between
empirical investigation
and ethical values.
1798
1748
C
D
a
m
p
v
1813
Francois Magendie
nd
ve
1783
1700
1725
1750
1775
Determined that many bodily
processes resulted from the
cofunctioning of several organs.
This was the basis of modern
physiology and set in train
numerous invasive animal-based
experiments.
1800
1825
1875
l Hall
The UK House of Commons was presented with a Bill aimed at regulating
vivisection, and a contrary Bill allowing for a regulation-free environment
resulting in the appointment of a first Royal Commission of Inquiry to
investigate laboratory procedures involving animals which found no
instances of animal abuse but recommended that animal experimentation
be regulated. The Cruelty to Animals Act received royal assent in 1876.
d welfare issues from within
by proposing that physiologiedures be regulated in a way
into consideration the
of animals.
1824
Charles Darwin
UK Royal Society
for the prevention
of Cruelty to
Animals founded.
Publication of The
Descent of Man and
Selection in Relation
to Sex and Expression
of the Emotions in
Man and Animals.
1871
omte
ent of Positivism:
tion between
nvestigation
al values.
1847
1871
1906
RSPCA changed its
position to objection
to painful procedures
being performed on
animals.
British Association for the
Advancement of Science
published guidelines that aimed to
minimize suffering and discourage
conducting experiments of
dubious scientific merit.
Continuous lobbying by anti-vivisection societies
resulted in the Second Royal Commission on
Vivisection. However, due to medical advances
described and the advent of World War I which
focused the UK society’s attention in other
directions, the public were less keen to condemn
all experimentation.
Crawford Long
1902
Discovered the
anaesthetic
properties of
ether.
Extraction
of the first
hormone.
1842
Claude Bernard
1882
1920
Demonstrated that a precise
approach to experimentation
must involve the study of one
parameter while holding other
variables constant.
Discovery of bacterium
responsible for tuberculosis.
Frederik Banting
Charles Best
Discovery of diphtheria
antitoxin which reduced
infant mortality from
40% to 10% .
813
ns.
n
Isolation of insulin.
William Morton
1909
Further work on the
anaesthetic properties of
ether led to technically
sophisticated surgical
procedures.
Chemical
treatment
for syphilis.
ased
1847
1825
1850
1875
1900
1925
1950
Appendix B - Standard Operation Procedures: Mouse
Microisolator Cage, or Individual Ventilated Cage,
Change (Extracted from an actual SOP) Back to Module 06
Purpose: To describe the procedures for changing mouse static microisolator cages, or individual
ventilated cages from a ventilated rack.
General Information
All persons working with animals must adhere to the following standard operating procedure. All
personnel who work with microisolator caging will be trained by ACS (Animal Care Services) to ensure
that the barriers are properly maintained. Entry into the facility is limited to those persons that have
been previously trained and have permission from the ACS Director.
Mice that are taken out of the facility for any reason will not be allowed to return.
All mice entering the facility must have complete health records accompanying the shipment. Mice
shipped into the SPF (Specified Pathogen Free) facility must arrive in undamaged filtered crates to be
acceptable. Shipping crates must be opened only by ACS staff for transfer to microisolator caging.
Cage Examination
Examine cages prior to each use to ensure that they are in good repair (e.g., no cracks). Stainless
steel lids should be free of any sharp edges or loose bars. The microisolator filter tops must be intact
and dry.
Equipment and Materials
•
•
•
•
•
Laminar Flow (Class II Biological Safety Cabinet) Cage Change Station
Spray bottle with disinfectant - Clidox® mixed as per instructions
Sterile supplies of diet, bedding and waters
Sterilized waste containers, paper towels and other required supplies
Specific equipment required for intended procedures, pre-sterilised
Changing Cages
NOTE: All manipulation of mice and cages must be performed in the laminar flow hood (Class II
Biological Safety Cabinet).
•
•
•
•
•
•
Put on booties, gown, face mask, hair bonnet, and gloves upon entering each room.
Prepare a container of disinfectant following label instructions for dilutions.
Make sure that the laminar flow hood is turned on and operational. Check the gauges to make
sure.
Prior to use of the hood for any reason, spray all surfaces inside the hood with Clidox®.
Place a container of disinfectant inside the hood for dipping hands between dirty and clean
cages.
Bring the necessary pre-sterilized cages complete with bedding and feed on a clean cart to the
change hood.
Change one cage at a time as follows: remove dirty cage from the rack and place it in the hood; place
a clean cage beside the dirty cage in the hood; remove the microisolator bonnet from the dirty cage;
place the dirty bonnet bottom side down under the hood; remove the cage card holder from the dirty
cage and place it on the clean cage; remove the dirty water bottle from the wire bar lid and remove
the wire bar lid; transfer the mice to the clean cage using a disinfected forceps; place the wire bar lid
on the clean cage; fill the hopper with feed and place fresh water bottle on cage; replace filter bonnet
on cage.
Submerse hands in the disinfectant solution, and repeat for next cage.
Change gloves between each group or type of animal (e.g., between different investigators, different
strains, or different protocols).
Housekeeping
•
•
•
Daily: Empty trash and replenish supplies as needed, on a daily basis. Keep the room neat,
clean, and clutter-free. Record all activities on the room logbook.
Weekly: Disinfect animal room floor with disinfectant once per week.
Monthly: Disinfect walls, light fixtures, cabinets, and cage racks. Change air filters.
Receiving New Shipments
Unopened cartons of animals will be taken directly to the facility. Prior to entering a room, the exterior
of the carton will be sprayed with Clidox® until wet. Allow the container to sit for 10 minutes before
taking it into a room.
Spray the shipping crate again with Clidox® and put into the laminar flow change hood. Gently make
an opening in the top of the shipping carton with scissors. Using forceps that have been disinfected
pick up the individual mice by the base of their tails and transfer them from the shipping carton into
the sterile cage. Follow the standard cage changing procedure.
Appendix C - Animal Identification Brochure
Back to Module 06
Animal Identification
It is important, in most cases, to be able to identify individual animals in a study. In a few situations,
small group identification may be enough if all of the animals in the group are to be treated in the
same manner and if individual variations in response to a treatment are not to be accounted for
separately.
Ideally, the method should ensure a permanent, indisputable identification of an individual and should
not place a burden on the animal. Letters and numbers are often used to identify animals and these
should not be so complex as to increase the risk of human error in the recording of data particularly in
the transcription process.
Ear tags are commonly employed in farm animals (e.g., sheep, cattle) and sometimes in small
laboratory animals, including mice. However, for these very small animals, the ear tags are heavy and
distort the ears and are not recommended. In addition, it is often the practice to use two tags so that
if one is lost, the animal will still be identified. Wing clips and leg bands are commonly used to identify
birds. Collars with identification tags may be used for dogs and cats.
Tattooing, either with numbers and letters or with other codes, provides a good means of
identification. There is some risk that the tattoos will fade and become illegible so they should be
checked on a regular basis. Tattooing may be done on any part of the body but consideration should
be given to a location where the tattoo may be read without excessive handling of the animal. Microtattoos which are small tattoo dots on specific parts of the body, can be useful particularly in albino
animals.
Temporary or short term marking with various dyes is possible in some birds and animals, especially
those with white areas of fur or feathers, or on the tails of some rodents. Marking pens may be used
to apply the marks but as with other systems, the marks must be clear and the identity of the animal
unmistakable. Depending on the type of marker used, renewal may be required as often as daily to
ensure reliability.
Ear notching is another way of identifying small rodents and pigs, although tattooing is preferred.
There is a universally recognized notching scheme for small rodents but others have devised their own
pattern of coding. It is important to place the punches and notches accurately so that identification of
the animal is indisputable.
The use of implantable microchips has increased since these provide a unique and tamperproof means
of permanently identifying animals. These chips are glass encased passive transponders, i.e., they
must be energized from outside for the information encoded on them to be released. This information
is usually just an identification number but some of the newer microchips will also indicate the body
temperature of the animal. The microchips and scanners are relatively expensive but are very useful
where positive identification of valuable animals is required. The passive integrated transponder (PIT)
tag is being widely used in salmonid aquaculture to identify tagged fish as they pass interrogation
sites during their migration. The PIT microchip is implanted in the body cavity of the fish, rather than
subcutaneously as in mammals.
Photo of a microchip injector and a microchip
Caption: Microchip with needle and injection device.
Photo of a microchip scanner
Caption: Hand held scanner. Data may be downloaded to a computer.
Several of the techniques described above may be associated with pain to the animal (e.g., ear
tagging, ear punching, microchip insertion, tattooing). This may necessitate the use of an analgesic.
Small animals (e.g., mice) may be anesthetized with a volatile anesthetic which allows for quick
recovery from the procedure. In larger animals, a local anesthetic may be sufficient to allow the
procedure. For some animals, the use of a systemic analgesic like butorphanol may be sufficient to
alleviate the pain.
Several other methods of identification have been used in the past but have limited if any application
in the laboratory. Toe clipping to identify newborn rodents in a litter or for some wild animals (e.g.,
salamanders) is now considered inappropriate. Branding is also not necessary for the identification of
cattle, horses, etc., as there are more humane ways to identify them. However, cattle or horses
purchased from farm animal sources may have been branded previously.
Fish are often identified by clipping fins or by means of small tags on the gills or on the back.
Field biologists are interested in identifying animals to follow their activities in their home ranges or on
migratory pathways. Radio transmitters are placed on a wide variety of animals from moose to small
birds. These transmitters emit a radio signal that allowed the investigators to locate the source of the
signals. Satellite and global positioning technology are also used. Some of these transmitters are
applied externally to the feathers or fur and are designed to fall off the animal after a certain period of
time. On birds, for example, the transmitter will be lost during the bird's annual molt.
Appendix D - Wild mouse versus Laboratory mouse
Back to Module 07
In each of the categories give a score for each mouse, either plus or minus. You may feel that
you do not know enough about either mouse to judge properly but you should allocate a score
based on what you think each mouse experiences. Consider the laboratory mice to be living in
an animal facility and that they are not part of an experiment.
The Five Freedoms
Freedom from hunger and thirst
Freedom from discomfort
Freedom from pain, injury and disease
Freedom to express normal behaviours
Freedom from fear and distress
Total
Wild
Laboratory
A second system of looking at the well-being of the two mice considers in more detail some of
the parameters that affect their lives. The scoring system is the same as for the five freedoms.
Wild
Laboratory
Food
ß Quality
ß Variety
ß Availability
Water
ß Quality
ß Availability
Activity
ß Social
o Variety
o Stress
ß Physical
o Variety
o Extent
Environment
ß Climate
o Variation
o Extremes
ß Space
o Size
o Complexity
Health
ß Infections
ß Injuries
ß Deficiencies
Total
Appendix E – Buprenorphine
Back to Module 10
Name: Buprenorphine (buprenorphine hydrochloride)
Trade Names: Temgesic, Buprenex
Preparation: Sterile injectable liquid packaged in 1ml vials, in boxes of 5 vials. Each 1ml contains
0.3 mg buprenorphine, in pH adjusted water.
Description: Buprenorphine is an opioid analgesic (narcotic agonist/anatagonist) classed as a
narcotic under the Controlled Substances Act.
Availability: Buprenorphine is a controlled substance classified as an experimental drug in Canada,
and must be obtained through a licensed veterinarian. Regulations regarding security and record
keeping apply.
Indications: For relief of moderate to severe pain, as in the post-surgical period.
Dosage: Consult the laboratory animal veterinarian.
Routes of administration: Intramuscular, subcutaneous, intravenous, oral
Duration of action: Six to twelve hours depending on the species.
Mechanism of analgesic action: Buprenorphine is a m agonist, and binds to m opiate receptors in
the central nervous system.
Clinical pharmacology: Pain relief begins about 15 minutes after intramuscular injection and
persists for 6 hours or longer. Some sedation also occurs. Buprenorphine is metabolised by the liver
and clearance is related to hepatic blood flow.
Physiological effects:
Cardiovascular: May cause a decrease in heart rate and blood pressure.
Respiratory: Depression of respiration is observed.
Drug Interactions: Buprenorphine in the presence of other narcotic analgesics, sedatives or
tranquilisers, or general anesthetics may cause increased CNS depression. When used in such
combinations the dose of one or both agents should be reduced.
Notes: Buprehorphine has been incorporated into jello and fed to rats. Oral administration requires
higher doses because a significant amount of the drug is metabolised during the first pass through the
liver.
References:
Pain Management in Animals. 2000 Flecknell P and Waterman-Pearson A (eds). WB Saunders,
London 184pp
Laboratory Animal Anesthesia. 1996. Flecknell P. Academic Press, London. 274 pp.
Appendix F – Butorphanol
Back to Module 10
Name: Butorphanol (butorphanol tartarate).
Trade Name(s): Torbugesic (injectable), Torbutrol (tablets).
Preparation: Sterile injectable liquid in 10ml or 50 ml mulitdose vials. Each ml contains 10mg of
butorphanol tartrate.
Description: Butorphanol tartrate is a synthetic, centrally acting, opioid agonist-antagonist analgesic
classed as a narcotic under the Controlled Substances Act.
Availability: Butorphanol is a controlled drug and must be obtained through a licensed veterinarian.
Regulations regarding security and record keeping apply.
Indications: For the relief of mild to moderate, deep or visceral pain, and for chronic pain. Some
sedation accompanies the analgesia.
Dosage: Consult the laboratory animal veterinarian.
Routes of administration: Intramuscular, subcutaneous, or intavenous
Duration of action: About 4 hours
Mechanism of analgesic action: Butorphanol is a synthetic opioid agonist-antagonist analgesic
acting at the m and k receptors.
Clinical pharmacology: Butorphanol tartrate is a member of the phenanthrene chemicals. The
chemical name is morphine-3, 14-diol, 17-(cyclobutylmethyl)-, (-),
(S-(R*,R*))-2,3-dihydroxybutanedioate (1:1). In animals, butorphanol has been demonstrated to be
4-30 times more potent than morphine. Butorphanol also has potent antitussive activity.
Physiological effects:
Cardiovascular: Cardiopulmonary depressant effects are minimal.
Respiratory: Cardiopulmonary depressant effects are minimal.
Drug Interactions: used with caution with other sedative or analgaesic drugs as these are likely to
produce additive effects.
Notes:
References:
Pain Management in Animals. 2000 Flecknell P and Waterman-Pearson A (eds). WB Saunders,
London 184pp
Laboratory Animal Anesthesia. 1996. Flecknell P. Academic Press, London. 274 pp.
Compendium of Veterinary Products, 6th Ed. 1999. Canadian Animal Health Institute. North
American Compendiums, Hensall, Ontario
Appendix G – EMLA
Back to Module 10
Name: EMLA
Trade Name(s): EMLA Cream
Preparation: EMLA (Eutectic Mixture of Local Anesthetics) is a eutectic mixture of lidocaine and
prilocaine bases, in 30 gm tubes. Each gram of cream contains 25 mg lidocaine and 25 mg prilocaine
as an oil/water emulsion.
Description: An emulsion of two local anesthetics.
Availability:
Indications: For use as a topical anesthetic for skin.
Dosage: Cream is applied to area of skin at least 30 minute and ideally one hour before analgesia is
needed at the site. Cream is left in contact with the skin for 5-10 minutes.
Routes of administration: Apply topically on the skin in the area where analgesia is required.
Duration of action: Local analgesia of intact skin is achieved after a 60 minute application under an
occlusive dressing. Analgesia increases up to 120 minutes. The duration of analgesia after I-2 hour
application is at least 2 hours and decreases after 5 hours.
Mechanism of analgesic action: Lidocaine and prilocaine are amide-type local anesthetic agents.
They stabilize the neuronal membrane preventing the initiation and conduction of nerve impulses
Clinical pharmacology: Lidocaine and prilocaine are amide-type local anesthetic agents. They
stabilize the neuronal membrane preventing the initiation and conduction of nerve impulses.
Physiological effects:
Cardiovascular:
Respiratory:
Drug Interactions:
Notes:
References
Pain Management in Animals. 2000 Flecknell P and Waterman-Pearson A (eds). WB Saunders,
London 184pp
Laboratory Animal Anesthesia. 1996. Flecknell P. Academic Press, London. 274 pp.
Compendium of Pharmaceuticals and Specialities 36th Ed. 2001. Canadian Pharmacists Association,
Ottawa, Ontario
Appendix H – Flunixin
Back to Module 10
Name: Flunixin (flunixin meglumide).
Trade Name(s): Banamine.
Preparation: Flunixin is an aqueous injectable solution containing 50 mg flunixin per ml, packaged in
50 ml and 100 ml multidose bottles.
Description: Flunixin is an injectable non-steroidal anti-inflammatory (NSAID) - analgesic.
Availability: Flunixin is a prescription drug.
Indications: It is useful as a medium duration analgesic and anti-inflammatory drug. Like other
NSAIDs, it also has some anti-pyretic properties. It is used for visceral or musculoskeletal pain in
horses.
Dosage: Consult your laboratory animal veterinarian.
Routes of administration: Intravenous or intramuscular.
Duration of action: Up to 12-24 hours.
Mechanism of analgesic action:
Clinical pharmacology : Flunixin meglumine is a potent, non-narcotic, nonsteroidal, analgesic agent
with anti-inflammatory activity (NSAID). Antipyretic activity has been demonstrated in laboratory
animals.
Physiological effects :
Cardiovascular:
Respiratory:
Drug Interactions: Drug interactions have not been studied.
Notes: Not to be used in food producing animals.
References
Compendium of Veterinary Products, 6th Ed. 1999. Canadian Animal Health Institute. North
American Compendiums, Hensall, Ontario
Pain Management in Animals. 2000 Flecknell P and Waterman-Pearson A (eds). WB Saunders,
London 184pp
Laboratory Animal Anesthesia. 1996. Flecknell P. Academic Press, London. 274 pp.
Appendix I – Ketamine
Back to Module 10
Name: Ketamine (ketamine hydrochloride)
Trade Name(s): Ketaset, Ketalean, Rogarsetic, Vetalar
Preparation: Ketamine hydrochloride is supplied as a injectable solution at a concentration of 100
mg/ml. The solution is slightly acid (pH 3.5-5.5). It is supplied in 10 ml and 50 ml bottles.
Description: Ketamine is a rapid acting anesthetic producing an anesthetic state termed "dissociative
anesthesia" because it appears to selectively interrupt association of the brain before producing
sensory blockade. It also produces analgaesla. Pharyngeal - laryngeal reflexes are maintained, as is
skeletal muscle tone.
Availability: Ketamine is a prescription drug.
Indications: Useful as a rapid onset, short acting general anesthetic in a wide range of species. It is
best used for short surgical procedures or for chemical restraint for minor procedures.
Dosage: A wide range of doses have been used in a wide range of species. Consult your laboratory
animal veterinarian.
Routes of administration: Ketamine can be administered parenterally by intramuscular,
subcutaneous or intraperitoneal injection.
Duration of action: Following injection of recommended doses, most animals become ataxic in about
five minutes; anesthesia lasts about 30 minutes. Recovery is generally smooth if the animal is not
stimulated or handled. Complete recovery usually occurs within four to five hours.
Mechanism of action: Ketamine produces an anaesthetic state termed "dissociative anaesthesia"
because it appears to selectively interrupt association of the brain before producing sensory blockade.
Clinical pharmacology: Ketamine hydrochloride is a nonbarbiturate general anaesthetic in the
cyclohexylamine group, with the chemical name 2-(o-chlorophenyl)-2-methylamino cyclohexanone
hydrochloride.
Physiological effects:
Cardiovascular: There is mild cardiac stimulation, blood pressure and heart rate are usually
moderately and transiently increased.
Respiratory: There is respiratory depression; the respiratory rate is usually decreased.
Drug Interactions: Ketamine can be used in effective combinations with a variety of sedatives or
tranquilisers.
Notes: Many protective reflexes are not abolished, and so monitoring the depth of anesthesia is not
similar to other general anesthetics. The eyes remain open with the pupil dilated so lubricating eye
ointments should be instilled.
References:
Compendium of Veterinary Products, 6th Ed. 1999. Canadian Animal Health Institute. North
American Compendiums, Hensall, Ontario
Pain Management in Animals. 2000 Flecknell P and Waterman-Pearson A (eds). WB Saunders,
London 184pp
Laboratory Animal Anesthesia. 1996. Flecknell P. Academic Press, London. 274 pp.
Appendix J – Ketoprofen
Back to Module 10
Name: Ketoprofen
Trade Name(s): Anafen
Preparation: Anafen injection is supplied in two concentrations, 10 mg/ml for small animals and 100
mg/ml for large animals, in multidose vials. It is also available as 5mg, 10mg and 20mg tablets.
Description: Ketoprofen is a nonsteroldal anti-inflammatory (NSAID) and analgesic drug of the
propionic acid subclass of carboxylic acid derivatives. Other propionic acid derivative NSAIDS Include
ibuprofen, naproxen and fenoprofen.
Availability: Ketoprofen is a prescription drug
Indications: Ketoprofen has potent anti-inflammatory activity against acute, subacute and chronic
infflammation, and is useful for management of post-surgical pain, and for relieving chronic pain (e.g.,
arthritis, etc.) and for the symptomatic treatment of fever.
Dosage: Consult the laboratory animal veterinarian.
Routes of administration: Ketoprofen can be injected by intramuscular, intravenous or
subcutaneous routes, or given orally (tablets).
Duration of action: Ketoprofen has a long duration of action and can usually be administered once a
day. Onset of activity occurs within 1/2 hour after parenteral injection and within 1 hour of oral
administration.
Mechanism of analgesic action: The primary mechanism of action is through inhibition of
prostaglandin synthesis by interfering with the cyclo-oxygenase pathway of arachidonic acid
metabolism.
Clinical pharmacology: Ketoprofen is propionic derivative; 2-(3-benzoyl phenyl) propionic acid.
Other propionic acid derivative NSAIDS include ibuprofen. Like other NSAIDs, ketoprofen produces
analgesic, anti-inflammatory, and antipyretic effects.
Notes: Like other NSAIDs, ketoprofen may cause irritation of the gastrointestinal tract, and should
not be used if there is impaired liver and/or kidney function, or coagulation disorders.
References:
Compendium of Veterinary Products, 6th Ed. 1999. Canadian Animal Health Institute. North American
Compendiums, Hensall, Ontario
Pain Management in Animals. 2000 Flecknell P and Waterman-Pearson A (eds). WB Saunders,
London 184pp.
Laboratory Animal Anesthesia. 1996. Flecknell P. Academic Press, London. 274 pp.
Appendix K – Xylazine
Back to Module 10
Name: Xylazine
Trade Name(s): Rompun, Anased, Xylazine HCl Injection
Preparation: Sterile injectable liquid packaged in 20 ml vials with a concentration of 20 mg/ml, or 50
ml vials with a concentration of 100 mg/ml.
Description: Xylazine is an alpha-2-adrenergic agonist sedative with analgesic properties.
Availability: Xylazine is a prescription drug.
Indications: Used alone for short minor manipulations or minor surgical procedures. See below for
use in combination with other drugs for anesthesia.
Dosage: Consult the laboratory animal veterinarian.
Routes of administration: Intramuscular, subcutaneous, or intravenous
Duration of action: Sedation lasts a few hours; the analgesia is of shorter duration. ROMPUN is a
potent sedative and analgesic as well as a muscle relaxant. Its sedative and analgesic activity is
related to central nervous system depression. Its muscle-relaxant effect is based on inhibition of the
intraneural transmission of impulses In the central nervous system. Sedation develops within 10-15
minutes after intramuscular injection and within 3-5 minutes following intravenous administration.
Mechanism of analgesic action: Xylazine is an alpha-2-adrenergic agonist sedative with analgesic
properties. Its sedative properties relate to central nervous system depression. The muscle relaxant
properties relate to inhibition of the Intraneural transmission of Impulses In the central nervous
system.
Clinical pharmacology: The sedation and muscle relaxation produce a sleep-like state, with
decreased respiratory and heart rates. There is significant depression of respiration and heart rate.
The decrease in heart rate is related to a transient change in conductivity of cardiac muscle due to a
partial atrioventricular block.
Physiological effects:
Cardiovascular: Significant depession of heart rate
Respiratory: Significant respiratory depression
Drug Interactions: Should not be used in combination with other tranquilisers. There are additive
effects when used in combination with anesthetics or analgesics and then required doses would be
lower.
Notes: In dogs and cats vomiting may occur soon after administration of xylazine.
References:
Compendium of Veterinary Products, 6th Ed. 1999. Canadian Animal Health Institute. North American
Compendiums, Hensall, Ontario
Pain Management in Animals. 2000 Flecknell P and Waterman-Pearson A (eds). WB Saunders,
London 184pp
Laboratory Animal Anesthesia. 1996. Flecknell P. Academic Press, London. 274 pp
Glossary
Index: A | B | C | D | E | F | G | H | I | K | M | N | O | P | Q | R | S | T | U | V | W | Z
Ad hoc
An ad hoc committee is one that is set up for a particular purpose.
Adrenalectomy
Surgical removal of the adrenal glands.
Aesthetically
Tastefully; concerned with appearance.
Agonist
When talking about drugs, this refers to a compound that stimulates or enhances activity of the cell
receptors.
Air lock
In biosafety facilities airlocks are usually two sets of doors in a corridor which when closed form a series of
air locks to prevent air moving in or out of the area. Only one door should be opened at one time.
Air pressure gradient
Different air pressures in an animal facility's ventilation system can be used to help create barriers to
contain or exclude microorganisms. A clean, biocontainment room or area, for example, would have a
higher air pressure than the corridor, so that when the door is open, the air flows out rather than in.
Albumin
Albumin is one of the major plasma proteins. It has many functions in the blood plasma including carrying
molecules (and some drugs) throughout the body.
Allergen
Any substance capable of producing a type 1 allergic reaction.
Alpha2 adrenergic receptor agonists
This is a group of pain relieving drugs that includes xylazine and medetomidine.
Alveoli
The alveoli in the lungs are the small grape-like clusters of outpouchings at the end of the lung's air ducts,
where gas exchange (oxygen, carbon dioxide) takes place.
Anaphylaxis
A generalized allergic reaction (also called anaphylactic shock) in sensitized individuals that results in lifethreatening symptoms which may include vascular collapse, shock and respiratory distress.
Anoxia
Anoxia means a total lack of oxygen. Often used interchangeably with hypoxia, which means a reduced
supply of oxygen.
Antagonist
When talking about drugs, this refers to a compound that opposes the activity of the cell receptors.
Anthropomorphic
This term is used to describe a person's attitude when he/she ascribes human attributes to an animal, or
to the animal's experiences or perceptions.
Anti-rejection drugs
People who have received an organ transplant need medication every day to prevent organ rejection.
Drugs called immunosuppressants or anti-rejection drugs help suppress the immune system to prevent or
reverse rejection. At the same time, these drugs may have side effects. A number of drugs are commonly
used for this purpose.
Anxiolytic
Removing or diminishing anxiety.
Arthritis
Inflammation of the body joints.
Asthma
Asthma is a condition of the lungs caused by constriction of the airways and mucus secretion. These
interfere with normal air movement in the lungs and cause wheezing.
Association of Universities and Colleges of Canada (AUCC)
AUCC is the national, non-governmental, not-for-profit organization that represents 93 Canadian public
and private not-for-profit universities and university-degree level colleges. They provide a forum for
discussion and a framework for action at the national level, facilitate development of public policy on
higher education, and encourage cooperation among universities. The AUCC is a member organization of
the CCAC, with four representatives on Council. http://www.aucc.ca/en/index.html
Atopy
Atopy is an inherited, familial tendency to develop some form of allergy such as hay fever, asthma,
eczema.
Autoclave
A machine used to sterilize by a combination of steam and pressure.
Axenic
Completely germ free. An axenic rat in an isolator would be one that is free of all other microorganisms.
Barbiturates
Barbiturates are a group of sedative/anesthetic drugs. Some of these have a high potential for abuse, and
all are controlled drugs.
Benzocaine hydrochloride
Benzocaine is a local anesthetic, often used topically in human medicine. It is also used to kill amphibians
and fish.
Bradycardia
Slowing of the heart rate.
Bradykinin
One of a group of small proteins that actively affect smooth muscle contraction in blood vessel walls, and
so have effects on blood pressure. Bradykinin dilates blood vessels, and also stimulates pain receptors.
Bronchoconstriction
Narrowing of the air passages in the lungs.
BSE Bovine Spongiform Encephalopathy
Bovine Spongiform Encephalopathy, also known as "Mad Cow Disease", is a chronic, degenerative disorder
affecting the central nervous system of cattle, which was first diagnosed in Britain in 1986.
[ Back to Index ]
Canadian Federation of Humane Societies (CFHS)
The Canadian Federation of Humane Societies is a national charitable body comprised of animal welfare
organizations and individuals, whose purpose is to promote compassion and humane treatment for all
animals. CFHS is a national voice on animal welfare issues, representing its member societies and
branches across the country, and is a member organization of the CCAC, with three representatives on
Council. The CFHS is committed to ending the suffering of animals by working with the public,
government, industry, the scientific community, educators and the media on both the national and local
levels. http://www.cfhs.ca/index.htm
Carcinogen
A carcinogen is a chemical, physical, or biological substance that is capable of causing cancer. Often used
in reference to chemicals or pollutants. Some carcinogens are used to produce cancer in research models.
Cardiac arrhythmia
Irregular beating of the heart.
Catecholamines
A group of compounds with active roles in the sympathetic and parasympathetic nervous systems. This
group includes adrenalin (epinephrin is another name for the same compound) which is a hormone
secreted by the adrenal gland, and noradrenalin (norepinephrin is another name for the same compound).
Effects include blood vessel constriction and increase in blood pressure, and increased heart rate.
Cerebral ischemia
Ischemia refers to a lack of adequate blood flow to an area. Cerebral ischemia refers to a lack of adequate
blood flow to the brain, which may be the result of a blood clot, blood vessel constriction or a hemorrhage.
Cervical dislocation
A physical euthanasia technique where pressure is applied to the neck to dislocate the spinal column from
the skull, normally only conducted on small animals.
Chemical restraint
Chemical restraint is the use of sedatives or anesthetics to control an animal's activity and thereby allow
certain procedures to be done with minimal stress to the animal.
Chloramphenicol
Chloramphenicol is an antibiotic once in common use in veterinary medicine but now banned in food
producing animals, due to the potential for bone marrow depression of blood cell production.
Chronic Wasting Disease (CWD)
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy of elk and other deer, first
recognized the 1960s in Colorado and identified as a prion disease in the late 1970s. Deer with CWD have
been found in game farms, and wild elk and deer with CWD have been found in the USA and Canada.
Class II Biosafety Cabinet
Class II biological safety cabinets are designed to have air flow drawn around the operator, into the front
of the cabinet to protect the person. Inside the cabinet a downward laminar flow of HEPA-filtered air
protects the product being handled. There are several types of Class II biosafety cabinets.
CNS
The Central Nervous System consists of the brain and spinal cord, in vertebrates.
Commensal
Commensal means a microorganism that normally lives in close contact with a human or animal without
causing any harm. This would include many of the bacteria normally present on a person's or animal's skin
or intestine.
Conjunctivitis
Inflammation of the membranes of the eyelids and around the eye.
Controlled drug
Controlled drugs are those listed in the schedule to the Narcotic Control Regulations of the Controlled
Drugs & Substances Act, and can only be obtained by a licensed doctor, dentist or veterinarian, or by
special permit for research purposes. The list includes the barbiturates, and many opiates.
Corticosteroids
Corticosteroids are hormones produced by the adrenal cortex that affect many body processes. Their
properties include anti-inflammatory actions, breakdown of protein and fat, activation in the nervous
system, the body's salt and water balance, regulation of blood pressure, and so on. Synthetic
corticosteroids are used in many treatments prescribed by physicians and veterinarians.
CT
Computed Tomography, also called Computerized Axial Tomography (CAT) scanning, is a diagnostic
medical tool that uses x rays to obtain a series of cross-section images of the body which are then
integrated using a computer.
Cystitis
Inflammation of the urinary bladder.
Cytokines
Cytokines are a group of compounds that are secreted primarily from white blood cells when the immune
system is activated. They stimulate both the humoral and cellular immune responses, and activate other
cells of the immune system. Some of the cytokines are called interleukins.
Cytolytic
Cyto - cells; lysis - destruction. Cytolytic ability or capacity refers to the ability to destroy cells, by a
microorganism, for example.
[ Back to Index ]
Dander
Small scales from hair or feathers which flake off and can become airborne.
Demeanor
Demeanor refers to a person's bearing ,or behavior towards others.
Discomfort
Discomfort is viewed as a mild form of distress.
Distress
Distress is a state associated with invasive procedures conducted on an animal, or with restrictive or other
conditions which significantly compromise the welfare of an animal, which may or may not be associated
with pain, and where the animal must devote substantial effort or resources to the adaptive response to
challenges emanating from the environmental situation.
Diurnal rhythm
Most animals (and plants) have a daily rhythm of activity. This is the diurnal rhythm.
Doppler equipment
A Doppler transducer is one that uses ultrasound to evaluate blood flow inside the body. The instrument
looks like a microphone, and sends and receives silent, high frequency sound waves.
[ Back to Index ]
Edema
Edema is the term used to describe the presence of abnormally large amounts of fluid in a tissue or organ.
Encephalitis
Refers to infection or inflammation of the brain. Ascending encephalitis refers to an infection of the
nervous tissue that travels up towards the brain and eventually affects the brain too.
Endorphins
Endorphins are a group of small proteins naturally occurring in the brain around nerve endings, that bind
to opiate receptors and thus can raise the pain threshold. Enkephalins are included in this group of
compounds.
Endpoint
The term "endpoint" can be defined as the point at which an experimental animal's pain and/or distress is
terminated, minimized or reduced by taking actions such as humanely killing the animal, terminating a
painful procedure, or giving treatment to relieve pain and/or distress.
Eosinophils, neutrophils, lymphocytes
These are different cell types in the white blood cell series. Their numbers change in many conditions such
as disease, distress, toxic states. White blood cell counts are useful for diagnostic purposes.
Epidural
The epidural space is the space around the spinal cord. Epidural anesthesia is produced by injecting the
analgesic drug (usually a local anesthetic, but it could also be an opioid) into the epidural space, usually in
the lumbar region. The drug directly affects the spinal cord or the nerve roots arising from the cord.
Euthanasia
To kill an animal painlessly, and without distress.
Eczema
A skin reaction typically resulting in itchiness, reddening, thickening and possibly oozing.
Exsanguination
Generally, the excessive loss of blood. As a secondary technique for euthanasia, it may involve
deliberately removing as much blood as possible from an animal.
Ex vivo
Outside the living body, for example removing a liver for studies in a perfusion apparatus.
[ Back to Index ]
First pass metabolism
First-pass metabolism refers to the phenomenon of a drug taken orally which is absorbed through the
intestinal wall and goes directly to the liver through the portal vein system and gets metabolized there
before reaching the target organ. This is avoided by giving the drugs by other routes (e.g., intramuscular,
intravenous).
Flexible film isolators
Isolators used for housing germ free (axenic) and gnotobiotic animals - a system with a complete barrier
against contamination.
Flight zone
The flight zone is an animal's "personal space". The size of the flight zone varies with the tameness of the
animal, and other animal-related factors. Wild animals have a much larger flight zone than most domestic
animals that are used to humans.
Fomite
Any object, that is not harmful itself, but may be capable of carrying an infectious microorganism on it and
thus transmit disease.
Formalin
Formalin, or formaldehyde, in solution is used as a tissue preservative and fixative for tissue specimens.
[ Back to Index ]
Garbage in garbage out (GIGO)
This term refers to the fact that computers will process nonsensical, faulty or incomplete input data and
produce nonsensical, faulty or incomplete output.
Gnotobiotic
A gnotobiotic system is a completely closed biological environment in which all organisms are known. For
example, a gnotobiotic mouse could be a mouse that has only one species of bacteria in its intestine.
[ Back to Index ]
Hantavirus Pulmonary Syndrome (HPS)
Hantavirus Pulmonary Syndrome (HPS) is characterized by a sudden onset of fever, pain, vomiting, and
onset of respiratory distress and prostration. Mortality rates are high despite symptomatic treatment.
Harried
A person or animal who/that is harassed or bothered.
HEPA filter
HEPA filter stands for High Efficiency Particulate Air filter. Such a filter will be at least 99.97% efficient at
removing all particles in the air down to a size of tenths of a micrometre.
Hepatic
Associated with the liver.
Hepatitis
Inflammation of the liver.
Histamine
A compound found throughout the body, but in highest concentrations in the white blood cell types that
are active in inflammation and allergy responses. Release of histamine and its effects on tissues are
responsible for some of the symptoms of allergic reactions.
Hypertension
High blood pressure.
Hypophysectomy
Surgical removal of the pituitary (also know as the hypophysis) gland at the base of the brain.
Hypotension
Low blood pressure.
Hypothalamic-pituitary-adrenal (HPA) axis
The HPA axis refers to the hypothalamic-pituitary-adrenocortical axis. It is the internal neuroendocrine
system that responds to stress and results in production of corticosteroid hormones that affect the brain,
the cardiovascular system, and other systems in getting the body ready for what is known as the "fight or
flight" mechanism.
Hypoxia, hypoxemia
A state of reduced oxygen in the blood or organs or tissues.
[ Back to Index ]
ICCVAM
ICCVAM, the Interagency Coordinating Committee on the Validation of Alternative Methods, is an agency
of the US National Institute of Environmental Health Sciences (NIEHS), working to develop and validate
new safety test methods, and to establish criteria and processes for the validation and regulatory
acceptance of toxicological testing methods.
Immunocompetent
An animal whose immune system is functioning normally and capable of mounting an antibody or cellmediated immune response, is immunocompetent.
Immunocompromised
An animal whose immune system is not functioning normally and that may be incapable of mounting an
antibody or cell-mediated immune response, is immunocompromised. Some genetic models are immune
deficient (immunocompromised). Anti-rejection drugs also result in compromise of the immune system.
Incubation period
A disease incubation period is the period of time between the actual infection and the first signs of
disease.
Infrastructure
This is the basic supporting structure of an organization. Physical infrastructure in this context refers to
the buildings and animal facilities used to house experimental animals.
In situ
In situ refers to something that is in its natural or original position.
Intercostal
Situated between the ribs.
Intraperitoneal
Within the peritoneal cavity. An intraperitoneal injection is made into the peritoneal cavity in the
abdomen.
Intravenous
Within a vein. An intravenous injection is made into a vein.
In vitro
In an artificial environment, for example in a test tube.
In vivo
Within the living body.
Irradiated food
There are several types of radiant energy used to sterilize foods. Gamma irradiation is commonly used to
sterilize laboratory animal feeds. The amount of irradiating energy is controlled to kill all microorganisms,
without affecting the quality of the food.
[ Back to Index ]
Keratin
The primary protein of skin, hair, and nails.
[ Back to Index ]
Macaque Monkeys
The macaques are a subgroup of the old world monkey and baboon family, that are widely distributed in
Africa, central and southeast Asia, southern China and Japan. This group includes the rhesus and
cynomolgus monkeys – two types of macaques used in biomedical research.
Malaise
A vague feeling of bodily discomfort or tiredness.
Malignant hyperthermia
An inherited condition in humans and some animals that results in an uncontrolled increase in body
temperature upon exposure to certain anesthetics. Halothane is a potent trigger of malignant
hyperthermia in susceptible animals or people. At one time, the genetic trait was common in commercial
pigs, but it has been almost eliminated through genetic selection.
Mannequin
An animal mannequin is an anatomical model used for teaching.
Mastitis
Inflammation of the mammary glands.
Metazoan
Multicellular organism (e.g. worms). A protozoan is a unicellular organism.
Microisolator (cages)
The term "microisolator" describes laboratory animal cages that have an air filter in a frame covering the
entire top of the cage, used to minimize contamination of the animals in the cage.
MRI
Magnetic Resonance Imaging (MRI) is a medical diagnostic technique that creates images of the body
using nuclear magnetic resonance. When a patient is placed into the cylindrical magnet, the process
follows three basic steps. First, MRI creates a magnetic steady state within the body using a magnetic field
30,000 times stronger than the earth's magnetic field. Then MRI stimulates the body with radio waves to
change the steady-state orientation of protons. It then stops the radio waves and “listens” to the body's
electromagnetic transmissions at a selected frequency. That signal is used to construct detailed internal
images of the body using a computer program.
MSDS
Material Safety Data Sheets (MSDS) are the internationally standardized way to document the hazardous
properties of chemicals and other hazardous agents. Chemical companies provide such data sheets, and
collections of MSDS sheets are available from several sources.
Mucosa
The mucosa is the membrane that lines body cavities including the intestine.
Mu receptors
Opioid drugs bind to the mu receptors in the brain and nervous tissue .
Muscarinic actions of acetylcholine
The muscarinic actions of acetylcholine include slowing the heart, increasing secretions from the salivary
gland and respiratory tract. Thus, anti-muscarinic drugs used as preanesthetics block secretions that
might clog up the respiratory track during anesthesia, and reduce the slowing of the heart rate. Muscarine
is a mushroom-derived alkaloid that mimics certain actions of the neurotransmitter acetylcholine, hence
the term "muscarinic" actions of acetylcholine.
[ Back to Index ]
NMDA Receptors
N-methyl-D-aspartate (NMDA) receptors are important for the transmission of some aspects of pain in the
central nervous system. In particular, they appear to be involved in the development of hypersensitivity
that accompagnies injuries or inflammation
Neuroleptanalgesia
Neuroleptanalgesia is defined as a state of quiescence, altered awareness, and analgesia produced by the
administration of a combination of a neuroleptic agent and a narcotic (opioid) analgesic.
Neuroma
A neuroma is a tumor growth of nerve cells and fibres. It may occur at the end of an injured nerve fibre.
[ Back to Index ]
OECD - Organisation for Economic Co-operation and Development
The Organisation for Economic Co-operation and Development (OECD) is made up of a group of 29
member countries sharing a commitment to democratic government and the market economy. Its work
covers economic and social issues, produces internationally agreed-upon instruments, decisions and
recommendations that allow for rules where multilateral agreements are necessary for individual countries
to make progress in a globalized economy. In the area of safety/toxicity testing of products, the OECD
guidelines set standards for such testing, including when animals are used.
[ Back to Index ]
Pain
Pain is an unpleasant sensory and emotional experience associated with actual or potential damage or
described in terms of such damage.
Peritonitis
Inflammation of the lining of the peritoneal cavity (abdomen).
Phenothiazine derivative drugs
This group of drugs includes commonly used tranquilizers in both human and veterinary medicine such as
acepromazine, chlorpromazine.
pH
pH is the symbol given to the hydrogen ion concentration in a liquid. pH 7 is neutral; higher pH is alkaline,
lower pH is acidic.
Piloerection
Standing up of the body hair.
Pithing
Pithing is a physical method of rendering an animal brain dead by destroying the cerebral hemispheres. A
sharp probe is introduced into the brain cavity through the foramen magnum to accomplish this.
Placenta
The membranes surrounding the fetus in the womb.
Positive pressure ventilated suits
Positive pressure ventilated suits are worn by personnel who have to work in maximum containment (level
4 biocontainment). They are sealed suits with their own filtered air ventilation.
Pre-emptive analgesia
This refers to the administering of pain relieving drugs before the pain is expected to begin, for example
giving analgesics before anesthesia and surgery begin.
Prion
Prions are infectious agents which (almost certainly) do not have nucleic acid - a protein alone seems to
be the infectious agent. Prions are small proteinaceous infectious particles which resist inactivation by
procedures that modify nucleic acids. Prion diseases are often called spongiform encephalopathies because
of the post-mortem appearance of the brain with large "holes" in the cortex and cerebellum.
Prion Diseases
Prion diseases are often called spongiform encephalopathies because of the post-mortem appearance of
the brain with large "holes" in the cortex and cerebellum. Examples include: scrapie in sheep; CWD
(chronic wasting disease) in elk and other deer; BSE (bovine spongiform encephalopathy) in cattle.
Human prion diseases include: CJD (Creutzfeld-Jacob Disease); Kuru; Alpers Syndrome.
Prostaglandin
The prostaglandins are a group of fatty acid compounds that have many effects throughout the body,
including activity in inflammation, smooth muscle contraction, regulating body temperature, and effects
on certain hormones.
Protozoan
A unicellular organism (e.g. bacteria). A metazoan is a multicellular organism.
Pulse oximeter
A pulse oximeter is an external probe that uses light bounced off the blood vessels under the probe to
determine level of oxygenation of the hemoglobin in the blood cells, through a computer calculation. The
pulses of blood with the heart rate are detected.
Purpose-bred laboratory animals
Animals specifically bred for scientific purposes.
[ Back to Index ]
Quarantine
Refers to the confinement or isolation of animals which may be carrying an infectious disease, usually for
a specified period of time, to allow for testing.
[ Back to Index ]
Red light test
To accurately evaluate changes in the level of activity in rodents, the room lights can be turned off and
using only a red light, their “normal” activity level will emerge after about 5 minutes. This is the “red light
test”.
Reflex
Reflex usually describes an immediate involuntary response evoked by a stimulus, for example the cough
reflex.
Ringtail
Ringtail in the rat is a condition in young suckling rats and mice, believed to be caused by low relative
humidity (less than about 30%). There are annular constrictions on the tail that may progress to necrosis
and sloughing of the tail tip. Ringtail can be prevented by maintaining relative humidity at approximately
50%.
[ Back to Index ]
Scalpel
A scalpel is a small surgical knife with a handle onto which a blade is placed.
Scavenge, scavenger, scavenging
In a surgery room, the scavenging system is used to exhaust all waste anesthetic gasses out of the room
to minimize risk of exposure of people to the anesthetic gas.
Scrapie
Scrapie is a transmissible spongiform encephalopathy - a fatal, degenerative disease affecting the central
nervous system of sheep and goats, believed to be caused by prions.
Sentinel animal
A sentinel animal is an animal known to be susceptible to an infectious agent that is placed in the area
suspected of being contaminated, for example in a new shipment of laboratory animals under quarantine.
That animal is then tested to see if it became infected or developed antibodies to infectious agents.
SPF (Specified Pathogen Free)
Specified Pathogen Free is a designation used to describe the health status of animals. It means that a
specific list of potentially infectious organisms have been tested for, and not found in an animal or group
of animals.
Standard Operating Procedure (SOP)
Standard operating procedures are written documents that describe in detail, step-by-step, how a
procedure should be done.
Stereotypy
In animal behavior, stereotypies have been defined as behaviors that are repetitive, performed the same
way each time, and seem to serve no obvious purpose or function. They may be compulsive behaviors
that have become displaced, perhaps due to boredom, frustration, or unresolved stress.
Subarachnoid space
Injections of local anesthetic into the lumbar subarachnoid space are often called "spinal anesthesia",
because they affect the preganglionic fibers in the spinal cord. The intent is to produce complete spinal
cord "denervation" including all sensory and motor neurons in the area.
[ Back to Index ]
T cells
The T cells are cells derived from the thymus that play a major role in a variety of cell-mediated immune
reactions. (B cells are also important in immunity because they synthesize and secrete antibodies which
protect animals from infection, viruses, etc.)
Tetanus
The disease called Tetanus occurs when a wound becomes infected with bacterial spores of Clostridium
tetani. These spores grow and produce a very powerful toxin which affects the muscles by producing
rigidity, and in severe cases convulsions. Treatment is difficult, and vaccination is the only way to provide
safe, effective long-term protection against tetanus.
Thoracotomy
Surgery through the chest wall.
Thyroidectomy
Surgical removal of the thyroid glands.
Tidal volume
The Tidal Volume in the lungs is the amount (volume) of air inhaled and exhaled with each normal breath.
[ Back to Index ]
Ultrasonic
Sound waves that are too high for humans to hear are "ultrasonic". The hearing range of many animals,
including rodents, extends much higher than for humans.
[ Back to Index ]
Vagus nerve
The vagus nerve is the tenth cranial nerve. It is the longest of the cranial nerves and its name derives
from the Latin meaning "wandering". The vagus nerve wanders from the brain stem through organs in the
neck, chest and abdomen. It supplies both sensory and motor fibres to parts of the neck and chest, and to
both the sympathetic and parasympathetic nervous systems to organs in the chest and abdomen.
Vasodilation
Dilation of the blood vessels (veins, arteries). Usually is accompanied by a drop in blood pressure.
Vector
A carrier, particularly the animal (e.g. an insect or mite) that can transfer an infectious organism from one
host to another.
Virulence
The degree to which an infectious organism can cause serious disease or invade the host tissue.
[ Back to Index ]
WHMIS
The Workplace Hazardous Materials Information System (WHMIS) is Canada's hazard communication
system. The system includes labelling of containers of "controlled products", provision of material safety
data sheets (MSDS) and worker education programs. The system is implemented through legislation, and
administered by Health Canada. Employers are required to
ensure that controlled products used, stored, handled or disposed of in the workplace are properly
labelled, MSDSs are made available to workers, and workers receive education and training to ensure the
safe storage, handling and use of controlled products.
Windup
In some forms of ongoing pain, the spinal cord and brain receive pain signals over a considerable period of
time. There can be adaptation by the spinal cord that results in the incoming pain signal becoming
amplified in a phenomenon known as windup pain. This means the pain signal reaching the brain is
stronger than before, and the ability of other pathways to modify (decrease) the sensation is diminished.
[ Back to Index ]
Zoonosis
A zoonosis is a disease of animals that may, under natural conditions, be secondarily transmitted to
humans - a disease that is communicable between animals and humans.