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Essentials For Animal Research:
A Primer For Research Personnel

                    B.T. Bennet
                    M.J. Brown and
                    J.C. Schofield

                    Beltsville, Maryland
                    National Agricultural Library
                    April 1990

Chapter 1. . . . . . . . . . . .   Regulations and Requirements
                                   by B. Taylor Bennett

Chapter 2. . . . . . . . . . . . . .  Alternative Methodologies
                                   by B. Taylor Bennett

Chapter 3. . . . . . . . . . . . .  Animal Care and Use: A
                                   Experimental Variable
                                   by John C. Schofield and
                                   Marilyn J. Brown

Chapter 4. . . .  . . . . . . . .  Principles of Anesthesia and
                                   by Marilyn J. Brown

Chapter 5. . . .  . . . . . . . . .  Principles of Aseptic
                                   by John C. Schofield

Chapter 6. . . . . . . . . . . . . . .  Perioperative Care
                                   by Marilyn J. Brown and
                                   John C. Schofield

Chapter 7. . . . . . . . . . . . . . . .  Euthanasia
                                   by B. Taylor Bennett

Chapter 8. . . . . . . . . . . . . . .  The Animal Welfare
                                   Information Center
                                   by Jean A. Larson,
                                   Kevin P. Engler and
                                   B. Taylor Bennett

Chapter 9. . . . . . . . . . . . Organizations, Associations
                                   and Societies
                                   by  Marilyn J. Brown,
                                   John C. Schofield and
                                   B. Taylor Bennett

Chapter 10 . . . . . . . . . . . . .  General References
                                   by John C. Schofield,
                                   Marilyn J. Brown and
                                   B. Taylor Bennett

     This manual was developed from the outlines of a course
entitled Essentials for Animal Research, originally developed at
the University of Illinois at Chicago for graduate students who
wanted to learn more about the use of animals in research than
generally covered in the training received in their chosen area
of concentration.  From its inception, the course has constantly
evolved to remain current with everchanging regulations and an
increasing awareness by graduate students of the issues
concerning the use of animals in biomedical research, teaching
and testing.  The course introduces those elements which have
become essential requirements for using animals in research,
teaching or testing programs. These requirements primarily center
around the responsibilities one assumes when they intend to use
animals in their work.  The ultimate responsibility lies with the
Principle Investigator who must have a working knowledge of the
regulations, be familiar with the factors that affect the
selection, acquisition and maintenance of experimental animals
and be aware of the ethical and social issues involved with the
use of animals in biomedical research.

     The goals and objectives established for developing the
class lectures are applicable to the material presented in this
manual.  With these goals in mind, the authors developed the ten
chapters included in this manual.  Remember it was not the
authors' intentions to present an exhaustive treatise on key
elements essential for conducting animal research in a manner
which assures individual and institutional compliance with
pertinent regulatory requirements, but rather an introduction to
the subject matter in a manner which will hopefully encourage
additional reading where appropriate.

     In writing this manual it was the author's intent to provide
the reader with:

      An appreciation and basic understanding of the regulatory
process and the means by which compliance can be assured.  An
overview of those factors which can affect the selection,
acquisition and maintenance of animals used in biomedical

      An understanding of the basic principles of controlling
pain and distress, preventing intraoperative infection and
assuring a humane death in the animals used.

      An awareness of the responsibilities that one assumes when
choosing to use laboratory animals.  These responsibilities would
include, but not be limited to, those which involve an obligation
to the institution, regulatory and funding agencies, the public
and the animals.

     The manual has been organized into ten chapters, the first
seven are intended to cover the specific objectives described
above.  The last three chapters contain resource information on
the Animal Welfare Information Center of the National
Agricultural Library, a list of organizations from which
additional information can be obtained and a list of general
references covering topics of interest to the investigator who
utilizes animals in research, teaching and testing programs.

     The authors would like to thank Drs. James Harwell, Louis
Ramazzotto and Richard Simmonds for their input and support
during the review process of this project.  To the staff of the
AWIC, we send our appreciation for their enthusiastic support
throughout the project and for their assistance in the final
stages of transferring the electronic version of the manual to
the library.

     This manual was produced as joint effort of the USDA
National Library of Agriculture and the University of Illinois at
Chicago and supported by cooperative agreement number

                            Chapter 1
                    Regulations and Requirements

                B. Taylor Bennett, D.V.M., Ph.D.


     Since the ultimate responsibility for compliance with
regulations that affect the care and use of animals lies with the
investigator, it is important that he/she have a working
knowledge of the basic regulatory requirements.  In this manual,
the types of regulations will be discussed under two broad
general headings: 

     1.   Involuntary
     2.   Voluntary

     Involuntary regulations can be defined as those required by
law or set forth as a condition of funding.  There are four types
of regulatory controls which can be considered as involuntary:

     1.   The Animal Welfare Act (AWA)
     2.   The Public Health Service Policy
     3.   The Good Laboratory Practices Act
     4.   The Requirements of Private Funding Agencies

     Voluntary regulations can be defined as those that an
individual or institution adheres to as part of their overall
commitment to research and academic excellence.  There are two
types of regulatory controls which can be considered as

     1.   Accreditation by the American Association for the
Accreditation of Laboratory Animal Care (AAALAC)
     2.   Requirements of Individual Users


Animal Welfare Act

     The Animal Welfare Act was first passed August 24, 1966, as
PL-89-544.  It was entitled the "Laboratory Animal Welfare Act"
and authorized, "The Secretary of Agriculture to promulgate such
rules and regulations, and orders as he may deem necessary to
effectuate the purposes of this Act."  The purposes of the
original act were to:

     1.   Protect the owners of dogs and cats from theft of such
     2.   Prevent the sale or use of dogs and cats which had been
     3.   Insure that certain animals intended for use in
research facilities were provided humane care and treatment.

     In charging the Secretary, Congress specifically prohibited
the promulgation of rules, regulations, or orders which would
interfere with the conduct of actual research.  Determination of
what constituted actual research was left to the discretion of
the research facility.

     The original Act covered non-human primates, guinea pigs,
hamsters, rabbits, dogs and cats.  Humane treatment was required
while they were at the dealers or research facility and while
being transported by dealers.  Dealers were required to be
licensed.  Research facilities which used, or intended to use,
dogs or cats and either purchased them in commerce or received
any federal funds were required to be registered.

     The Secretary also established regulations and standards for
the implementation of unannounced facility inspections and for
the maintenance of specific records by dealers and research
institutions.  Responsibility for administering the Act was
delegated within the United States Department of Agriculture
(USDA) to the Administrator of the Animal and Plant Health
Inspection Service (APHIS).  Enforcement duties are the
responsibility of the APHIS Deputy Administrator for Regulatory
Enforcement and Animal Care (REAC).  The actual inspections are
conducted by 42 Veterinary Medical Officers working under one of
the five REAC Sector Supervisors.  The Sector offices are located
in Fort Worth, Texas, Tampa, Florida, Annapolis, Maryland,
Minneapolis, Minnesota and Sacramento, California.

     In 1970 the original Act was amended (PL- 91-579) and
renamed the Animal Welfare Act.  The amended Act covered broader
classes of animals and included those used in exhibitions and
sold at auction and regulated anyone involved in these
activities.  The definition of an animal was expanded to include
all warmblooded animals.  The definition of a research facility
was expanded to include those institutions using covered live
animals and not just dogs and cats.  These facilities were
required to file an annual report.  Civil penalties were also
added for refusing to obey a valid cease and desist order from
the Secretary.  The term "handling" was added to the basic
categories for which standards were to be created and the
phrase "adequate veterinary care" was broadened to include the
appropriate use of anesthetics, analgesics and tranquilizers.

     The intent of the original Act to prohibit interference with
research was clarified and the Secretary was enjoined from
directly or indirectly interfering with, or harassing in any
manner, research facilities during the conduct of actual research
or experimentation.  The determination of when actual research
was being done was still left to the discretion of the research
facility itself.

     In 1976, the Animal Welfare Act was further amended to
enlarge and redefine the regulation of animals during
transportation and to combat the use of animals for fighting.
Essentially the Act was broadened to include all forms of
commercial transportation of animals and required all carriers
and intermediate handlers who were not required to be licensed
under the Act to register with the USDA.  It also expanded the
definition of a dealer and extended the record keeping
requirements to carriers and intermediate handlers.

     In 1976, the Secretary also promulgated regulations which
specifically excluded rats, mice, birds, horses and farm animals
from the definition of an animal.  This exclusionary language
effectively excludes over 80 percent of the animals currently
used in research, teaching and testing from coverage under the
Animal Welfare Act.

     In 1985 the Act was further amended with the passage of the
Food Security Act of 1985 (PL-99-198) which contained an
amendment entitled the "Improved Standards for Laboratory Animals
Act."  This amendment when fully implemented will strengthen the
standards for providing laboratory animal care, increase
enforcement of the Act, provide for collection and dissemination
of information to reduce unintended duplication of experiments
using animals and mandate training for those who handle animals.

     The most recent amendment to the AWA also includes
development of standards: for the "exercise of dogs," for
"provision of a physical environment which promotes the
psychological well-being of primates," for limitation of multiple
survival surgeries, and to require the investigator to consult
with a veterinarian in the design of experiments which have the
potential for causing pain to insure the proper use of
anesthetics, analgesics and tranquilizers.  Each research
facility will have to show upon inspection, and include in their
annual report, assurances that professionally acceptable
standards for the care, treatment and use of animals are
being used during the actual research or experimentation.  As
part of these standards, the investigator is required to consider
alternative techniques to those which might cause pain or
distress in the experimental animals.

     The 1985 amendment requires the Chief Executive Officer of
each research facility to appoint an Institutional Animal
Committee consisting of at least three members including a doctor
of veterinary medicine and one member who is not affiliated with
the institution.  The regulations promulgated to implement the
amendment designate this committee as the Institutional Animal
Care and Use Committee (IACUC) and charge it to act as an agent
of the research facility in assuring compliance with the Act.
The Committee is required to inspect all animal facilities and

study areas at least once every six months, and to review the
condition of the animals and the practices involving pain to the
animals to insure compliance with the regulations and standards
promulgated under the Act.  The Committee is also required to
review once every six months the research facility's program to
assure that the care and use of the animals conforms with the
regulations and standards.

The Committee must file a report of its inspection with the
Institutional official of the research facility.  If significant
deficiencies or deviations are not corrected in accordance with
the specific plan approved by the Committee, the USDA and any
Federal funding agencies must be notified in writing.

     The Committee must also review and approve all proposed
activities involving the care and use of animals in research,
testing or teaching procedures and all subsequent significant
changes of ongoing activities.  As part of this review, the
Committee must evaluate procedures which minimize discomfort,
distress and pain and that when an activity is likely to cause
pain that a veterinarian has been consulted in planning for the
administration of anesthetics, analgesics and tranquilizers and
that paralytic agents are not employed except in the anesthetized
animal.  The IACUC must also determine that animals which
experience severe or chronic pain are euthanized consistent with
the design of study, that the living conditions meet the species
needs, that necessary medical care will be provided, that all
procedures will be performed by qualified individuals, that
survival surgery will be performed aseptically and that no animal
will undergo more than one operative procedure that is not
justified and approved.  Methods of euthanasia must be consistent
with the definition contained in the regulations.

     The IACUC must also assure on behalf of the research
facility that the principal investigator considered alternatives
to painful procedure and that the work being proposed does not
unnecessarily duplicate previous experiments.  To provide this
assurance the Committee must review the written narrative
description provided by the investigator.  This description must
include the methods and sources used in determining that
alternatives were not available.

     In reviewing proposed activities and modifications, the
IACUC can grant exceptions to the regulations and standards, if
they have been justified in writing by the principal

     In addition to the above requirements, the research facility
is required to provide training in the following areas to
scientists, animal technicians and other personnel involved with
animal care and treatment:

   1.   Humane practice of animal maintenance and

   2.   Research or testing methods that minimize or eliminate
the use of animals or limit pain or distress.
   3.   Utilization of the information service of the National
Agricultural Library.
   4.   Methods whereby deficiencies in animal care and treatment
should be reported.
   The regulations require that each research facility establish
a program of adequate veterinary care that includes: appropriate
facilities, personnel and equipment; methods to control, diagnose
and treat diseases; daily observation and provision of care;
guidance to personnel on the use of anesthetic, analgesic and
euthanasia procedures and pre and post-procedural care.  Specific
requirements for maintaining records and filing annual reports
are included in the regulations along with a miscellaneous
section containing a variety of requirements to which a research
facility must adhere.

Public Health Service Policy

     The Public Health Service Policy on Humane Care and Use of
Laboratory Animals can be found in Chapter 4206 of the NIH Manual
and Chapter 1-43 of the PHS Manual.  The NIH originally initiated
the Policy in 1971.  It was extended to all PHS activities
January 1, 1979, and was revised in the spring of 1985 with
implementation to be effective January 1, 1986.  With the passage
of the Health Research Extension Act of 1985 (PL-99-158), the
Policy was further revised and the Director of the NIH was
required by law to establish guidelines which heretofore had only
been a matter of PHS policy.  An additional revision was released
in September 1986 which reflected the changes required by this

     Under the PHS policy, each institution using animals in
PHS-sponsored projects must provide acceptable written assurance
of its compliance with the Policy.  In this Letter of Assurance
the institutions must describe:

   1.   The Institutional Program for the Care and Use of
   2.   The Institutional Status.
   3.   The Institutional Animal Care and Use Committee (IACUC).

   The Institutional Program must include a list of every branch
and major component, the lines of authority for administering the
program; the qualifications, authority and responsibility of the
veterinarian(s), the membership of the Institutional Animal Care
and Use Committee and the procedures which they follow must be
stated.  The employee health program must be described for those
who have frequent animal contact.  A training or instruction
program in the humane practices of animal care and use must be
available to scientists, animal technicians and other personnel
involved in animal care, treatment and use.  The gross square
footage, average daily census and annual usage of each animal
facility must be listed.

   The Institutional Status must be stated as either Category one
(1) (AAALAC accredited) or Category two (2) (nonaccredited). 
Institutions in Category two (2) must establish a reasonable plan
with a specific timetable for correcting any departures from the
recommendations in the Guide for the Care and Use of Laboratory
Animals (86-23).

   The IACUC must be appointed by the Chief Executive Officer and
consist of at least five members; one of whom is a veterinarian
with program responsibility, a practicing scientist, an
individual whose expertise is in a non-biological science and an
individual who is not affiliated with the institution.  This
Committee must use the Guide to review the animal facilities and
the institutional program for humane care and use of animals at
least once every six months and prepare reports of these
evaluations for the responsible institutional official.  The
Committee must review and approve animal-related components of
proposals and significant modifications made in ongoing
activities involving the care and use of animals.  The Committee
is responsible for reviewing concerns involving the care and use
of animals and making recommendations to the institutional
official regarding any aspect of the animal program, the
facilities, or the personnel training.  They are also
authorized to suspend activity involving the care and use of
animals as set forth in the PHS Policy.

   In reviewing the animal care and use component of a proposal,
the IACUC must confirm that the project will be conducted in
accordance with the AWA and consistent with the recommendations
in the Guide.  In addition, all procedures are reviewed to assure
that pain or distress will be minimized and that (when necessary)
appropriate anesthetics, analgesics and tranquilizers will be
used.  The living conditions and medical care available must be
appropriate for the species used, and personnel conducting the
procedures must be appropriately trained and qualified.  Methods
of euthanasia should be consistent with the recommendations of
the American Veterinary Medical Association Panel on Euthanasia.

     The investigator is responsible for completing a proposal in
accordance with recommendations in the PHS Policy and the
instructions contained in the PHS 398 application packet.  As of
October 1988, the instructions for completing 398 can be found in
two locations within the application package.  On page 6 the
research investigator's responsibilities for assuring the humane
care and use of animals are clearly addressed.  Detailed
instructions for completing Section F of the Research Plan which
describes the use of Vertebrate Animals can be found on page 21.

     The institution is responsible for maintaining all the
necessary records to document compliance with the PHS Policy and
for filing annual reports which detail any changes in the program
and indicate the dates of the semi-annual inspections and
programmatic reviews.

     The PHS Policy described above is intended to implement and
supplement the "U.S. Government Principles for the Utilization
and Care of Vertebrate Animals in Testing, Research and
Training."  The nine principles are published in the PHS Policy
and in the Appendix of the Guide.  All those responsible for the
design, supervision and review of the animal care and use
component of a proposal should be familiar with this document.

Good Laboratory Practices

     In 1978 the Food and Drug Administration adopted the Good
Laboratory Practices rules which applied to all regulated parties
who conduct nonclinical safety assessment studies.  The rules
require the creation of Standard Operating Procedures for all
aspects of the study including animal care and use.  A Quality
Assurance Unit must be established to conduct internal inspection
of practices and records to insure compliance with established
policies and procedures.  In general the recommendations
contained in the Guide would suffice in terms of animal care when
adherence is properly documented.

Private Funding Agencies

     In recent years the requirements of many private funding
agencies which fund research projects involving the care and use
of laboratory animals have changed.  It is important to obtain
the requirements from the agency before spending time preparing a
proposal.  Some of these agencies not only require review of the
proposal by the IACUC, but require proof of accreditation by
AAALAC.  In many instances, the proposals must be reviewed and
approved prior to submission.


            American Association for the Accreditation of
                  Laboratory Animal Care (AAALAC)

  AAALAC was originally chartered April 30, 1965, as a voluntary
organization that accredited institutional programs of animal
care and use. AAALAC is governed by a Board of Trustees composed
of representatives of 32 professional organizations.  An
18-member Board-appointed Council on Accreditation makes
recommendations based on the results of site visits to evaluate
an institution's compliance with the recommendations contained in
the Guide.  This is a peer review process in which standards are
being continually upgraded to reflect current knowledge in
laboratory animal medicine and science.  In its accreditation
program the AAALAC Council uses the Guide more as a compilation
of regulatory "standards" and not as a set of "recommendations."

     Since the AAALAC accreditation program and the Guide are so
closely linked, a brief review of the Guide's history and its
current contents are warranted. In 1963 the first Guide for
Laboratory Animal Facilities and Care was published by the
Institute for Laboratory Animal Resources (ILAR) under a contract
from NIH.  Since its original release the Guide has been revised
in 1965, 1968, 1972 (when the title was changed to the Guide for
the Care and Use of Laboratory Animals, 1978 and 1985.  In the
most recent revision, the organization of the chapters was
changed to reflect the increasing role and responsibility of the
institutional program in establishing acceptable standards for
the care and use of laboratory animals.  The first chapter is now
Institutional Policies.  The remaining four chapters are
Laboratory Animal Husbandry, Veterinary Care, Physical Plant and
Special Considerations.  Prior to an AAALAC site visit, each
institution is required to prepare a description of the
institutional facilities and programs using the AAALAC Outline
for Description of The Institutional Animal Care and Use Program,
which follows the Guide's chapter headings.

     Once accredited, an institution must submit an annual report
describing changes in the program and facilities and documenting
the annual usage of animals.  Site visits occur at least every
three years and these visits consist of an inspection and review
of policies, procedures and facilities which comprise the animal
care and use programs inclusive of selected animal usage areas.
Should deficiencies be identified in a previously accredited
program, the institution is either granted a probationary period
in which to make specified changes, or if the deficiencies are
major, accreditation could be withdrawn.

Individual Users

     The instructions for completing PHS 398 clearly define the
roles and responsibilities of the investigator in assuring proper
care and usage of laboratory animals.  In addition to this
requirement, it should be understood that any type of care or use
of an animal which results in the creation of nonexperimental
variables can potentially compromise the integrity of an entire
project.  As part of their commitment to scientific excellence,
users should provide the impetus for setting and maintaining high
standards for the care and use of laboratory animals within their
individual and collective institutions.  Failure to do so invites
increased internal and external regulatory requirements which can
drain limited institutional research resources.  Good animal care
is good science; the practice of good science should be the
primary goal of all who have chosen careers in the scientific


   In summary, the regulations that affect the use of animals in
research, teaching and testing programs are numerous.  A working
knowledge of the applicable regulations is necessary if the
principal investigator is to insure that proposals for funding
contain the necessary information and to assure that the conduct
of all research proposals is in compliance with the requirements
of the regulatory and funding agencies.  While the ultimate
responsibility for compliance rests with the principal
investigator, institutional policies should be designed to
provide those responsible for compliance with the necessary
resources to do so.


     Application for Public Health Service Grant, PHS, 398.
Revised October, 1988. OMB No. 0925-001.

     Animal Welfare Act (Title 7 U.S.C. 2131-2156), as amended by
PL-99-198, December 12, 1986.

     Guide for the Care and Use of Laboratory Animals, NIH
Publication No. 86-23.

     Public Health Service Policy on Humane Care and Use of
Laboratory Animals.  Revised as of September 1986.

     Non-Clinical Laboratory Studies.  Good Laboratory Practice
Regulations.  Register, December 22, l978, Part II, pp.

     Public Law 99-198. Code of Federal Regulations, Title 9,
Subchapter A, Animal Welfare l989.

     Townes, J.  Federal Regulations an Overview, Lab Animal,
July-August (9)4:l6-22 (1980).

                           Chapter 2
                      Alternative Methodologies

                   B. Taylor Bennett, D.V.M., Ph.D.


   In the regulations promulgated to implement the Animal Welfare
Act as amended in 1985, the research facility must provide
assurances that the principal investigators considered
alternatives techniques to painful procedures and to provide
guidance concerning research and testing methods that limit the
use of animals or minimize the animals' distress.  In this
chapter the reader will be introduced to the classical concept of
alternatives with a brief discussion of each major category
including a limited number of examples.  For more indepth
coverage of the subject, the reader is encouraged to obtain the
latest bibliography on alternative techniques available from the
Animal Welfare Information Center of the National Agricultural
Library (see Chapter 8).

   In recent years the term alternative techniques has come into
common usage in the current controversy involving the use of
animals in research, teaching and testing.  It is a term that has
different meanings to different people and this difference
largely depends on which side of the issue one is found.  To many
biomedical researchers, alternative techniques refer to those
which can be used in addition to the more traditional animal
models.  These techniques can focus on specific biological
functions and in many cases reduce the numbers of animals used. 
Therefore these
methods are an adjunct to the more commonly used animal models. 
the so-called abolitionist who seeks the immediate end to all
animal research, teaching and testing, the term alternative
to those techniques which can entirely replace the use of
The dictionary, defines alternative as: "offering or expressing a
choice."  The dictionary also defines technique as "a method of
accomplishing a desired aim."  By combining these definitions,
term alternative technique becomes "one which offers a choice in
accomplishing a desired aim."

     In designing an experiment which involves the use of animals
to confirm or refute a theory, one should consider all the
techniques that could be used to gather the necessary data.  From
this review, choose the method which offers the best chance of
generating the necessary information in the most economical
manner.  Economy, in this context, refers to time, actual cost
and the number of animals used.  By considering the choices that
are available for accomplishing the desired aim of the experiment
and choosing the one that offers the best chance for success, one
has met the requirements of this literal definition of
alternative techniques.

     Since a literal definition provides a rather simplistic
approach to dealing with our responsibility for reducing the
potential pain and suffering of animals that must be used, it is
necessary to develop a working definition of the term.  In Dr.
Rowan's book, Of Mice, Models and Men, he defines the term
alternatives to refer to those techniques or methods that replace
the use of laboratory animals altogether, reduce the numbers of
animals required, or refine an existing procedure or technique so
as to minimize the level of stress endured by the animal.  Since
stress can be difficult to describe and quantitate, for the
purpose of this manual it will be replaced by the term distress.
The working definition of alternative techniques thus evolves to
"those techniques which replace the actual use of animals, reduce
the numbers used, and/or refine the techniques to minimize the
potential for the animal to experience pain or distress."

   This concept of the 3 R's is not new.  It first appeared in a
book by Russell and Burch published in 1959 entitled The
Principles of Humane Experimental Technique.  In the original
work, the authors defined the 3 R's as follows:

 "Replacement means the substitution for conscious living higher
animals of insentient material.  Reduction means reduction in the
numbers of animals used to obtain information of given amount and
precision.  Refinement means any decrease in the incidence or
severity of in-humane procedures applied to those animals which
still have to be used."

   In this text the authors included nonrecovery techniques in
anesthetized animals, as well as tissue culture, as replacement
methods.  Reduction included statistical techniques which were
designed to reduce the actual numbers needed in the study.  The
use of better animals was also encouraged as a means of reducing
actual numbers used.  Refinement referred to techniques that
reduced the potential for pain and distress.  This approach still
holds today.  It is the principles of Replacement, Reduction and
Refinement that will be covered in this chapter.  To attempt to
address these issues for all the uses of animals that fall under
the general rubric of research, teaching and testing is far
beyond the scope of this manual.  Therefore the comments that
follow will address only broad issues with some specific examples
for the purpose of clarification.

   Prior to discussing the replacement of animals with non-animal
models, the word animal must be defined.  On the surface this
appears an easy task.  Common sense would tell us that an animal
is one of the two major kingdoms of living organisms.  The
dictionary defines an animal as "any of a kingdom of living
beings typically differing from plants in capacity for
spontaneous movement and rapid motor response to stimulation."
In the Definition of Terms promulgated to implement the amended
Animal Welfare Act an animal is defined as:

   "any live or dead dog, cat, nonhuman primate, guinea pig,
hamster, rabbit, or any other warm blooded animal, which is being
used or is intended for use for research, testing,
experimentation, or exhibition purposes or as a pet.  This term
excludes: birds, rats of the genus Rattus and mice of genus Mus
bred for use in research, and horses and other farm animals such
as but not limited to livestock or poultry used or intended for
use as food or fiber, or livestock, or poultry used or intended
for use for improving animal nutrition, breeding, management, or
production efficiency, or for improving the quality of food and

     The PHS Policy defines an animal as "Any live, vertebrate
animal used or intended for use in research, research training,
experimentation, or biological testing or for related purposes."
On the other hand the Guide defines an animal as "any warm
blooded vertebrate animal."  For the purposes of this manual, and
to be consistent with most approaches to discussing alternative
techniques, an animal will be any living vertebrate, with the
caveat that any model system which moves down the phylogenetic
scale from the generally acceptable animal model will be
considered an alternative.


     Alternatives which replace animal models can be classified
into the following broad general categories:

                    Use of Living Systems
                    Use of Nonliving Systems
                    Use of Computer Simulation

Use of Living Systems

In Vitro Techniques - The most commonly recognized nonanimal
living systems are those which fall into the broad category of in
vitro methods such as organ, tissue and cell culture.  These
techniques afford the investigator the greatest control of the
"test subject's" environment.  Since these systems will not work
when the incorrect combination of atmosphere, humidity,
temperature, pH and nutrients are provided, they tend to minimize
the effects that nonexperimental variables can have on the final
outcome of a study.
Generally, when suboptimal environments are provided for an in
vitro system, the problem becomes one of loss of all experimental
results and not just the production of compromised results.  The
most commonly used of the in vitro methods are cell culture
techniques for monoclonal antibody production, virus vaccine
production, vaccine potency testing, screening for the cytopathic
effects of various compounds and studying the function and make
up of cell membranes.  The potential uses of in vitro techniques
are almost limitless and will continue to expand as more is
learned about the various organs and their component tissues and
cells, and as the technology of maintaining in vitro environments

Invertebrate Animals - Invertebrates are another type of living
system which can be used to replace the more commonly used
laboratory animals.  Over 90 percent of the animal species thus
far identified are invertebrates.  An invertebrate which has long
been used in biomedical research is the fruit fly, Drosophila
melanogaster -- a classic model for the study of genetics.  This
species also can be used for detecting mutagenicity,
teratogenicity and reproductive toxicity.  The marine
invertebrates represent different species which have not been
widely investigated.  However in neurobiology a number of
different marine species have been well characterized and used to
study the physiology of the nervous system.

Micro-Organisms - The micro-organisms represent a third living
system which has been used to replace traditional animal models.
The Ames mutagenicity/carcinogenicity test uses Salmonella-
typhimurium cultures to screen compounds that formerly required
the use of animals.  Such systems allow for an almost limitless
number of compounds to be tested which can create an interesting
dilemma.  The more compounds that can undergo screening, the more
compounds that will be potentially available to test in animals.
Alternative techniques can replace the number of animals at a
given step in the screening process.   However, use of
alternatives may increase the number of compounds that must be
finally tested in intact animals.

Plants - Plants offer another alternative living system which can
be used to replace animals in studies of basic molecular
mechanisms.  There is very little morphological and functional
difference between the organelles isolated from plants and those
isolated from animals.  The rigid cell wall of plants, however
limits their applicability for use as undisrupted cells.

Use of Nonliving Systems

Chemical Techniques - The most widely used nonliving model system
involves the use of modern chemical techniques.  This is
particularly true of the analytical techniques which can be used
to identify substances and to determine their concentration or
potency.  Immunochemical techniques use the binding capacity of
highly specific antibodies to seek out minute quantities of
antigen.  A classical example of this technique can be
demonstrated by the currently used techniques for identifying
bacterial toxins.  Toxin identification previously required the
injection of as many as several hundred mice with supernatant
from cultures of suspected contaminating bacteria.  These new
antibody techniques save animals and speed up confirmation of a
tentative diagnosis.  By adding a color marker to the Enzyme
Linked Immunosorbent Assay system (ELISA), the whole process
becomes a commercially available test kit such as those used in
home pregnancy detection.  A test that previously required the
use of a rabbit now can be performed using an over-the-counter
test kit.  There are a variety of chemical techniques that can be
used to determine the presence of a particular chemical reaction
or the presence of an enzyme necessary for a specific reaction.
At the most basic level, the identification of a particular
chemical structure in a compound can provide a great deal of
insight into the potential reactivity and thus the resulting
toxicity of a given substance.

Physical and/or Mechanical Systems - The use of physical and/or
mechanical systems to replace living animals of even the highest
order has application in teaching specific skills and/or
reactions to a well defined set of predetermined circumstances.
The use of computer-linked mannequins in teaching basic
principles of medicine and applied techniques can be best
illustrated by the mannequins used to train people in
cardiopulmonary resuscitation.

     Historical data can be used for analyses in a variety of
databases commonly used in the field of epidemiology.  However,
while the body of potentially useful information that already
exists in a variety of sources is immense, it may not always be
in a format which permits ready accessibility for evaluation.
For this reason, retrospective epidemiological studies are often
the subject of fairly heated debates.  Yet with the increasing
access to historical data available on existing computer
programs, this problem may to a large extent be overcome in the

Use of Computer Simulation

   The standout in the alternative techniques controversy is the
claim made for computer simulation as a means of virtually
replacing the use of living animals. In order for a biological
phenomena to be adapted to a computer model, the basic processes
must be expressed in a mathematical formula.  Once a formula is
developed then an enormous number of variables can be introduced
and swiftly processed.  The key element for success is the
generation of a program from the mathematical formula.  The more
complete the formula, the more useful the program.  The problem
is that many of the questions being asked of an animal model are
not  defined well enough to develop the necessary mathematical
model.  As the core knowledge of the biological processes expands
so will the opportunities to use computer simulation to replace
the number of live animals being used.


   In discussing the ways to reduce the numbers of animals used,
the definition of an animal and the principle of moving down the
phylogentic scale must also be kept in mind.  The four broad
categories for reducing the number of animals used are:

                    Animal Sharing
                    Improved Statistical Design
                    Phylogenetic Reduction
                    Better Quality Animals

Animal Sharing

     Sharing of animals can significantly reduce the number of
animals used within a given institution.  Between institutions,
sharing is more difficult, but can be effective as demonstrated
with the Primate Supply Information Clearinghouse, Regional
Primate Research Center (SJ-50) University of Washington,
Seattle, WA 98195.  This service has reduced the total number of
primates used by helping to optimize the usage of those already
in facilities throughout the country.

     Sharing can be as simple as allowing someone to practice a
surgical approach on an animal that has been, or is to be
euthanized for other purposes, or providing organs or tissues at
the time of necropsy.  Sharing becomes more complicated when
attempting to maximize the use of control animals, but it can
significantly reduce the number used within an institution.  If
two studies involve the need to perform a sham operation, the
administration of compounds by identical routes, the use of
standard control diets or the need to condition animals to a
particular environment, control animals could be shared within
the institution.  Animal sharing would require some form of
centralized clearing process within the Institutional Animal Care
Program to match the needs of the various investigators and their

Improved Statistical Design

     Anyone who has ever taken a course in experimental design or
applied statistics has been bombarded with the importance of
consulting with the statistician during the design phase of the
experiment and not when the data collected needs to be analyzed.
Improper design of experimental protocols and/or the failure to
use appropriate statistical methods can result in the usage of an
inappropriate number of experimental animals.  A variety of
design strategies are available which can reduce the number of
animals needed in a given study. Experimental protocols which
utilize serial sacrifice, group sequential testing and crossover
designs can significantly reduce the numbers of animals required.

     The availability of low cost statistical packages for almost
every computer on the market permits more and more investigators
access to sophisticated data management and analysis.  This
accessibility makes possible the use of design criteria and
complicated statistical analysis which heretofore have been
largely confined to institutions with large statistical support
units.  With this ability at their finger tips, investigators
should be able to maximize the analysis of the data generated
from each animal used, thus reducing the total numbers of animals
necessary for a particular set of data.

Phylogenetic Reduction

     Projects which can be designed to use one of the myriad of
invertebrate species instead of a non-human primate species
represent a type of phylogenetic reduction which was discussed as
a replacement technique. Such broad jumps across the phylogenetic
scale are not always possible, but less dramatic shifts can
significantly reduce the numbers of higher species being used in
research, teaching and testing.  In many instances, the theory of
phylogentic reduction has been blurred by a species's use as a
companion animal with little regard for phylogenetic ranking.
The animals chosen for project usage should be the least advanced
from a phylogenetic standpoint that will provide the necessary

     The principle of phylogenetic reduction is generally well
accepted as a way to reduce the number of animals used, but it
often brings many hidden difficulties. As one descends the
phylogenetic scale, the available information on the maintenance
and use of these animals in a biomedical setting often becomes
difficult, if not impossible, to obtain.  When choosing a study
model, it is critical that the principal investigator take into
account the ability of the institution to provide appropriate
care for the species chosen.  Phylogenetic reduction is an
important means of decreasing the number of animals used, but
should be practiced carefully and with the full knowledge of the
requirements of the species chosen.

Better Quality Animals

   It is a rare study in which the initial cost of the animals to
be used represents the single most expensive aspect of the study.
For this reason it can often be false economy to select the
source of the animal based on cost alone.  When purchasing
laboratory animals, it is important to keep in mind that cost and
quality are usually directly correlated.  By choosing the best
quality animal in terms of health status, the possibility that
animals will be lost or data compromised by the intrusion of a
concurrent disease condition is minimized, if not eliminated.
Choosing the best quality of animal, in terms of genetic status,
will virtually insure the consistency of animals from study to
study.  This requires an institutional commitment to the use of
animals of defined health status and limits the investigators to
the animal sources approved by the institution.  Mixing of
animals of different health status is a disaster waiting to
happen and may negate all the benefits derived from the use of
quality animals.

     The role of the investigator and their staff in assuring the
integrity of an animal colony cannot be overemphasized.  In
choosing a source of animals, a veterinarian should be consulted
to insure that the best animals that can be effectively
maintained in the institution are purchased.  Animals of
different or unknown health status should never share the same
environment nor common equipment in the animal facility or in the
research laboratory.


     Refinement refers to techniques which reduce the pain and
distress to which an animal is subjected. For the purpose of this
manual these techniques can be classified into the following
broad categories:

                    Decreased Invasiveness
                    Improved Instrumentation
                    Improved Control of Pain
                    Improved Control of Techniques

Decreased Invasiveness

     A hallmark of most of the new diagnostic and therapeutic
techniques used in human medicine is the minimal degree of
invasiveness that is required to successfully perform a procedure
to obtain a given set of data. In many instances these techniques
are applicable in the research environment and can be adopted for
use in animals.  A sophisticated example could be the use of
Magnetic Resonance Imaging for results that formerly required
euthanasia of multiple animals along a time curve to obtain assay
tissue.  Today one animal can provide all the information along a
given curve.  A less dramatic example is the vascular access
device which permits repeated samples or injections in a single
animal instead of using several animals.  Invasiveness reduction
methods are available in almost every area of biomedical
research, and in project design, it is important to identify and
use these methods wherever possible.  Not only do they represent
an alternative technique, but they generally provide much more
consistent and reproducible data.

Improved Instrumentation 

Monitoring Animals -In this age of microelectronics, fiber optics
and laser instrumentation, the potential for refining techniques
used in animal experimentation seems almost limitless.  Improved
instrumentation can minimize animal distress by reducing the
level of restraint and/or manipulation necessary to obtain
biological samples.  Included in this category are the use of
tethers in a variety of species to allow continuous access to the
various organ systems, while permitting the animal virtually
unrestricted movement within its primary enclosure.  The
advantages of these systems are numerous, not the least of which
is minimizing a variety of nonexperimental variables associated
with prolonged restraint.

Analyzing Samples -Once obtained, samples can be analyzed in very
small volumes for a multitude of parameters. Examples of this can
be found in the commercially available diagnostic laboratory
equipment which require only micro-liter blood samples to perform
a variety of diagnostic tests.  The use of smaller sample sizes
permits the use of smaller animal species and prevents the need
to euthanize many of these species to obtain the necessary volume
of blood. It is now possible to obtain serial blood samples from
small laboratory rodents which reduces the number of animals
necessary to obtain data over the length of the study.

Improved Control of Pain

     The Animal Welfare Act requires "that the principal
investigator consider alternatives to any procedure likely to
produce pain or distress in an experimental animal" and in any
practice which could cause pain to animals that a doctor of
veterinary medicine is consulted in the planning of such
procedures for the use of tranquilizers, analgesics and
anesthetics.  Since appropriate anesthetic and analgesic agents
can minimize the potential pain and distress experienced by
animals, an entire chapter of this manual is devoted to the
principles of using these agents.  Suffice it to say, that of all
the possible ways that the 3 R's can be utilized this is an area
where the laboratory animal veterinarian can often be of most
help to the investigator.

Improved Control of Techniques

   Proficiency in the handling and restraint of animals makes it
easier to perform a variety of routine procedures with minimal or
no pain or distress to the animals involved.  Animals are
creatures of habit and when proper handling is part of their
regular routine, the degree of distress caused by the procedures
is minimized.  Animals can be trained or conditioned to accept a
variety of procedures which if suddenly forced upon them can be
Almost every animal commonly used in the laboratory responds
positively to a little tender loving care.  It's inexpensive,
readily portable, safe even at the highest doses and spreads
rapidly through the staff.  To develop the proper techniques and
gain confidence in their use requires training by someone with
appropriate experience. This can be the veterinarian, a member of
the animal care staff or a fellow investigator.  Whomever it may
be should be sought out before a new species or technique is
incorporated into the study.  This will reduce the potential
distress of all animals involved in the study up to and including
the principal investigator.


     In this chapter, the use of alternative techniques has been
defined in terms of the present regulatory requirements and the
principles of Replacement, Reduction and Refinement were
introduced.  In summary, the reader should consider a fourth
R--Responsibility.  The use of animals in teaching and research
brings with it a responsibility to minimize animal pain and
distress.  The adoption of the 3 Rs as part of the process of
planning and conducting projects using laboratory animals will go
a long way toward implementing Responsibility--the fourth R.


     Animal Welfare Act (Title 7 U.S.C. 2131-2156) as amended by
PL 99-198, December 23, 1985.

     Guide for the Care and Use of Laboratory Animals, NIH
Publication No. 86-23.

     Models for Biomedical Research: A New Perspective, l985.
National Academy Press, Washington, DC; l985.

     Navian, J.B.  Animal Models in Dental Research.  The
University of Alabama Press.

     Paton, William. Man and Mouse Animals in Medical Research.
Oxford University Press, New York, 1984.

     Public Health Service Policy on Humane Care and Use of
Laboratory Animals.  Revised as of September 1986.

     Public Law 99-198. Code of Federal Regulations, Title 9,
Subchapter A, Animal Welfare, 1989.

     Rowan, A.N.  Of Mice, Models, and Men: A Critical Evaluation
of Animal Research. State University of New York, 1984.

     Russel, W.M.S. and Burch, R.L.  The Principles of Humane
Experimental Technique, Methuen and Co, Ltd., London, 1959.

    U.S. Congress, Office of Technology Assessment. Alternatives
to Animal Use in Research, Testing, and Education. (OTA-BA-273,
Feb. 1986)

     Webster's Ninth New Collegiate Dictionary, Merriam-Webster,
Inc., Springfield, MA; 1986.

     Wessler, S. l976. Animal Models of Thrombosis and
Hemorrhagic Diseases, NIH Publication No. 76-982.

                          Chapter 3
          Animal Care and Use: A Nonexperimental Variable

             John C. Schofield, B.V.Sc., M.R.C.V.S.
                 Marilyn J. Brown, D.V.M., M.S.


     The response of a laboratory animal to an experimental
variable is influenced by a variety of genetic and environmental
factors.  An understanding of these factors is necessary to
control their affects and minimize the potential influence of
nonexperimental variability on the final outcome of a given
experimental protocol.  Minimizing nonexperimental variability
can optimize the use of animals in a given study.

     Since the 1930's, the concept of genetic makeup, or genotype
of an animal, combining with the developmental environment to
produce the phenotypic expression of the animal had been well
accepted.  A useful concept concerning the relationship of
genetic and environmental factors--dramatype--was proposed by
Russell and Burch in 1959.  They defined dramatype to be the
pattern of performance in a single physiological response of
short duration relative to the animal's life time.  It is
determined by phenotype and the immediate environment in which
the response is elicited.  This concept distinguishes between the
developmental environment, which directly interacts with genetic
factors, and the proximate or immediate environment, which acts
upon the combined system.  Simplified, genotype plus
developmental environment equals phenotype and phenotype plus the
immediate environment equals dramatype.  This concept stresses
the interrelation of the genetic background of the animal, the
environment in which it is raised and housed and the laboratory
environment in which the animal is used or tested.

     Genotype may be controlled through the use of genetically
defined animals produced in structured breeding systems or by
genetic engineering.  This is easiest to accomplish through the
purchase of genetically defined animals from reputable suppliers.
In-house breeding programs are difficult and time consuming to
maintain in a manner which assures genotypic integrity.  If such
colonies must be used, it is advisable to consult a geneticist to
design a breeding program that produces animals of defined
genetic characteristics.  A genetic monitoring program might also
be required to define the genetic makeup of the animals produced.
This can be an expensive proposition and requires some expertise
to perform.  The phenotype can be influenced by regulating
environmental conditions in which the animals are reared.  For
uniform dramatype, the environmental conditions in which the
animals are tested must be controlled.

     This chapter will deal with three broad categories of
nonexperimental variables: physical factors, chemical factors and
microbial factors.  Physical factors which will be discussed
include: cage design and construction, temperature, humidity,
ventilation, light intensity and photoperiodicity, noise,
bedding, watering systems, feeding, housing systems, shipping and
handling.  Chemical factors to be discussed will include
contaminants of food, water, bedding, and air.  Microbial factors
will be discussed in terms of some of the common viral, bacterial
and parasitic diseases that can affect laboratory animals.  The
total of all of the components included in these three broad
categories combines with the animal's genetic background to
constitute Russell and Burch's concept of phenotype and
dramatype.  It is important to appreciate that our knowlege of
the effects of nonexperimental variables is rapidly expanding and
the purpose of this chapter is to introduce the reader to this
subject rather than present an exhaustive or complete treatise.

                         PHYSICAL FACTORS

     The physical environment of laboratory animals may be
considered to consist of the animal room, or macroenvironment,
and the primary enclosure (cage), or microenvironment.  Cage
design and composition influence the interaction between micro
and macro environment.  Therefore the temperature, humidity,
airflow, concentration of waste gases, illumination and noise
levels within the cage may be quite different from that monitored
at the room level.  Each of these factors represents an important
nonexperimental variable that will be discussed in more detail.

Cage Design

     Cage design and construction material can influence the
study results.  Galvanized caging material or rubber bottle
stoppers can serve as a source of trace minerals which could
affect the results of studies where the level of these compounds
is being controlled.  Other important considerations include
whether contact bedding can be used or if animals must be housed
on a wire floor.  The type of sample collection may require the
use of a metabolic cage, or observation studies may require the
use of clear rather than opaque caging.  The behavioral
characteristics of the animal will also dictate the type of cage
design used.  For example, some animals require perches, nesting
boxes or hiding places, and others require built-in restraint
devices such as the squeeze mechanisms often found in primate
caging.  Reproductive needs may require specific caging features.
In some species the male must have a method of escape from an
overaggressive  female.  Many neonates have inadequate
homeothermic mechanisms and will become hypothermic if not
protected by contact bedding or nesting material placed in the

Temperature and Humidity

     The temperature and humidity in the animal room
(macroenvironment) should be monitored and maintained within
published acceptable limits.  The temperature and humidity in the
microenvironment is more difficult to monitor and control.
Variations in temperature and humidity are influenced by such
factors as filter tops, hanging wire or solid bottom caging,
population density, animal activity level, cage location, and
temperature and humidity in the animal room itself. Variations in
temperature and humidity can have a variety of effects.  For
example, exposure to high temperatures will frequently cause
rabbits to lick their fur which can predispose them to the
formation of hairballs.  Very low humidity has been associated
with a rodent lesion called ring tail which is characterized by
annular constrictions and can result in loss of the tail.  More
subtle temperature and humidity effects include: altered drug
metabolism, increased disease susceptibility and decreased
reproductive efficiency.  These examples serve to illustrate the
need for controlled temperature and humidity in the animals'
micro and macroenvironment and the vital role it plays in the
generation of consistent, reliable data.


   Ventilation in animal rooms can have significant impact on the
health status of the occupants. Excessive odor is often the first
indication of a ventilation problem in an animal room.  The 
concentration of waste gases at the cage level is usually higher
than those detected at the room level.  Furthermore, the
concentrations capable of causing pathology are much less than
human sensory threshold levels.  Many design features affect room
ventilation including the location, number, and configuration of
supply and exhaust ducts.  Cage-level ventilation is further
affected by the presence and/or type of filter top on the cage as
well as the design and location of the cage relative to the room
airflow pattern.  Ventilation should be such that it keeps the
concentration of waste gases to a minimum, reduces the spread of
disease, provides a stable temperature and humidity and avoids


     Light intensity and photoperiodicity in animal rooms can
affect reproductive function and animal vision.  The
recommendation of the Guide for light intensity in animal rooms
is 75-125 footcandles (fc).  However, prolonged exposure to such
levels can cause irreversible retinal degeneration in albino
rodents and 25 fc has been suggested as a more appropriate
intensity for these species.  Variable light intensity control
devices such as dimmer switches or multiple bank lighting can be
installed to facilitate adequate light for observation and
husbandry yet provide lower intensity light for general animal
housing.  Cage position on a rack can be an important factor and
an 80-fold difference in light intensity can exist between the
upper and lower shelf locations.  Photoperiods or light/dark
cycles (usually given in hours as L:D) can modify reproductive
behavior and circadian rhythms. A daily light cycle which has 12
to 14 hours of light is usually recommended for most species. It
is important to keep the light intensity and periodicity
constant. Animal rooms should be equipped with automatic light
timers.  The presence of windows, either to the outside or to the
corridor, can affect reproduction in some animals.  Corridor
windows may be desirable for observational purposes, but they can
provide enough light to affect circadian rhythms in nocturnal
animals.  As with all environmental factors, the special
characteristics of the animal should be taken into consideration
when planning light cycles.  Duration and type of light can
affect estrus behavior.  Animals can have their reproductive
cycles manipulated by changing the light cycle.  This technique
has been used in several rodent species, cats, and farm animals.
Reversed light cycles can be used to accommodate circadian
rhythm, sleep and breeding studies within the normal working
hours in an institution.  Individual room timers provide a
facility with more flexibility to meet a variety of experimental


     Excessive noise can also disrupt animal breeding behavior.
Noise at excessive levels can cause mechanical damage to the
auditory system in both animals and man.  Some effects of noise
in animals include audiogenic seizures, eosinophilia, increased
serum cholesterol levels and increased adrenal weights. It is
recommended that noise levels in animal facilities not exceed 85
decibels (db).

Caging Accessories

   In addition to the microenvironmental effects of the physical
configuration of the primary enclosure as discussed above, other
aspects of the cage environment should be considered.  The
presence or absence of bedding material is dependent on the
species and situation.  For example, many breeding programs
utilize some form of bedding to improve neonatal survival.  An
ideal bedding material should be dustfree, nonpalatable,
absorbent, and free of microbial and toxic contaminants.  The
choice of watering system depends on species, experimental
design, and management factors.  Automatic watering systems are
expensive to install but can pay for themselves in labor savings
over time.  Automatic watering systems should be flushed daily
when used with low flow rates, such as in rodent rooms, to avoid
stagnation and minimize bacterial buildup.  When the study
protocol requires delivery of a compound in the water, or
measurement of daily intake is needed, water bottles or
pans are often used.  Choice of feeder and type of food is also
species and situation dependent.  Some species such as the
hamster are frequently fed on the floor of the cage because their
broad muzzle can make obtaining food from some rodent feeders
difficult.  Some species such as the rabbit do not readily
tolerate sudden changes in diet composition or formulation.  When
designing a study, it is important to consult someone
knowledgeable in the biology and husbandry requirements of the
species to be used, so that wherever possible, species variations
are taken into consideration.

Cage Size - Occupancy Standards

     Consideration should also be given to the cage size.  There
are specific cage size requirements set forth in the Guide for
the Care and Use of Laboratory Animals and by the Animal Welfare
Act.  Cage size requirements depend upon the species, weight or
size of the animal(s), number of animals in the cage and breeding
status.  In addition to the floor space requirements the
behavioral characteristics of the species, strain, and sex must
be considered when group-housing animals.  For very social
animals, individual housing may cause stress.  Even among social
animals, the formation of new groups can result in fatal trauma
from fighting.  Male mice will often fight when group housed,
whereas male rats usually do not.  Aggressive behavior can be
strain specific; for example, F344 male rats and C57BL mice are
generally considered to be more aggressive than other commonly
used strains.  Even in docile animals, overcrowding can lead to
fighting, cannibalism and stress.  Breeding activity can be
significantly modified by group housing arrangements.  For
example, group-housing female mice can lead to anestrus with
subsequent estrus synchronization with the introduction of a male


     The shipping of animals can be a significant physiological
stress.  Studies  have documented the effects that prolonged
transport, high ambient temperatures, lack of water and the
potential for microbial contamination may have on the research
data collected from animals exposed to such factors.  The
provision of climate-controlled transport vehicles and filtered
crates decreases these stresses.  Even under optimal shipping
conditions, it has been shown that it takes 1-5 days for the
immune system and body weights to return to normal.  It is also
important to remember that changes in feed, water, and housing
conditions can markedly affect newly arrived animals.  Animals
should be given an adequate period of time to equilibrate after


     The frequency and type of handling an animal receives is
another nonexperimental variable.  Investigators and technicians
should be familiar with and skilled in the correct techniques for
handling and restraining the species involved.  This can prevent
injury to either the animal or the handler.  Daily husbandry
routines may need to be scheduled around the research needs.
Close communication between the investigator and the animal care
staff can minimize handling stress.  For example, collection of
biological samples may be performed during routine cage changing.
This is particularly useful when chemical restraint is required
for either function.  Since many animals are creatures of habit,
regular handling may reduce stress.


     Chemicals found in the animal's environment may be
inherently toxic or their metabolism may result in the formation
of toxic products.  They may directly injure cells or interfere
with cellular homeostasis.  The possible effect of a chemical
depends on the concentration, the agent's physiochemical
properties, as well as the duration, frequency and route of
exposure and potential interactions.  These chemicals can
influence various body systems.  For example, it has been
demonstrated that chemicals can affect hepatic microsomal enzymes
which have many functions, including the biotransformation of
drugs and chemicals and regulation of oxygen radical removal.
Such chemical sources include: softwood bedding, room
deodorizers, insecticides, and ammonia.  Chemicals can also
target the immune system.  Some insecticides cause lymphopenia.
Heavy metals can decrease resistance to disease by the reduction
of antibody formation, altered  phagocytic capacity of
polymorphonuclear cells and macrophages, and suppression of
interferon production.

Food and Water

   Food and water can serve as sources for chemical contamination
of research animals.  Drinking water may be contaminated with
synthetic organic solutes such as pesticides. Trihalomethanes are
often found in water supplies as a result of the chlorination
process.  Some facilities hyperchlorinate or acidify water to
decrease microbial contamination; however, these techniques can
affect the immune response. Inorganic contaminants may include
heavy metals and nitrites.  Diets can also be a source of
contaminants such as estrogenic compounds, aflatoxins,
insecticides, and preservatives.  These compounds may occur
naturally in plant materials, remain as residues from
agricultural use, or be the result of contamination in storage or
the processing procedures.  Commercial diets assayed prior to
shipment are available and the results of this assay are printed
on the tag attached to each bag.


     Drug therapy, prior to or during a study, can compromise the
data obtained.  For example, tetracycline alters the immune cell
function through its ability to depress chemotaxis and
phagocytosis.  Aminoglycosides can have neuromuscular blocking
properties, and can have negative inotropic effects on cardiac
and arterial muscle.  Other agents having neuromuscular
depressant activity include tetracycline, lincomycin, and the
polymyxins.  It is important that investigators and the animal
care staff communicate about the effect that any medications may
have on study animals prior to the initiation of treatment.
Similarly, anthelmintics or insecticides given by the animal care
staff to treat parasitism problems, could affect research results
and must be considered in protocol design.

     Anesthetic agents are frequently part of experimental
protocols.  The researcher should balance appropriate levels of
analgesia, anesthesia, and chemical restraint with the possible
effects of these agents on the experimental results. For example,
the dissociative agent ketamine hydrochloride is widely used in
anesthesia and restraint because it is easy to administer, is
effective in a wide range of species and has a wide margin of
safety.  Besides the better known cardiovascular effects of
ketamine hydrochloride, this drug also has been shown to affect
intracellular cyclic AMP, cellular permeability and calcium
channels.  A pharmacologic knowledge of these drugs will aid in
selecting those best suited for each experimental protocol and
allow for more informed interpretation of results.  Consultation
with the institutional veterinarian regarding the use of
anesthetics and analgesics during the planning of potentially
painful procedures is now a legal requirement.

                        MICROBIAL FACTORS

     Pathogenic microbial agents can affect research by causing
clinical disease, lesions and death.  However, in laboratory
animals, infection more frequently is asymptomatic with carriers
who develop overt disease when stressed by shipping or
experimental manipulation.  Animals with latent infection may
show no overt disease but research results may be compromised
through subtle physiological, biochemical or histological

Bacterial Diseases

     Species-specific mycoplasmal and bacterial diseases are well
documented. There are a number of these pathogens associated with
commonly used laboratory animal species.  For example,
mycoplasmosis is an endemic disease in some conventional rodent
colonies.  It can cause respiratory and genital tract infections
thereby affecting exercise tolerance, sensitivity to anesthetic
agents, increased susceptibility to other respiratory pathogens,
decreased reproductive efficiency and a variety of immune system
anomalies.  The investigator using rabbits should be aware of the
incidence and significance of pasteurellosis as a cause of acute
and chronic disease.  Pasteurella multocida is very common in
conventional rabbit colonies and can cause upper and lower
respiratory tract infections, subcutaneous abscesses, middle and
inner ear infection and reproductive tract infections.  Some
species may serve as asymptomatic carriers of bacterial
infections which can cause severe clinical disease in other
species; therefore different species should not be mixed.
Bordetella bronchiseptica can often be isolated from clinically
normal rabbits and rarely causes disease in that species but it
can be a significant cause of respiratory disease in guinea pigs.
In addition to the species-specific organisms, post-operative
infections can be caused by a myriad of bacterial contaminants
normally present in the animal's environment.  It is important
that invasive surgical procedures be done aseptically to minimize
the potential affects of these opportunistic organisms.

     Although not experimental variables, there are several
bacterial diseases of laboratory animals which can be transmitted
to man and therefore are of possible concern to those using
animals in research.  These may include tuberculosis,
salmonellosis, campylobacterosis, and shigellosis.  The
investigators whose studies involve substantial animal contact
should be familiar with institutional guidelines and policies
regarding the prevention of zoonotic disease.  These should
include a program of periodic physical examination, an
educational program for personnel, immunization where appropriate
and the use of protective clothing.

Viral Diseases

     Viral infections in laboratory animals can often be
asymptomatic.  As with bacterial and mycoplasmal infection,
clinical viral disease can occur when an animal is stressed.
These viruses can be particularly devastating because the effects
on research data may not be recognized, yet still be significant.
The effects of these latent viruses have been best defined in
rats and mice.  Barrier housing of commercially available
specific pathogen-free rodents will help eliminate these viruses
from a colony.  Contaminated tissues, particularly murine tumors,
have been implicated in many outbreaks of disease.  Tissues
should be screened for the presence of contaminants prior to
their use in a research facility.  It is beyond the scope of this
chapter to review all the research implications of viral
pathogens currently known; however, a few examples will be
briefly mentioned.  There are key viral diseases of most common
laboratory animals and it is important for the investigator to
work with the institutional veterinarian to become familiar with
those viruses and learn how they might affect a particular
research project.

   Sendai virus, a common viral contaminant in conventional mouse
and rat colonies, can cause histopathologic changes in the
respiratory tract, immunosuppression, and decreased reproductive
efficiency.  It can also act synergistically with other
respiratory pathogens.  A viral disease of mice which is often
asymptomatic but serious is Mouse Hepatitis Virus (MHV).  This
virus has been implicated in wasting syndromes in nude mice.  It
can cause respiratory, hepatic, and enteric disease.  Even in
asymptomatic animals, it can cause profound immunological
disturbances.  Some diseases of laboratory animals are often
associated with clinical disease and affect a research study due
to high morbidity and mortality rather than the subtle effects of
the latent viruses.  Canine distemper, feline panleukopenia and
measles in macaques are examples of these types of viral
infections.  Although not as prevalent as bacterial zoonoses,
some viruses of laboratory animals can be transmitted to man.
Examples of these include; lymphocytic choriomeningitis, Herpes
virus simiae and rabies.

Parasitic Diseases

     Parasites of laboratory animals have also been implicated as
nonexperimental variables in research.  Some parasites such as
Trichosomoides crassicauda of rats are capable of causing tumors
which could significantly obscure results of a carcinogenicity
study.  Skin mites of mice have been shown to affect immune
parameters.  Parasites are also capable of causing significant
clinical disease such as the rectal prolapses seen with pinworms
in rodents and bowel perforation seen with Prosthenorchis elegans
in non-human primates.  Some parasites of laboratory animals can
also be transmitted to man.  Examples of these parasites are
Hymenolepis nana and Entamoeba histolytica.

   It is important to remember that while laboratory animals may
not show clinical signs of microbial infection, the infections
can have profound effects on research results.  Investigators
studying immunological function should be particularly familiar
with the otential effects of microbial agents on their research.
Transmission of contaminants can occur in tumor or tissue
inoculation, from direct transmission or via fomites in the
laboratory. Animals of different health status should be strictly
isolated from one another and all biologic material should be
screened for the presence of viral and other contaminants.


   The concepts of Russell and Burch-refinement, replacement, and
reduction-are generally well accepted in the research community.
Adherence to these concepts includes attempting to minimize the
nonexperimental variables introduced in this chapter.  The
maintenance of healthy laboratory animals and the reduction of
nonexperimental variables is the responsibility of the animal
care facility and the investigator working together in an
atmosphere of open communication and cooperation.


     Allert, J.A.; Adams, R.A.; and Baetjer, A.M. l968. Role of
environmental temperature and humidity in susceptibility to
disease. Ach. Environ. Health l6:565-570.

     Broderson, J.R., et al.  l976.  The role of ammonia in
respiratory mycoplasmosis of rats. American Journal of Pathology

     Davis, D.E.  l978.  Social behavior in a laboratory
environment. pp 44-63 in Laboratory Animal Housing. Proceedings
of a symposium organized by the ILAR Committee on Laboratory
Animal Housing. Washington, DC; National Academy of Sciences.

     Guide for the Care and Use of Laboratory Animals, NIH
Publication No. 86-23.

     Greenman, D.L.P., et al. l982. Influence of cage shelf level
on retinal atrophy in mice.  Lab Animal Science, 32(4):353-356.

     Lang, C.M. and Jessell, E.S. l976. Environmental and Genetic
Factors affecting laboratory animals; impact on biomedical
research.  Federal Proceedings Vol. 35 No. 5-8, ll23-ll65.

     Lindsey, J.R., et al.  Physical, chemical and microbial
factors affecting biologic response, pp. 3-43, In:  Laboratory
Housing. Proceedings of a symposium organized by the ILAR
Committee on Laboratory Animal Housing.  Washington, DC; National
Academy of Sciences.

     Pakes, S.P. et al., Factors that complicate animal research,
Laboratory Animal Medicine, Chap. 24.  Fox, J.G. (ed.), Academic

     Public Law 99-198. Code of Federal Regulations, Title 9,
Subchapter A, Animal Welfare, 1986.

     Russell, W.M.S. and Burch, R.L. The Principles of Humane
Experimental Technique, Methuen and Co., Ltd., London, 1959.

                          Chapter 4
             Principles of Anesthesia and Analgesia

                 Marilyn J. Brown, D.V.M., M.S.


   It is important that all scientists using animals in research
meet their ethical and legal responsibilities to avoid
unnecessary pain and distress to the animal.  Studies involving
unavoidable pain and distress must be justified by the
investigator in accordance with Federal regulations and
institutional policies.  This chapter will cover some of these
legal responsibilities as well as try to help the investigator
meet these responsibilities through knowledge of the basic
principles of anesthesiology.  Included in these principles are
an understanding of some of the basic terms used in the field of
anesthesiology, the types of variables that can affect an
animal's response to an anesthetic agent, the effect of a given
anesthetic protocol on an experiment, some general considerations
and the recognition of pain.  Also mentioned in this chapter are
anesthetic monitoring and some fundamentals of anesthetic crisis
management.  Controlled drugs and their use are briefly
discussed.  This chapter is not meant to be a complete treatise
on the subject of laboratory animal anesthesiology but to give an
introduction to stimulate further reading in areas of specific

   Anesthesiology is not an exact science.  Recommendations and
dosages given in textbooks should be taken as guidelines.  An
investigator contemplating a procedure requiring anesthesia,
tranquilization or analgesia should not neglect the resource of a
veterinarian who can often provide valuable assistance.  In fact
the Animal Welfare Act requires that "in any practice which could
cause pain to animals . . . a doctor of veterinary medicine is
consulted in the planning of such procedures."

     There are many variables affecting an animal's response to
anesthesia. Because the absorption and biotransformation of drugs
differs between species, it is nearly impossible to develop a
single anesthetic or analgesic protocol that applies to all
laboratory animals. Morphine can cause profound CNS depression in
the rat and rabbit but can cause tremors and convulsions in mice
and cats.  The dosage of xylazine needed to sedate a ruminant is
one-tenth that necessary to sedate a dog.  These are but two of
many examples.  A common mistake is to extrapolate dosages across
animal species or from man to animals.  The strain of animal used
is also a variable to consider. Some rat strains are sensitive to
nitrous oxide.  Some breeds of dogs (whippets and greyhounds) are
more sensitive to barbiturates than other breeds.  The size and
even the sex of the animal can make a difference in the response
to anesthetics.  In rats, females are more sensitive to
barbiturates but in mice, barbiturate narcosis lasts longer in
males.  The temperament of the animal can change the way it
responds to a given agent.  Some tranquilizers will cause a
vicious dog to become even more difficult to handle.

     Fat does not play a key role in the initial absorption of an
anesthetic agent, but it does affect the body weight upon which
the dosage is based.  Fat can later serve as a repository for the
agent, thus prolonging recovery.  The age of the animal also must
be considered.  Since very young animals require frequent
feedings, prolonged recoveries can present a formidable problem.
There are also age-related changes in liver enzyme functions
which affect biotransformation of anesthetic agents.  Older
nimals can present an anesthetic challenge due to impaired renal
or hepatic function.

   The animal's physical condition can affect its responses.  The
presence of pre-existing disease will increase an animal's
anesthetic risk.  Respiratory diseases can often be asymptomatic
in the uncompromised animal even though it is endemic in many
rodent populations.  Even less obvious is the effect of diet and
environment.  Rats fed an inadequate diet are more resistant to
barbiturates, yet fasted mice have an increased barbiturate sleep
time.  Abnormal environmental temperatures and humidity cause
stress which can result in a compromised animal and variable
anesthetic responses.  High temperatures sensitize rats and
rabbits to anesthesia.

   Various factors will influence anesthetic choice.  The use of
concurrent drugs changes an animal's response to anesthetic
agents.  For example, some antibiotics potentiate barbiturates.
The type of experimental procedure planned may impact on the
anesthetic protocol.  In an obstetric procedure, the effects on
the fetus must be considered.  When surgery involves the head and
face, there is limited access to the animal so the anesthetic
protocol should be planned to facilitate monitoring under these


   Minimizing pain and distress in research animals is an ethical
responsibility, produces better scientific results and is the
law.  The Public Health Service Policy on Humane Care and Use of
Laboratory Animal states that "Procedures that may cause more
than momentary or slight pain or distress to the animals will be
performed with appropriate sedation, analgesia, or anesthesia
unless the procedure is justified for scientific reasons in
writing by the investigator."  The NIH further addresses the
subject of anesthesia in the Guide for the Care and Use of
Laboratory Animals.  This document states that the proper use of
anesthetics and analgesics is necessary for humane and scientific
reasons and recommends that the veterinarian provide guidance for
their usage.  The Animal Welfare Act (AWA) requires standards for
animal care, treatment, and practices in experimental procedures
to ensure that animal pain and distress are minimized, including
adequate veterinary care with the appropriate use of anesthetic,
analgesic, tranquilizing drugs or euthanasia.  It prohibits the
use of paralytics in painful procedures without anesthesia and
states "that the withholding of tranquilizers,  anesthesia,
analgesia or euthanasia when scientifically necessary shall
continue for only the necessary period of time."  Exceptions to
such standards may be made only when specified by the research
protocol and any such exception shall be detailed and explained
in full in a report filed with the Institutional Animal
Committee.  And as previously noted, it further requires that if
practices could cause pain to animals, a doctor of veterinary
medicine be consulted in the planning of such procedures.


     As with all branches of science, there are certain terms one
needs to be familiar with in order to communicate effectively
about anesthesiology.

The following is a list of the most common terms:

Analgesia            Insensibility to pain without loss of

General              Temporary, controllable and reliable loss of
Anesthesia           consciousness induced by intoxication of the

Sedation             Calm state usually accompanied by

Tranquilization      Calmness without drowsiness or
                     unconsciousness.  Analgesia is usually not
                     a feature.

Time to Peak        Time between initial administration and onset
                    of the effect maximum expected effect.

Duration of Effect   Length of time peak effect can be expected
                     to last after a single administration of an
                     anesthetic dose.

Time to Recovery     Time between initial administration and the
                     ability to stand unaided.


   When anesthesia, analgesia, or chemical restraint is used, it
may be advisable to ascertain any distortion of results by
anesthetics through limited trials.  Check the literature and
package inserts for the effect of the agent on the systems being
experimentally evaluated.  These changes need to be taken into
consideration when evaluating the effect of an experimental
manipulation. Choose the agent which has the least effects on the
systems under investigation.  General anesthetics often depress
the cardiovascular and respiratory systems, alter blood gases,
lower metabolism, decrease body temperature, and alter tissue
perfusion.  Anesthetics can also produce histopathologic changes.


   Whenever possible, try a new anesthetic protocol in a limited
number of animals before depending on it for surgical or painful
procedures involved in an experiment.  This allows determination
of suitability for the anticipated protocol and allows necessary
changes to be made before it effects the data being collected.
It also facilitates familiarization with the anesthetic method to
minimize problems later, when attention is often focused on
surgical procedures or data collection.

     Pay particular attention to the health of the animal before
using it in an experiment.  A preanesthetic checkup is a good
idea.  To minimize anesthetic risks, only use healthy animals and
allow them to acclimate to the facility before an anesthetic
procedure.  Consider the general adaptation syndrome: alarm
increases basal metabolic rate which may increase the amount of
anesthetic needed; however, this is often followed by an
exhaustion phase when less anesthetic is required.

     Use the minimal degree of CNS depression necessary for the
procedure that is compatible with the animal's welfare.  The
degree of depression required for procedures such as radiographs
or blood withdrawal is not the same as that needed for a
thoracotomy or orthopedic procedure.  Remember, during painful
procedures, the use of paralytics without anesthesia is
prohibited by law.

   Consider if, and to what extent, the anesthetic protocol will
affect the validity of experimental results and how it will react
with other drugs being used.  For example, if studying
catecholamine effects, halothane should be avoided since its
combination with catecholamines can cause severe cardiac

     Even in the absence of sophisticated equipment, try to have
some basic items available to insure adequate ventilation.  This
includes a source of oxygen, the use of endotracheal tubes when
feasible, and aspiration suction to remove excessive oral
secretions, and/or vomitus.

     Regard the conservation of heat as an integral part of
anesthetic management.  This is particularly important in small
or young animals.  A rectal thermometer can help monitor the
animal's body temperature.  More sophisticated thermal monitors
are also available.  Maintenance of body temperature is enhanced
through the use of external heat sources such as hot water
bottles, thermal blankets and heating pads.  Care should be taken
to avoid thermal burns from external heating sources; i.e.,
electric heating pads.

     Administer warm, balanced salt solutions by continuous I.V.
drip whenever possible. This is not always possible in very small
animals but is especially important for prolonged procedures or
when significant blood loss is expected.  Fluids often come in
bags which are easy to handle and when warmed can double for hot
water bottles.

     Pay particular attention to post-anesthetic care.  The
anesthetist's responsibility does not end when the animal is
taken off the table.  Allow animals to recover in an environment
approaching the normal body temperature of the species.  Maintain
intravenous fluid infusions when possible and have an
endotracheal tube in place until the swallowing reflex is
recovered.  Be sure the animal is protected from injury, either
self-inflicted or by other animals, during recovery.

   Consider the implications for laboratory safety.  Scavenging
systems should be used with gaseous agents.  Avoid carcinogens
such as urethane and chloroform.  Consider flammability when
using ether.


   In the Definition of Terms developed to implement the amended
Animal Welfare Act, a painful procedure is defined as, ". . any
procedure that would reasonably be expected to cause more than
slight and momentary pain or distress in a human being."  In both
humans and most animals the total pain experience results from an
interaction between sensory pathways and the affective system,
which provides the motivational and emotional component of pain.
This varies  considerably between species and individuals within
a species.

     Understanding the degree of pain involved in various
experimental procedures allows a prediction of animal pain or
distress.  Physiological responses to pain can include increased
blood pressure and heart rate, pupillary dilation, increased
respiration, and an arousal response on the electroencephalogram.
If baseline values are known for these variables, they can be
monitored for changes.

   To detect behavioral signs of pain, one must be familiar with
the animal's normal behavior.  Behavioral responses to pain vary
between species, within species, and even within the same animal.
General behaviors to evaluate include:  sleeping, feeding,
drinking, locomotion, grooming, exploration, performance in
learning and discrimination tasks, mating behavior, social
interactions, and dominance/subservience responses within the
social system.

     Typical behavioral signs of acute pain include:

          - protecting the painful area
          - vocalizing (especially when handled or moving)
          - licking, biting, scratching, or shaking the painful
          - restlessness
          - lack of mobility
          - failure to groom
          - abnormal postures
          - lack of normal interest in surroundings.

     Unless there is evidence to the contrary, assume that a
procedure that causes pain in humans will cause pain in animals.
Points to remember are:

   -   Abdominal surgery appears to be less painful in animals
than humans, probably because most animals do not use their
abdominal muscles for postural support.

   -   Lumbar and thoracic spine surgery in animals also appears
to be less painful than in man, probably due to man's postural
requirements.  However procedures involving the cervical spine
seem to be more uncomfortable in animals.

   -   In animals, chest surgery involving the sternum appears to
be more painful than surgery using a lateral intercostal

   -   Surgery on the eye, ear or surrounding structures seems to
distress most animals.  Signs such as head tilt or shaking, or
pawing or rubbing the area may be seen.  Perirectal procedures
also seem to produce discomfort.  In addition to analgesia,
protection of the affected areas is indicated.

   -   Surgery of the femur or humerus also seems to be painful
to most animals, which may be due to large muscle mass trauma.

     Pain perception can be influenced by drugs and/or
environmental and behavioral factors.  Recovery in familiar
surroundings may help to relieve pain and distress.
Acclimatization prior to a procedure may also facilitate
recovery.  The environment should be kept stable, minimizing
stimuli that evoke a fearful response in the animal.  When
appropriate, interact with the animal through talking or petting.
Always handle the animal in an appropriate manner.

     Various analgesics are available to the investigator.  These
can be divided into two main categories: the centrally acting
agents such as morphine, butorphanol and buprenorphine; and the
peripherally acting agents such as the anti-inflammatories,
aspirin and phenylbutazone.  The short half-lives of many of
these agents may cause a labor-intensive analgesic protocol for
the investigator, but creative delivery systems (such as the
osmotic minipumps and tethering systems) and the development of
new drugs such as buprenorphine with longer half-lives (12 hours)
should facilitate meeting the analgesic needs of most laboratory
animals.  When designing an analgesic protocol, the investigator
should consult with a veterinarian who is experienced in
laboratory animal medicine.  This will help avoid problems with
species specific responses such as morphine sensitivity in cats
and mice or the unusually short duration of meperidine in the
dog.  Interaction of the analgesic with concurrently used drugs
and the effect of the agent on study results (such as the effect
of aspirin on healing or clotting time) must be taken into
consideration when choosing the best agent for a given situation.
Although there is much information available on the use of
various agents in animals, it is not always easily referenced and
may be difficult to find without some guidance.

                      ANESTHETIC MONITORING

   During an anesthetic procedure, the physiologic state of the
animal and the depth of anesthesia should be monitored.  This
allows the anesthetist to adjust the depth of anesthesia and to
anticipate impending complications.  The degree of jaw tone is an
indication of muscle relaxation.  This is easily monitored by
trying to open the animal's mouth -- taking care to avoid the
animal's teeth.

   Pulse quality is an indication of cardiovascular function.  It
can be checked in several areas but is commonly felt in the
inguinal region.  This "hands on" evaluation of the animal also
gives the anesthetist a crude indication of the animal's body
temperature so that hypo- or hyper-thermic states can be
detected.  Capillary refill is also an indication of
cardiovascular function.  This is checked by pressing firmly on
the mucous membranes of the gums until they blanche and then
releasing the pressure and  noting the time it takes the normal
color to return.  Full color should return in less than two
seconds.  A slow capillary refill time is suggestive of sluggish
blood flow and may be an early indicator of shock.  While
checking capillary refill, also note mucous membrane color.
White may indicate shock, while blue may indicate poor
oxygenation.  In small rodents, the foot pads or ears offer other
areas to check for color.

     Another method for monitoring cardiovascular and respiratory
function is through auscultation of the chest.  This takes more
experience and is difficult in small rodents. 
Electrocardio-graphic monitors are also available to aid in
anesthetic monitoring.

     Keeping written records of your anesthetic monitoring and
administration is important for several reasons.  They serve as a
permanent record of the procedure and of any complications and
when they occurred.  This can help explain unexpected
experimental data later.  Written records also help to visualize
significant trends which could lead to anesthetic complications.
In addition, written records represent the best method to clearly
document compliance with the AWA.

     The aim of anesthesia is to prevent the perception of
painful stimuli without undue depression of physiologic
functions.  One of the criteria used to monitor the depth of
anesthesia is the animals' response to stimuli or their reflex
responses.  Responses vary with the type of anesthetic used, the
species and health status of the animal, and the use of
concurrent drugs, particularly paralytics.

     The first reflex lost is usually the righting reflex.  This
reflex may be checked by turning the animal over on its back and
watching to see if the animal rolls back over onto its sternum.
Obviously an animal that can right itself is not at a surgical
level of anesthesia!

     The next reflex usually lost is the swallowing or laryngeal
reflex. It is the loss of this reflex that allows placement of an
endotracheal tube after induction.  Once in place, slight
manipulation of the tube will cause the animal to swallow, if it
is waking up.  With some commonly used anesthetics such as the
dissociative, ketamine, the laryngeal reflex may be present even
when a surgical level of anesthesia is obtained.

     The palpebral or eyelid reflex is an easy one to monitor.  A
light touch to the medial canthus or brush of the eyelashes will
cause eyelid movement if the reflex is present.  It may be as
obvious as a blink or just a slight muscle movement.  An overly
aggressive touch may cause movement that is not induced by the
animal and can lead to erroneous interpretation.

     The reflex most commonly used to determine if the animal is
feeling deep pain is the pedal or paw pinch reflex.  The toe is
firmly pinched between the fingers to elicit a withdrawal
response by the animal.  A forcep may also be used but care must
be taken not to cause tissue damage.  Pinching the ear can also
be used especially in rodents and rabbits.  If the animal draws
its head away or shakes its ear, it is still capable of feeling
deep pain and is not ready for any surgical manipulations.

     The pupillary reflex can also be monitored but it can be
affected by many things.  Common preanesthetic agents often make
the pupil unresponsive to light.  A dilated pupil can indicate
either very light anesthesia and the perception of pain or
dangerously deep anesthesia if the pupil is fixed and dilated.

   The corneal reflex is usually the last to go and it is usually
not necessary to get to this depth of anesthesia.  This reflex is
checked by very gently touching the animal's cornea and watching
for movement of the eyelid.


   General anesthesia is divided into stages and planes.  Stage
one is characterized by analgesia.  In stage two, excitement can
be seen.  Signs include struggling and erratic movement.  It is
preferable to avoid this stage.  Stage three is a surgical level
of anesthesia.  It is further divided into planes.  Plane one is
characterized by a loss of the palpebral reflex.  In plane two,
eyeball movement ceases and the animal exhibits deep, regular
respirations.  This is usually a good level at which to do
surgery.  With plane three comes paralysis of the intercostal
muscles and short, jerky, gasping diaphragmatic efforts.
Artificial ventilation is essential at this plane.  Stage four is
one to avoid as it is characterized by total loss of respiratory
movements, cyanosis and cardiac arrest.

                        SPECIFIC AGENTS

   It is not within the scope of this chapter to give a detailed
pharmacologic description of all the anesthetic agents and
regimes used in research animals.  However, a brief description
of the advantages and disadvantages of some of the most commonly
used agents will be given.  The reader is referred to the list of
references and a veterinarian when help is needed to design an
appropriate anesthetic protocol for a given research project.


     Preanesthetics are usually given as an anesthetic agent
adjunct to ameliorate some of the deleterious side effects and/or
to decrease the required dose of the primary anesthetic agent.
Atropine or its analogs are commonly given.  They depress
secretory activity making them especially useful in animals with
profuse oral secretions such as ruminants and guinea pigs.  These
agents also help maintain heart rate by counteracting the vagal
slowing of the heart rate induced by some anesthetic agents and
some surgical procedures.  Atropine causes pupillary dilation,
therefore this reflex cannot be used to monitor anesthetic depth
in the atropinized animal.

     Other commonly used preanesthetics are tranquilizers and
sedatives.  Use of these agents helps provide a stress-free
subject for the induction of anesthesia.  Acepromazine produces
good tranquilization, indirectly suppresses the emetic center,
potentiates the analgesic effects of other agents and provides
muscle relaxation.  Hypotension can be a serious side effect of
this agent. It is often used in combination with the dissociative
anesthetic agents such as ketamine.  Xylazine is a potent
hypnotic, muscle relaxant, and analgesic.  Use of this agent can
reduce the necessary barbiturate dose by 50 percent.  Like
acepromazine, xylazine is often used in combination with
ketamine.  Bradycardia and hypotension can be seen with xylazine.
Premedication with atropine can help prevent cardiac
dysrhythmias.  Respiratory rate can be decreased but increased
tidal volume usually maintains normal blood gases.  Xylazine can
cause abortion in late pregnancy in ruminants.  Diazepam is a
potent tranquilizer which also has muscle relaxant and
anticonvulsant properties.  It is useful in combination,
particularly with Innovar-VetR in rodents.  Although diazepam can
cause some respiratory depression, it has little effect on
cardiac output or blood pressure.  Morphine is a narcotic
analgesic sedative.  Anesthetic doses can be decreased as much as
50 percent after morphine administration.  Morphine depresses the
central nervous system, particularly the respiratory center, as
well as peristalsis. In dogs, morphine frequently causes emesis.
Morphine is generally contraindicated in the cat and mouse.

General Anesthetic: Injectable

     General anesthesia is delivered by two basic methods:
injection and inhalation.  It is usually preferable to give
injectable agents by the intravenous route (I.V.) -- given to
effect; however, intraperitoneal (I.P.), subcutaneous (S.C.) or
intramuscular (I.M.) techniques are sometimes necessary or even
preferable.  The advantages of injectable anesthetic agents are
ease of administration, low cost and lack of need for
sophisticated equipment.  The major disadvantage is that once the
drug is given, it is in the body until it is metabolized or

     Innovar-Vet(R) is a veterinary drug which combines fentanyl,
a morphine derivative, and droperidol, an alpha adrenergic
blocker.  Because it is a combination drug, doses are usually
given in ml/kg rather than mg/kg.  It is a potent analgesic.  The
cardiac depressant effects can be counteracted with atropine and
the respiratory depressant effects can be reversed with naloxone.
Innovar-Vet(R) is a poor muscle relaxant.  It is not recommended
for use in horses, ruminants, or cats.

    Ketamine is a commonly used dissociative anesthetic.  It is
short acting and produces variable analgesia.  It is often
combined with other agents to improve its muscle relaxation and
analgesic properties as well as provide a smoother recovery.  It
can be given I.V., S.C., or I.M.  It does not cause cardiac
depression and may even stimulate the cardiovascular system;
however mild respiratory depression may be seen.  The swallowing
reflex is maintained making intubation under ketamine alone
difficult.  The palpebral reflex is lost, so it is necessary to
use ophthalmic ointment to prevent corneal drying.

     The most commonly used injectable anesthetic agents are the
barbiturates.  There are two classes of barbiturates:
oxybarbiturates of which pentobarbital or nembutal is the most
common; and thiobarbiturates which are much faster acting and
which include thiopental and thiamylal.  Barbiturates are
potentiated by acidosis such as that which can be seen with
respiratory depression or diarrhea.  Many drugs potentiate the
effect of barbiturates.  Glucose or epinephrine cause prolonged
recovery times.  Barbiturates are controlled substances as
defined by the Drug Enforcement Agency.  Therefore a license is
required for purchase and records must be kept.  If possible,
barbiturates should be given to effect which is difficult when
administered I.P.  They have an accumulative effect, which means
two subsequent doses combined have a greater effect than the two
doses given alone.  Barbiturates are considered poor analgesics.
Respiratory depression can lead to hypercarbia.  Cardiovascular
effects include bradycardia, hypotension, myocardial depression,
and increased peripheral vascular resistance.  Use of
barbiturates is contraindicated in animals with liver or kidney
disease.  Lower doses should be used in young animals.  When
small doses must be given, it is often helpful to dilute stock
barbiturate solution.  Preanesthetics should be used when
possible to decrease the amount of barbiturate needed.

General Anesthetics: Inhalation

   Inhalation anesthesia has the advantages of rapid induction
and recovery.  Depth of anesthesia can be rapidly changed.
Typically animals are initially anesthetized with an I.V.
injection of an ultrashort acting barbiturate, or administered
the inhalation agent by mask or by use of an induction chamber.
When using gaseous anesthetic agents particular attention must be
paid to provide an adequate oxygen source and for the removal of
carbon dioxide.  This can be done through the use of a properly
maintained gas anesthesia machine.  If possible, it is preferable
to intubate the animal for the most efficient delivery system and
to help assure a patent airway.  This takes practice, especially
in rodents.  If the anesthesia is administered by mask, avoid
placement of the mask over the entire face as these agents are
irritating to the eye.  Also avoid direct contact of the liquid
form of the agent with the animal's skin or mucous membranes.
Scavenging systems should be in place to minimize personnel

   Nitrous oxide is often used in conjunction with an anesthetic
gas due to its potentiating effect.  It is always used in
combination with oxygen, usually at a 50:50 or 60:40 ratio. It is
quite safe, since it is neither flammable nor explosive, allows
rapid induction and causes little cardiovascular disturbance.  It
is also a very good analgesic. It enters air-filled cavities much
faster than it leaves them which could be a problem with a
pneumothorax or a large gas-filled bowel.  Oxygen should be
administered alone for a few minutes at the end of a procedure to
prevent diffusion anoxia.

   A commonly used gaseous anesthetic agent is ether.  Ether has
a slow induction and recover period.  It is highly flammable and
forms explosive mixtures with oxygen and nitrous oxide.  It is a
potent CNS depressant and analgesic.  It is extremely irritating
to the mucosal lining of the respiratory tract and may induce
laryngospasms, especially in cats and rabbits.  Respiratory
secretions are stimulated which can predispose or exacerbate
respiratory infections.  The respiratory depression caused is
usually only a problem in guinea pigs and chinchillas.  Ether
causes some myocardial depression.  Since ether is inexpensive
and can be administered without the use of sophisticated
equipment, it is very popular.  To minimize explosive hazards and
personnel exposure, ether should be used under a fume hood.

     Three other commonly used inhalation agents are halothane,
isoflurane and methoxyflurane.  Halothane is nonflammable and
nonexplosive.  It is a good muscle relaxant and adequate
analgesic.  It allows rapid, smooth induction and recovery.
Halothane depresses the cardiovascular system and sensitizes the
heart to dysrhythmias.  It also depresses the respiratory system
which can lead to acidosis.  Halothane requires special
vaporizers and equipment.  Isoflurane is also a stable,
nonflammable agent. Induction and recovery are rapid.  Arterial
blood pressure is decreased due to lowered peripheral vascular
resistance; however,  perfusion is maintained.  Other
cardiovascular functions are well maintained, but respiratory
function is depressed.  Isoflurance also requires special
vaporizers and equipment.  Methoxyflurane is very stable and
because it does not reach high concentrations at room
temperature, it has a good margin of patient safety.  It is a
good muscle relaxant and an excellent analgesic.  Like the other
inhalation agents, it does cause some respiratory depression and
hypotension can also be a problem.  Induction and recovery are
slower than with the other agents which may be an advantage by
keeping the animal quieter immediately post operative as well as
providing longer acting analgesia.


     When anesthetizing small rodents, particular care must be
taken to avoid hypothermia. The airway is easily obstructed so be
sure the neck is adequately extended and secretions are aspirated
as necessary.  Fasting is not necessary unless gastrointestinal
surgery is planned and even then only a 6-hour fast is necessary.
Water should not be restricted.  Loss of the toe pinch reflex
indicates surgical anesthesia in the mouse. In the rat and guinea
pig, the ear pinch is more sensitive.  Rodents are difficult to
intubate.  If they are intubated, care must be taken to minimize
dead space in the tubing.

     Rabbits are probably the most difficult laboratory animal to
anesthetize.  Their respiratory center is particularly sensitive
to anesthetics and a lot of individual variation in response
exists.  Rabbits should be fasted 6 hours prior to anesthesia.
Water should not be restricted.  The rabbit trachea is very
delicate and rabbits are predisposed to pulmonary edema with
prolonged inhalation administration.  A normally small lung
capacity combined with enzootic pulmonary  disease further
complicates the situation.  The best indicator for surgical
anesthesia is the loss of the ear pinch reflex.  Intubation in
rabbits is difficult due to lack of visualization of the larynx,
but it can be mastered with practice.

   Dogs are usually not difficult to anesthetize.  Large, easily
accessible veins make I.V. injection of agents quite easy.
Intubation is not difficult due to the easily visible larynx.
Administration of preanesthetics, particularly in large dogs, may
make induction easier.  Dogs should be fasted for 12 hours prior
to nesthetic administration.

     Cats are also relatively easy to anesthetize; however, they
are easily stressed when restrained so preanesthetics become even
more important.  The larynx is easy to visualize, but
laryngospasms can make intubation difficult.  Cats should be
fasted for 12 hours prior to an anesthetic procedure; however if
necessary, xylazine can be given to induce vomiting and serve as
a tranquilizer.  As noted previously, narcotics can cause severe
convulsions in cats and should be avoided.

     There are several considerations when anesthetizing swine.
The pig heart is smaller in proportion to body size than is the
heart of other domestic animals, which is a disadvantage during
periods of anesthetic stress.  Size and temperament can make
restraint difficult and the use of preanesthetics essential.
Pigs are predisposed to ventricular fibrillation and some breeds
exhibit malignant hyperthermia when exposed to halothane.  The
anatomy of the larynx and soft palate predispose pigs to
respiratory distress if not intubated; however, this same anatomy
combined with laryngospasms can make intubation difficult.  The
ear vein is the most readily accessible for I.V. injections.
Pigs should be fasted 12-18 hours prior to anesthetic

     The temperament and size of ruminants present a challenge to
the anesthetist.  Again, preanesthetics are desirable; however,
ruminants are very sensitive to xylazine, so only small doses are
needed. They are also very sensitive to barbiturates. Food should
be withheld for 24-48 hours with water withheld for 6 hours prior
to the procedure.  Gastric bloat can be minimized by passing a
stomach tube in the anesthetized animal once it is on the table.
Avoiding prolonged procedures and recoveries will also help
minimize bloat as well as decrease the incidence of pressure
myositis.  Thick pleura and extensive pulmonary supportive tissue
necessitate the use of high ventilation pressures.  Excessive
salivation is difficult to control even with the generous use of
atropine.  The jugular vein is the easiest to use for the I.V.
anesthetic administration.

     The use of sedatives and tranquilizers as preanesthetics in
nonhuman primates presents a risk to the handler because the
animal may present a false appearance of sedation in the cage and
become quite active when aroused!  Ketamine, given I.M. in a
monkey restrained in a squeeze cage, is the most common form of
preanesthesia.  This is often followed with an I.V. injection of
an additional anesthetic agent with maintenance accomplished with
additional injectable agents or inhalation anesthetics.  This
procedure minimizes the hazards of bites or scratches to
personnel or escape by the patient.  Monkeys should be fasted for
12 hours prior to an anesthetic procedure; however, ketamine
given alone usually does not cause emesis.  Monkeys are usually
not difficult to intubate after a little practice.


     Anesthetic emergencies are usually caused by human error. 
This may be due to inappropriate selection of agents or doses,
failure to recognize and treat inadequacies of respiration or
circulation before collapse, neglect in checking equipment or the
use of unhealthy animals.

     Respiratory failure is often caused by airway obstruction or
barbiturate overdose.  Airway obstruction can occur because of
positioning of the animal, secretions in the trachea, or
misplacement of the endotracheal tube.  Barbiturates are
particularly potent respiratory depressants and they must be used
with care and "to effect."  Signs of respiratory failure include
gasping, exaggerated chest movements and cyanosis.  Gasping
movements can be misinterpreted to be voluntary movements
indicating inadequate anesthesia causing the inexperienced
anesthetist to actually give more anesthetic agent.

     When respiratory failure occurs, the first thing to do is
discontinue anesthetic administration.  Then check for airway
patency.  Artificial ventilation can be performed through the
nostrils or through the endotracheal tube by compressing the
rebreathing bag on the anesthetic machine or the use of a manual
resuscitator bag.  An ear syringe can make a good rodent
resuscitator, as it fits right over the nose of larger rodents.
If the failure was caused by a narcotic, reversal agents may be
used.  Other drugs such as doxapram can be used to stimulate the
respiratory system.

     Causes of circulatory arrest include drugs, hypoxia,
hypercapnia, changes in the vascular volume or bed, deleterious
reflex responses, obstruction of venous return, severe
electrolyte imbalance, and primary cardiac pathology.  Careful
maintenance of ventilation is one way to avoid hypoxia and
hypercapnia.  Changes
in vascular volume can be minimized through the use of good
hemostasis by the surgeon and adequate I.V. fluid volume
replacement by the anesthetist.  Surgeons must be careful when
moving abdominal contents around, not to place too much pressure
on the posterior vena cava and thus impede blood flow return to
the heart.  Electrolytes can be monitored and imbalances
corrected during surgery before they get to the life threatening
stage.  In some cases the presence of primary heart pathology may
be identified in a routine presurgical physical exam.

     Signs of cardiac failure are white or cyanotic mucous
membranes, no pulsation in major arteries, no wound bleeding and
no palpable heart beat.

     Treatment of cardiac arrest begins the same way as that for
respiratory arrest and includes discontinuation of anesthetic
administration, checking for a patent airway, and the
administration of oxygen.  If possible, also lower the cranial
end of the animal by 30 percent.  Closed chest massage can be
done by compressing the thorax by one third to one half its width
or depth at a ratio of 5 compressions to each ventilation.  Fluid
replacement should occur as rapidly as possible. Drugs such as
epinephrine, sodium bicarbonate, prednisolone sodium succinate,
calcium chloride and lidocaine can be used but vary with
different situations which may be hard to define without the use
of an electrocardiogram.

     Anyone performing frequent anesthetic procedures should have
a well-stocked emergency kit handy with such items as
endotracheal tubes, a manual resuscitator bag, syringes and
needles, and some or all of the drugs mentioned above.  It is
helpful to have a card in this kit which list all the dosages for
these drugs to insure proper usage during the rare occasion when
they are needed.  Frequent emergencies are an indication of
improper anesthetic or surgical techniques and should be reviewed
with the veterinarian to ascertain a possible cause and implement
a potential solution.

                      CONTROLLED SUBSTANCES

   Many anesthetics, analgesics and tranquilizers are controlled
substances. They are divided into five schedules based upon their
abuse potential.  Schedule I drugs are those with a very high
abuse potential for which there is no medical use.  Schedule II
drugs also have a high abuse potential but are accepted for
medical use.  This schedule includes agents with narcotic,
stimulant or depressant actions such as morphine, codeine,
meperidine, oxymorphone, pentobarbital, cocaine and opium.
Schedule III includes some of the barbituric acid derivatives.
Schedule IV has phenobarbital, chloral hydrate, and diazepam.
Schedule V agents are those with narcotics in limited quantities
such as antitussives and antidiarrheals.  This is only a partial
list of the drugs in each schedule.  For a more complete list
refer to the Drug Enforcement Administration.

     Controlled substances can only be purchased by someone with
a narcotics license which is obtained from the Drug Enforcement
Administration.  Controlled substances must be stored under lock
and key, preferably in a safe.  Permanent records must be
maintained and should not be stored with the drugs.


     This chapter reviewed the principles of anesthesiology and
highlighted examples of animal and anesthetic variations.  The
need to carefully choose and evaluate an anesthetic protocol
cannot be overemphasized.  Improper use of anesthetic agents can
result in loss of valuable research data at the very least, and
misuse of the animal at the very worst.  Prior to using an
anesthetic protocol, ascertain its species-specific effects,
interactions with other agents and effect on experimental data.
When the use of analgesics is indicated, the animal's response to
potential painful stimuli must be evaluated in terms of its
normal behavior and the effect of the agent on the species.  When
using anesthetics and analgesics in laboratory animals, advice
from a veterinarian should be obtained during the planning stages
if the projects.  In designing an anesthetic or analgesic
protocol, remember if it would hurt you, it will probably cause
pain to an animal.  When in doubt, don't proceed without
carefully evaluated trial runs.

     It is the principal investigators' legal responsibility to
minimize pain and distress in the animals they use, and a key
element in meeting this responsibility is the proper use of
anesthetics, analgesics and tranquilizers.


   Alternatives to Animal Use in Research Testing and Education.
U.S. Congress, Office of Technology Assessment.  U.S. Government
Printing Office, Washington, DC; OTA-BA-273; February 1986.

   Animal Welfare Act (Title 7 U.S. C. 2131-2156), as amended by
PL-99-198, December 23, 1986. Animal Welfare. USDA, Hyattsville,
MD; 1985.

   The Biomedical Investigator's Handbook.  Foundation for
Biomedical Research, Washington, DC; 1987.

   Clifford, D.H.  Preanesthesia, Anesthesia, Analgesia and
Euthanasia in Laboratory Animal Medicine. Fox, J.G.; Cohen, B.J.;
and Loew, F.M. (eds.), Academic Press, Orlando, FL; 1984:

   Flecknell, P.A.  Laboratory Animal Anesthesia, An Introduction
for Research Workers and Technicians.  Academic Press, London,

   Green, C.J. Animal Anesthesia. Laboratory Animals Ltd, London,

   Guide for the Care and Use of Laboratory Animals, Department
of Health and Human Services, NIH Pub. No. 86-23, Bethesda, MD;

   Hall, L.W.  Wright's Veterinary Anesthesia and Analgesia,
Sixth Edition.  Williams and Wilkins, Baltimore, MD; 1966.

   Lumb, W.V.  Small Animal Anesthesia.  Lea and Febiger,
Philadelphia, PA; 1963.

   Public Health Service Policy on Humane Care and Use of
Laboratory Animals.  Department of Health and Human Services,
Bethesda, MD; 1986.

   Public Law 99-198. Code of Federal Regulations, Title 9,
Subchapter A, Animal Welfare, 1989.

   Riebold, T.W.; Goble, D.O.; and Geiser, D.R.  Large Animal
Anesthesia, Principles and Techniques. Iowa State University
Press, Ames, IA; 1982.

   Sawyer, D.C. The Practice of Small Animal Anesthesia.  W.B.
Saunders Company, Philadelphia, PA; 1982.

   Short, C.E. Principles and Practice of Veterinary Anesthesia.
Williams and Wilkins, Baltimore, MD; 1987.

   Soma, L.R. Textbook of Veterinary Anesthesia.  Williams and
Wilkins, Baltimore, MD; 1971.

   Swindle, M.M. Basic Surgical Exercises Using Swine. Praeger,
New York, NY; 1983, pp. 19-26.

                             Chapter 5
                   Principles of Aseptic Technique

                John C. Schofield, B.V.Sc., M.R.C.V.S.


     The regulations promulgated to implement the amended Animal
Welfare Act require that all survival surgery be performed using
aseptic procedures.  This includes the use of surgical gloves,
masks, sterile instruments and aseptic technique.

     In this chapter, the Principles of Aseptic Technique will be
discussed with the emphasis on the practical application of these
principles in the laboratory setting. In centralized experimental
surgeries, a well-trained staff should be available to advise
those who use such facilities and oversee its operation to ensure
the maintenance of an aseptic environment for survival surgery.
When survival surgery is conducted outside such an environment,
it is the principal investigator's responsibility to ensure that
appropriate aseptic conditions and practices are maintained.
This chapter will provide the necessary information to carry out
this responsibility.

Prior to discussing the specific principles of aseptic surgery a
brief review of pertinent terminology is necessary.


Antimicrobial      An agent or action that kills or inhibits the
                   growth of micro-organisms.

Antiseptic         A chemical agent that is applied topically to
                   inhibit the growth of micro-organisms.

Asepsis            Prevention of microbial contamination of
                   living tissues or sterile materials by
                   excluding, removing or killing

Autoclave          A steam sterilizer consisting of a metal
                   chamber constructed to withstand the pressure
                   that is required to raise the temperature of
                   steam to the level required for sterilization.
                   Early models were termed "autoclaves" because
                   they were fitted with a self-closing door.

Bactericide        A chemical or physical agent that kills
                   vegetative (non-spore forming) bacteria.

Bacteriostat       An agent that prevents multiplication of

Commensals         Non-pathogenic micro-organisms that are living
                   and reproducing as human or animal parasites.

Contamination      Introduction of micro-organisms to sterile
                   articles, materials or tissues.

Disinfectant       An agent that is intended to kill or remove
                   pathogenic micro-organisms, with the exception
                   of bacterial spores.

Pasteurization     A process that kills nonspore-forming
                   micro-organisms by hot water or steam at

Pathogenic         A species that is capable of causing disease
                   micro-organism in a susceptible host.

Sanitization       A process that reduces microbial contamination
                   to a low level by the use of cleaning
                   hot water or chemical disinfectants.

Sterilant          An agent that kills all types of

Sterile            Free from micro-organisms.

Sterilization      The complete destruction of micro-organisms.

     Since the pioneering work of such surgeons as Joseph Lister,
who introduced the use of carbolic acid antiseptics in 1865, and
William Halstead, who advocated the use of surgical gloves in
1898, surgeons have strived to eliminate surgical infections
through the use of aseptic technique.  Potential sources of
contamination are well defined.  They include the patient and the
surgical environment: the surgeon and support staff, the
instruments, sutures, drapes and all other equipment which can
have contact with the surgical field.


     The basis for this discussion about facilities will be the
recommendations for Aseptic Surgery contained in the Guide for
the Care and Use of Laboratory Animals.  The Guide states:

     "Functional areas for aseptic surgery should include a
separate support area, a preparation area, the operating room or
rooms and an area for intensive care and supportive treatment of
animals.  The interior surfaces of this facility should be
constructed of materials that are impervious to moisture and
easily cleaned.  The surgical support area should be designed for
storing instruments and supplies for washing and sterilizing
instruments.  Items that are used on a regular basis, such as
anesthetic machines and suture materials, can be stored in the
operating room."

   "There should be a separate surgical preparation area for
animals.  An area equipped with surgical sinks should be close
to, but apart from, the operating room.  A dressing area should
be provided for personnel to change into surgical attire."

     The surgical facility should be located outside normal
facility traffic patterns.  This can help to minimize the
potential for surgical suite contamination by the movement of
personnel and equipment.  Personnel access to these areas should
be restricted to essential surgical support staff.

     Ideally, the operating room ventilation system should
provide a net positive pressure with respect to the surrounding
facilities.  The system should be regularly monitored.
Maintenance work should be performed when the surgery is idle.
Ventilation filters should be inspected and cleaned or replaced
at regular intervals.  If explosive anesthetics agents are to be
used, the Guide recommends that floors should be conductive and
electrical outlets should be explosion-proof and located not less
than 5 feet off the floor.  Dedicated surgical facilities should
be used for aseptic surgeries and the storage of essential
surgical equipment, not as general storage space.


   The equipment in areas used for aseptic surgery should be easy
to clean and portable to simplify sanitization of the area.  The
operating table should be constructed with a durable surface
material impervious to moisture which can be readily cleaned. 
Plastic or stainless steel is frequently used for this purpose.
Other useful table design features which assist patient
positioning nclude height and tilt adjustments, V-trough
configuration and restraint strap cleats.  A disadvantage of
stainless steel construction is that it predisposes animals to
hypothermia.  This can be corrected by the routine use of a
heating pad placed under the surgical patient.  Reusable, easy to
clean vinyl heating pads which recirculate hot water are
frequently used for this purpose.  Inexpensive short-term
alternatives include hot water bottles or heat lamps.  Any heat
source should be used with caution to prevent patient burns.

     Instrument tables provide the surgeon ready access to the
surgical instruments and minimize the risk of sterilized
instrument contamination by contact with non-sterile fields.
Commercially available instrument tables, such as Mayo stands,
consist of a stainless steel tray supported by a pedestal base
with a foot-operated height adjustment device, but any tray
arrangement may be used for this purpose.  The unit should be
easy to clean and simple to operate.  The drapes in an instrument
pack frequently include impervious table covers which can
minimize instrument contamination and allow the surgeon to
reposition the table without breaking aseptic technique during
the procedure.  Surgical buckets on wheels (kick buckets), which
can be readily positioned with the feet, are another recommended
piece of equipment.  They should be easy to clean and lined with
a plastic bag which should be changed at the end of the

     Adequate lighting is essential for performing surgical
procedures.  A variety of fixtures can be used to provide
sufficient light.  The commercially available surgical light
fixtures may be ceiling or wall-mounted or free standing.
Surgical lights are often positioned above the operative area and
should be regularly wiped with a moist towel prior to use to
minimize potential contamination of the sterile field below.
Light fixtures designed with detachable sterilizable handles
allow the surgeon to adjust the beam during surgery.  Wheeled,
height-adjustable intravenous drip stands should be available
when conducting major surgery.  Care should be taken to assure
that the I.V. tubing does not contaminate the sterile fields.
Positioning the I.V. tubing along the heating blanket helps warm
I.V. solutions before infusion.

     Surgical suction is another useful accessory.  Sterilized
tubing and suction tips are provided for use in the aseptic
field.  The tubing is connected to a non-sterile suction bottle
which in turn is connected to a built-in vacuum line. If built-in
vacuum lines are not available, portable electric vacuum pumps
are commercially available.

     Ancillary equipment such as respirators, electrosurgical
units and ECG monitors should be portable and included with the
light fixtures in a routine equipment cleaning schedule.
Specific details on such devices could be obtained from an
institutional veterinarian or surgical supervisor.

     Surgical instrumentation and pack preparation will vary with
the type and complexity of surgery to be performed.  Consultation
with an institutional veterinarian or surgical supervisor could
be helpful when selecting the appropriate surgical instruments
necessary to perform a proposed procedure.  Instrument packs
should be double wrapped.  Various commercial materials are
available for this purpose.  Although pack instrument preparation
will be discussed later, as many sterilizable items as possible
should be included.  These might include prepackaged surgical
blades, sponges, saline bowls and miscellaneous catheters.


     Aseptic technique requires careful attention to a series of
steps which begins with patient and instrument preparation and
ends at final wound closure.  Failure at any one step may result
in wound infection which could compromise the animal's health and
the experimental data derived from the animal.  Aseptic technique
designs all actions and motions to protect the sterile field from
contamination.  The surgeon and surgical support staff must be
adequately trained to perform each step correctly.  Acquiring and
developing the necessary skills to maintain aseptic technique
requires practice.  Personnel should receive instruction on the
indications for aseptic technique, the sources of potential
contamination, patient, instrument and equipment preparation,
sterilization systems, gowning and gloving techniques, and
intraoperative aseptic management.  Once this theoretical
knowledge is gained, trainees can rapidly learn by observing the
aseptic management techniques of a well-trained surgical support
staff.  Trainees should practice each step until correct
techniques become second nature.

     Assistance with employee training may be available from the
institutional veterinarian, a member of the animal care staff
and/or a member of a hospital surgery staff.


     Sterilization is the process that is intended to kill or
remove all types of micro-organisms.  There are two principal
sterilization methods:

     1)   Physical (dry heat or saturated steam)
     2)   Chemical (ethylene oxide gas or chemical liquids).

   Factors which determine the method to be used are the type of
micro-organisms involved, the nature of the article to be
sterilized and the time available for sterilization.

Physical Methods (Steam)

    Steam sterilization (frequently referred to as autoclaving)
depends on the use of steam above 100C.  Temperatures ranging
from 121-134C at pressures of 15-30 psi are generally
recommended.  The biocidal action of moist heat is a denaturation
of major cell constituents.  Many sterilizers are designed to
provide an automatic sterilization cycle.  In the first stage of
the cycle, air is evacuated and the chamber brought to the
pre-set sterilizing temperature, which is maintained for a
holding period sufficient to kill all microbial contaminants.
Minimum holding times for the sterilization of medical equipment
are 15 minutes at 121C, 10 minutes at 126C, and 3 minutes at
134C.  The steam is then removed and instrument packs are allowed
to dry or liquids cool.  The drying stage may be adjusted to suit
the load.  The chamber is then restored to atmospheric pressure
by the introduction of filtered air.

     The recommended periods of exposure vary with the nature of
the article to be sterilized and the method used to wrap the
article.  Specific details are available from the references at
the end of the chapter.

   Steam sterilization has the advantage of rapid penetration of
wrapped materials with the destruction of all viruses and
bacteria, including the most resistant spores.  The sterilization
of different supplies is more readily controlled than in other
types of sterilizers.  However oils, grease and powdered
substances cannot be sterilized by this method.  The steam
autoclave must be maintained in good repair and operated
correctly in order to perform to specifications.  Sterilization
failure can occur when machines are not regularly serviced.

     Steam autoclave function should be monitored continuously
using one or more of several commercially available indicator
systems.  The color change on a chemical dye impregnated
indicator strip placed within the pack can provide a convenient
and rapid visual check that the appropriate sterilization
conditions were reached.  Function should also be monitored on a
regular basis using commercially available biological indicators.
Spore strips of Bacillus stearothermophilus are placed within the
wrapped article prior to sterilization.  After sterilization the
strip is incubated at 57C for 48 hours.  The absence of growth
indicates effective sporicidal autoclave action.

Chemical Methods (Gas)

     Ethylene oxide gas is effective against all types of
micro-organisms.  The biocidal action of this gas is considered
to be alkylation of nucleic acids.  It is non-corrosive and safe
for most plastic and polyethylene materials.  However, it is not
applicable to liquids or to articles in impervious packaging
material.  It cannot be used to sterilize animal diets due to the
potential toxic effects of this gas.  It can also be a toxic
hazard for animals receiving prosthetic implants which have been
sterilized by this gas.  The operating pressures and temperatures
(45-60C and 10-12 psi) of ethylene oxide sterilizers are
considerably less than for steam units.  Articles should be well
aerated prior to use to minimize the potential for tissue
toxicity.  Aeration should be done in a manner which minimizes
exposure of personnel.  This can be accomplished through the use
of self-aerating sterilizers or separate aeration cabinets.

     Ethylene oxide gas is a potential carcinogen and mutagen and
represents a potential occupational health hazard for personnel
operating sterilizers.  Operation of gas sterilizers and aerators
should be in strict conformance with manufacturers'
recommendations and institutional policies.  Personnel exposure
should be minimized by appropriate ventilation of exhaust gas.  A
regular monitoring program for personnel should be in place.

   Gas sterilizer function should be monitored continuously using
one of several commercially available indicator systems.  The
color change on a chemical dye-impregnated indicator strip placed
within the pack can provide a convenient and rapid visual check
that the appropriate sterilization conditions were reached.
Function should also be monitored on a regular basis using a
commercially available biological indicator such as spore strips
of Bacillus subtilus which are placed within the wrapped article
prior to sterilization.  After sterilization the strip is
incubated at 37C for 24 hours.  The absence of growth indicates
effective sterilization.

     Temperature-sensitive adhesive tape used to secure packages
prior to sterilization only indicates that the package has been
exposed to the sterilizer; this tape does not monitor sterilizer

Chemical Methods (Liquids)

     The use of chemical solutions as a sterilization technique
for surgical equipment is frequently employed, but it should be
stressed that most solutions only disinfect and do not guarantee
sterility.  When the necessity for maintaining sterility is a
critical factor, as in the implantation of prosthetic devices,
indwelling catheters or vascular access ports, disinfection in
chemical solutions is not recommended.  Such prostheses should be
thoroughly sterilized by either gas or steam.  Chemical
solutions, however, offer the advantages of safety for delicate
and thermolabile plastics.

     Other limitations of chemical solutions should also be
appreciated.   Equipment must be thoroughly cleaned before
immersion, as chemical action is ineffective in the presence of
proteins or fats.  There are currently no indicators commercially
available to monitor the effectiveness of this sterilization

     Alcohols are neither sporicidal nor viricidal.  They are not
stable and lose effectiveness through evaporation.  Alcohols
cannot be used for instruments that have plastic or cemented

     The chlorine compounds exert their biocidal action by
oxidization.  The  formulations which require the mixing of acid
and base components with water to generate chlorine dioxide,
offer the advantages of wide spectrum biocidal action and a safe
alternative to the more hazardous phenols or formaldehydes.  The
active shelf life of mixed chemicals is reported to be 24-48

     If chemical sterilization of instruments is the method to be
used, it can be performed in covered trays containing fresh
solutions.  A two-tray system, one each for even-numbered and
odd-numbered days, will ensure that instruments have a full
24-hour contact time.


     The animals should be prepared in an area separate from
where surgery will be performed.  Preparation is facilitated by
first inducing anesthesia.  The stomach, rectum and urinary
bladder can then be evacuated as required at this stage.  Hair is
then removed from the surgical site using electric clippers
equipped with a fine blade.  A liberal area is clipped to
anticipate any enlargement of the initial surgical incision and
minimize wound contamination from adjacent unclipped areas.  In
rodents the need to minimize the loss of heat during surgery and
recovery must be balanced against the need to provide an adequate
aseptic field when clipping the animal.  Animal hair,
particularly rabbit hair, tends to clog clipper blades.  This can
be minimized by frequent cleaning of the blades and regular
lubrication with a commercial aerosol product between use.  A
vacuum can be used to clean up after clipping.  Depilatory
creams may be applied to the surgical site, but they may cause
contact dermatitis which may interfere with the healing process.

     Initial skin cleaning can be done prior to moving the animal
to the operating area. When the animal is moved to the operating
area, it should be positioned on a heating pad on the surgical
table.  To avoid burns heating pads should be wrapped to prevent
direct contact with the animal.  Inclined positioning with a tilt
table is indicated for some procedures and some species.  The
surgical approach will dictate actual animal position, however
some guidelines to consider are:

   a.   The animal's respiratory function should not be
compromised by overextension of forelegs stretched towards the
head, or by excessive body tilt which causes pressure from the
abdominal organs on the diaphragm.

   b.   Limbs should not be extended beyond their normal range of
motion and restraint straps should be padded as needed to prevent
impaired venous return in extremities.

   c.   After the animal has been secured, any monitoring devices
such as ECG electrodes and esophageal stethoscopes should be
placed and their function tested.

   d.   Ruminants are frequently positioned on slight incline
with the head dependent, to minimize the potential for aspiration
of rumen fluids. After intubation with a cuffed endotracheal
tube, a large bore stomach tube is also frequently placed down
the esophagus to remove rumen fluids and gas.

     The animal is now ready for final preparation of the
surgical site.   Personnel who perform the presurgical skin
preparation should wear a cap and mask when preparing the
surgical scrub supplies and when opening pre-sterilized sponge
and drape packs.  Skin preparation solutions may be applied with
a sterile sponge held by a pair of sterile forceps or by a hand
wearing a sterile glove.  A sterile surgical glove is put on one
hand, while the other hand is used to hold and manipulate
non-sterile bottles of surgical scrub solution.  A sterile sponge
held in the gloved hand is saturated with surgical scrub solution
and the surgical area is scrubbed beginning with the central
incision site and working progressively in a circular fashion to
the margins of the shaved area (see fig. 1).  The sponge is then
discarded and the process repeated, working from the center to
the outside to minimize contamination of the surgical site.

     Some of the most frequently used chemical solutions for
preoperative  surgical skin preparation are: chlorhexidine,
iodophors and povidone-iodine surgical scrubs.  Recommended
contact times vary from 2 to 4 minutes.

     Following removal of the scrub solution with a 70 percent
alcohol solution  using the same technique, an iodine skin
solution is painted on the site using the above technique and
left to dry.

     Drapes serve to isolate the surgical site and minimize wound
contamination. Drapes should be positioned without the fabric
dragging across a non-sterile surface.  There are two basic types
of drape systems used: fenestrated and four corner.

     Fenestrated drapes have a hole in them which is placed over
the surgical site.  Frequently used for smaller species, these
drapes are utilized for routine elective procedures.  The
fenestration should be just slightly larger than the intended

     The second alternative is the four corner drape system in
which a drape is placed at each of the four margins of the
surgical site.  Four corner drapes are applied one by one in a
clockwise or counterclockwise direction.  Each drape should be
carefully positioned with a 6 to 8 inch edge folded underneath at
the incision site (see fig. 2 A to D).  Small adjustments in
position can then be made without contaminating the underside of
the drape.  Drapes can be secured in place with towel clamps at
the four corners or aerosol adhesive applied to the margins of
the surgical site prior to draping.

   Some surgeons prefer to secure four corner drapes, then apply
a fenestrated drape as a second layer of protection (see fig. 2,
E and F).  Ideally, the patient and entire surgical table should
be draped, and the drape extended to the instrument table.  The
need to monitor the draped patient should always be considered.
The surgeon who has to work alone often has to assess eye and jaw
reflexes, mucous membrane or tongue color; therefore the head
should not be entirely covered by drape material.

     Self-adhesive backed paper drapes and clear plastic drape
material with one adhesive surface are also commercially


     A well-organized and consistent surgical pack preparation
system can avoid errors and facilitate surgery.  Instruments can
be cleaned by hand or with an ultrasonic cleaning unit.  After
cleaning, each instrument should be inspected to ensure that all
debris has been removed.  After physical cleaning, instruments
can be dipped in a commercial protective lubricant solution and
allowed to drain dry.  Items should be assembled on a tray and
arranged in a consistent order.  Materials should be placed in
sequential order so that items used first are placed on top (see
fig. 3).  Packs should not be too densely packed in the autoclave
to allow for adequate steam or gas penetration.  Indicator test
strips can be placed deep within the pack.  Packs should be
double wrapped, and the outer wrap should be secured with
adhesive indicator tape on which is recorded the date of
sterilization.  When applicable, the type or contents of pack
(e.g., laparotomy, thorocotomy) can also be noted on the tape.

     Note the following points when opening a sterilized surgical
pack.  The sterilization date should be checked; the shelf life
of wrapped instruments is generally considered to be up to 6
months.  The adhesive indicator tape should be noted for the
appropriate color change and the pack description should be
checked, when applicable.  Packs should be placed on a dry
instrument tray and the outer wrapping carefully unfolded by
touching only the corners of the outside drape surface.  The
operator should avoid reaching over the pack.  The packs should
not be opened too early.  The surgeon working without assistance
should open the pack immediately before scrubbing.  Any other
sterilized supplies which can be opened onto a sterile field
should be made ready at this time.


     In a laboratory setting, the extent of surgeon preparation
will depend on the facilities and the need for strict attention
to aseptic technique.  Well-equipped surgical facilities, in
which sophisticated survival procedures are performed, generally
require surgeons to wear appropriate surgical clothing and to
scrub, gown and glove.  Instruction in such procedures should be
done on a one-to-one or small group basis in appropriately
designed scrub rooms.  To augment the actual hands-on approach or
when necessary a video tape demonstration or pictorial diagrams
can be used.  Readers are advised to consult the references
quoted at the end of the chapter for instructional details.

     To minimize wound contamination potential, the surgeon
should change into surgical scrubs and shoes or wear shoe covers.
Head covers and face masks should cover all facial hair.  Remove
all rings, jewelry and wrist watches before scrubbing.
Finger-nails should be trimmed short and cleaned with a
disposable nail cleaner.  Scrub sinks equipped with leg or
foot-operated faucets are ideal.  Regular faucets must be turned
on, adjusted and not touched again.  The hands and forearms are
washed for 30 to 60 seconds with a surgical scrub soap.  Then a
sterile brush is used to methodically scrub all surfaces of the
hands, fingers and forearms down to the elbows.  Both arms are
rinsed and the process repeated starting with fingertips working
down to the elbows.  The definition of a "complete surgical
scrub" is controversial.  However, contact times of 3 to 15
minutes and/or 5 to 20 strokes per surface are frequently

     After rinsing, the hands are held together high and rinse
water allowed to drip from the elbows.  This minimizes the
contamination of hands by water dripping from the non-sterile
upper arm areas.  The surgeon should avoid touching anything at
this stage except to dry the hands with a sterile towel.  Next
the sterile gown is carefully removed from the pack to avoid
touching the outside of the gown.  It is held away from the body
and shaken out.  The sleeve hole is located and each arm inserted
in turn.  Correct gowning requires an assistant to tie the back
of the gown at the neck and waist (being careful to touch only
the inner gown surface).

     Sterile surgical gloves are packaged with the cuff of each
glove turned down.  This allows the gloves to be put on without
the bare hands ever touching the outside surface of the glove.
One glove is picked up by the turned-down cuff and pulled onto
the hand with the cuff left turned down (see fig. 4 - 1 and 2).
Using the gloved hand, pick up the remaining glove by inserting
the fingers into the cuff and pulling it onto the opposite hand
(see fig. 4 - 3).  Then the glove cuff is lifted over and onto
the gown cuff and the process repeated on the other hand (see
fig. 4 - 4,- 5,- 6).  This technique is known as "open gloving."
An alternative and more ifficult method is closed gloving,
descriptions of which can be found in general surgical texts.
Remove the powder on the outer glove surface by wiping the gloved
hands with a damp sterile gauze.  Arms and hands should be held
above the waist at all times.  Aseptic technique is maintained
when the gowned and gloved surgical team only touches sterilized
equipment within the sterile field.

     The surgeon working alone faces logistical problems when
attempting rigid aseptic protocol as defined above.  A proposed
practical sequence of steps to minimize errors is presented as

   1.   Assemble all sterilized supplies required.
   2.   Change into scrubs.
   3.   Set up table, heat pads and gas machines, check
   4.   Weigh animal, induce anesthesia.  Prepare animal by hair
clip and shave, catheters placed as required.
   5.   Position and secure animal on the table.
   6.   Connect to gas machine, connect accessory monitors.
Start I.V. lines as required.
   7.   Make certain that a stable anesthetic plane is attained.
   8.   Put on cap, mask.  Open sterile instrument and prep
   9.   Using one sterile glove, prepare surgical site with scrub
   10.   Put on new sterile glove and drape patient.
   11.   Remove gloves.  Recheck stable anesthetic state.  Open
glove and gown packs if not included in instrument pack.
   12.   Perform surgical scrub.
   13.   Put on gown and gloves.
   14.   Start surgery.


     The practice of aseptic technique, when performing survival
surgical procedures, minimizes the chances that animal health or
experimental data will be compromised by post-surgical
infections.  Aseptic techniques require that appropriate
facilities and equipment be available and that the personnel
involved be adequately trained.  The key element in maintaining
an aseptic environment is well-trained personnel who understand
the principles of aseptic technique and utilize this knowledge on
an ongoing basis.


    Animal Welfare Act (Title 7 U.S.C. 21 31-2156) as amended by
PL 99-198, December 23, 1980.

    Lang, C.M. Animal Physiologic Surgery. Springer-Verlag, New
York, 1976.

     Leonard, E.P. Fundamentals of Small Animal Surgery. W.B.
Sanders, Philadelphia, 1968.

     Knecht, C.D.; Allen, A.R.; Williams, D.J., et al.
Fundamental Techniques in Veterinary Surgery.  W.B. Sanders,
Philadelphia, 1981.

     Gardner, J.F. and Peel, M.M. Introduction to Sterilization
and Disinfection. Churchill Livingstone, Melbourne, 1986.

    McCredie, J.A. and Burns, G.P. (eds.), Basic Surgery.
MacMillan Pub. Co., New York, 1986.

    Banerjee, K. and Cheremisinoff, P.N. Sterilization Systems.
Technomic Publishing Company Inc., Lancaster, PA; 1985.
                             Chapter 6
                         Perioperative Care

                    Marilyn J. Brown, D.V.M., M.S.
                 John C. Schofield, B.V.Sc., M.R.C.V.S.


    Effectively managed perioperative care improves the animals'
recovery by minimizing their pain and distress thus improving the
well-being of the animal and the quality of research data which
can be derived from that animal.  For the purpose of this
discussion, the development of an effective perioperative care
program will be broken down into three overlapping phases;
preoperative planning, intraoperative management and
postoperative support.  Since the nature of the surgical activity
in an institution will largely determine the type of
perioperative program required, evaluation of this activity
should be an ongoing part of the institution's overall animal
care program.

     The investigator, animal care staff and institutional
veterinarian are all essential members of the perioperative care
team.  Communication between these team members is essential to
minimize patient distress and to create an environment in which a
perioperative care program, tailored to the institution's needs,
can be effectively managed.

     The following discussion will review some of the general
principles that should be considered in the establishment and
management of an effective perioperative care program.  Since an
effective perioperative program must be tailored to each
institution's needs, the three-phase approach to developing such
a program will be discussed in general terms.  For more specific
details, the reader is referred to the references included in
this manual.

                      PREOPERATIVE PLANNING

     Personnel who will be involved with perioperative management
and care should be identified with particular attention to assure
that they are appropriately trained.  These individuals need to
be able to identify problems immediately and be familiar with
their management.  The preplanning inclusion of the support
staff, animal care technicians, research technicians and
veterinarians helps assure timely treatment of complications.
The responsibilities of those involved with perioperative care
need to be well defined to assure effective care.  Anticipated
complications, such as pain, vomiting, and paresis, or special
maintenance requirements (e.g., special diets and dressing
changes), need to be thoroughly discussed to facilitate the
development of an effective perioperative management plan.  A
secondary plan to handle the unexpected or less likely
complications should also be established.

   Surgical success is optimized, and more reliable research data
is economically generated when animals in good physical condition
are used.  This starts with the purchase of disease-free
laboratory animals.  Some latent or enzootic diseases in
laboratory animals include: mycoplasmosis in rats, pasteurellosis
in rabbits, distemper in dogs, or Sendai or Mouse Hepatitis Virus
in mice.  Investigators can consult with the institutional
veterinarian or animal care supervisor to identify the most
appropriate source of healthy animals for their study.

   A presurgical physical exam is often appropriate and serves to
identify potential problems.  This may identify animals which
should be rejected from the study or which need some treatment or
special considerations prior to inclusion in the study.  Special
anesthetic or surgical support requirements may also be
determined at this time.  This exam should include visual
observation, and may also include palpation, auscultation, body
temperature, diagnostic laboratory and/or radiographic tests.

     The next step in the preoperative planning process is to
design the most appropriate anesthetic protocol.  Factors such as
species, type of surgery, duration, effect on parameters to be
measured during surgery, etc., should all be considered.  Minimal
central nervous system depression consistent with adequate
analgesia will hasten postoperative recovery and still provide
humane care for the animal.  Well planned anesthetic monitoring
performed by appropriately trained individuals will help avoid
complications.  The Animal Welfare Act requires that the
institutional veterinarian be consulted when designing a study
which has the potential for causing pain to the laboratory
animal.  This consultation can be of great assistance when
designing an anesthetic protocol.

     The necessity for presurgical fasting is species specific.
For example, rabbits should be fasted for 6 hours prior to
intra-abdominal surgery whereas ruminants should be fasted for 48

     The actual surgical procedure, including the following of
asceptic techniques, should be planned with a goal to avoid
postoperative complications.  When selecting a surgical approach,
the anatomy and normal body posture should be considered.  For
example, the dog tolerates lateral thoracotomy with minimal
evidence of discomfort whereas a sternal approach is likely to
cause significant postoperative pain and slower recovery.  In
larger animals such as ruminants, a paracostal approach to the
abdomen is frequently used instead of a ventral midline due to
the high incision tension caused by the heavy abdominal viscera
and the sternal resting position favored by these species.  The
type of suture and suture pattern should also be planned with the
species in mind.  Many animals will either bite at or rub an
incision line, so appropriate wound closure techniques should be
used.  For example, subcuticular sutures are often used in
nonhuman primates who frequently pick at exposed sutures.  The
inherent difficulty in keeping a wound clean and the capillary
action of some uncoated braided fibers which can combine to cause
infection of the surgical site should be considered when
selecting external suture materials.  Planning for the use of
intraoperative analgesics and/or long-acting local anesthetics
should be considered to minimize postoperative pain.

     Cadaver practice and nonsurvival trials can help train
investigators in the sophisticated surgical procedures planned.
This practice can minimize anesthetic and surgical time thereby
promoting uneventful recovery during the actual experiment.

     Plans to monitor the animal for signs of postoperative
infection should be made.  The humane and economic constraints of
research make preventable morbidity and mortality from sepsis
unacceptable.  If the use of antibiotics is anticipated, they
should be administered preoperatively to provide maximum blood
levels during the perioperative period. Dosages for antibiotics
should be appropriate for the species, with consideration given
to species-specific drug toxicity.  For example, penicillin is
contraindicated in guinea pigs.

     The actual location for postoperative recovery needs to be
predetermined.  Recovery in the laboratory may be adequate for
minor procedures; however, major surgeries may require a fully
equipped and staffed postoperative recovery room.  Transportation
to the recovery area also needs to be considered.  Care should be
taken to avoid injury to the animal during anesthetic recovery
(whether by cage mates or by self-inflicted trauma).  Position
the animal in the transport cage to prevent obstruction of the
airway.  Just as in the surgery and the postoperative recovery
cage, maintenance of body temperature is an important
consideration during transport.

                     INTRAOPERATIVE CARE

     To maintain homeostasis during anesthesia, the physiological
condition of the animal should be regularly monitored.
Cardiovascular function can be monitored using mucous membrane
color, auscultation or with electrocardiogram and blood pressure
monitors, depending on the situation and resources available.  In
addition, basic monitoring requires close attention to
respiratory function.  Mucous membrane color can also give an
indication of oxygenation.  Respiratory volume and rate can also
be observed.  Some situations may require the use of a blood gas
analyzer.  A source of oxygen should be available in case of
emergencies even when short, simple procedures are performed.
For longer procedures, periodic manual inflation of the lungs
will help prevent atelectasis.  Adequate cardiopulmonary function
during the operative procedure will facilitate a more rapid and
uneventful recovery.

     Body temperature should also be monitored during surgery,
and maintained through the use of heated water blankets, drapes
and underpads, hot water bottles, etc.  Warming intravenous
fluids prior to administration can also aid intraoperative
thermoregulation.  Hypothermia can be a major problem in animals,
particularly small animals whose larger surface area in relation
to body mass results in quicker relative heat loss.  Hyperthermia
is generally a species-specific phenomenom seen in some breeds of
pigs and families of dogs.

     The small total blood volume of some of the laboratory
animal species necessitates careful attention to hemostasis
during surgery, to prevent hypovolemic shock.  Prolonged surgical
procedures or those procedures with significant relative blood
loss, may require the use of intravenous fluids to maintain blood
pressure and prevent shock.  The use of blood transfusions may be
a useful adjunct in some situations.  Blood type matching is
generally not a practical consideration in many of the laboratory
animal species.

     Positioning of the animal on the table should be done to
avoid compromising cardiovascular or respiratory function.
Improper positioning can lead to other complications such as
aspiration pneumonia, tissue necrosis at pressure points or

     Strict adherence to the principles of aseptic technique is
necessary to avoid postsurgical infection.  These principles can
be reviewed in chapter 5.

     Careful handling of tissues during the surgery is another
factor that will help minimize postsurgical complications.
Traumatic handling of tissue with hands or instruments will delay
healing and may lead to such complications as paralytic ileus.
Careful replacement of viscera will help avoid complications such
as intestinal torsion.  Attention should be given to insuring
that exposed tissues do not become dessicated.  A sterile moist
gauze sponge placed over tissues is often used for this purpose.
Wound closure techniques with either staples or suture material
should be performed in a manner which minimizes tissue damage.
Skin sutures should allow for some tissue swelling or necrosis
may result.  Suture material should be chosen to minimize tissue
reaction and should be of the size appropriate for the location
and species.  It is important to remember that since most
laboratory animals are quadrapeds, the full weight of their
abdominal viscera is on a midline abdominal incision; therefore,
it is usually prudent to use an interrupted pattern in the
abdominal wall.  A subcuticular pattern in the skin may prevent
self-mutilation of skin sutures at the surgical site.

                   POSTOPERATIVE SUPPORT

     The postoperative period can be divided into three phases. 
The first phase is that of anesthetic recovery.  This may be the
most critical time as it is usually the time of greatest
physiologic disturbance and crises can arise quite rapidly.  For
that reason frequent observation is required. The second phase is
that of acute postoperative care when the animal is usually
maintained in the recovery area until adequate stabilization
allows removal to a more standard husbandry situation (i.e.,
eating and drinking has resumed and critical physiological
parameters are within acceptable ranges for the model created).
The third phase, and one most often neglected, is that of
long-term postoperative care.  This long-term management is
important to return the animal to as normal a physiological and
behavioral state as possible.  During this phase, routine
postoperative procedures such as regular observation of the
surgical site, suture removal, observation for return to normal
motor function, dressing changes, physical therapy if indicated,
etc., should be followed.

     Careful observation by trained personnel is the key to good
postoperative care.  Frequency of monitoring is determined by the
nature of the surgical procedure and the stage of recovery. 
Immediate attention needs to be given to the animal's vital
signs.  Cardiovascular and respiratory function must be checked
and maintained.  Specific details about monitoring can be found
in Chapter 4, Principles of Anesthesia and Analgesia.  Until the
animal has recovered from anesthesia, it should be rotated or
turned over every 30-60 minutes to facilitate respiration and
avoid dependent edema.

     Postoperative recovery is best accomplished in a dedicated
postoperative recovery room, ideally located adjacent to the
operating area and close to those persons responsible for
postoperative monitoring.  As in all animal rooms, this room
should be easy to sanitize, equipped with cages designed to avoid
injury to occupants and of appropriate size for the species
involved.  Usually animals should be individually housed during
recovery in cages that have been sanitized between usage.  Care
should be taken to physically separate species which could
transmit disease to one another.  Depending on the procedures,
this room should be equipped with a variety of items designed to
assist with maintenance of homeostasis.  Thermometers should be
available to monitor body temperature.  Hypothermia can be
managed with the use of heat lamps, heating pads, hot water
bottles, increased ambient room temperature, or heated cages.
Intravenous stands and fluids should be available.  Maintenance
of adequate respiratory function is imperative to good recovery;
therefore, a source of oxygen, endotracheal tubes and
laryngoscopes, resuscitation breathing bags and suction should be
available.  Emergency drugs, miscellaneous dressings and supplies
also should be readily available.  An additional light source may
assist in examination and treatment of postoperative patients.  A
place to write and maintain individual postoperative records
should be present.

   Pain, an undesirable aftereffect of surgery, can be difficult
to detect due to species and individual variation.  Therefore,
the investigator must be familiar with the animal's normal
posture and behavior.  Typical behavioral signs of pain include:
guarding the painful area, vocalizing, licking, biting,
self-mutilation, restlessness, lack of mobility, failure to
groom, abnormal posture, failure to show normal patterns of
inquisitiveness, and failure to eat or drink.  Understanding the
degree of pain involved in various experimental procedures allows
a prediction of pain to the animal.  Unless there is evidence to
the contrary, assume that a procedure or a condition painful for
humans will also be painful for animals. When in doubt as to an
animal's pain status, analgesics should be given.  Subsequent
improvement in the animal's condition suggests the previous
existence of pain.  In addition to the administration of
analgesics, parenteral fluids may be continued during the
postoperative period.  Administration of antibiotics may also be
initiated or continued.

     Food and water intake is usually restricted during the
immediate  postoperative period.  When food and water are
reintroduced to the animal, special diets may be indicated.
Intake should be monitored as it is very important to the success
of the recovery that the patient maintain an anabolic state.
Oral or parenteral supplementation may be necessary in some

     Quantity and quality of urine and feces should also be
monitored because changes may indicate one of several
postoperative complications such as paralytic ileus, renal
shutdown or irritation hypermotility.  Appropriate treatment can
then be initiated.  Body temperature should be regularly
monitored for signs of hypothermia or infection.  The wound site
should also be observed for signs of infection, incision
breakdown, or self-inflicted trauma.  Elizabethan collars and/or
bandages can be used to protect the surgical site from
self-inflicted trauma.  If Elizabethan collars are used, the
staff should assure that the animal can reach food and water.
Drains, collars and dressings need to be checked and changed

     Long-term postoperative maintenance may include continued
observation of incisions, dressing maintenance, suture removal,
regular checks to monitor weight loss, and observation for
decubital ulcers or edema.  Physical therapy may also be needed
in some cases for postoperative paresis or paralysis.

                       PROGRAM EVALUATION

     After a procedure and the subsequent postoperative periods,
the perioperative plan and its implementation should be evaluated
and changes initiated where indicated.  This review should have
the input of the investigator/surgeon, the research technicians,
the veterinarian and the animal care staff.  Modifications that
result from this evaluation need to be reviewed with all
personnel involved including the Institutional Animal Care and
Use Committee, where appropriate.  An investigator needs to be
prepared to make appropriate changes in a procedure to prevent
preoccurrence of avoidable perioperative complications.


     An effective, comprehensive perioperative care program
includes: preplanning involving all appropriate personnel;
careful performance of the operative procedure in accordance with
the predetermined plan; careful postoperative observation by
trained personnel during all phases of recovery; and regular
evaluation of the postoperative program in light of the
institution's overall animal care program.  It should be
understood that such a program is tailored to the research being
conducted within the institution and individualized to the
well-being of each animal involved.  The investigator, animal
care staff and institutional veterinarian are all essential
members of the perioperative care team.  Communication between
these team members is essential to minimize patient distress and
to create an environment in which a perioperative care program
can be effectively managed.


     Archibald, J. and Blakely, C.L.  "Surgical Principles",
Canine Surgery, 2nd Ed., Archibald, J. (ed.), American Veterinary
Publications, Inc., Santa Barbara, CA; 1974.

     The Biomedical Investigators Handbook.  Foundation for
Biomedical Research, Washington, DC; 1987.

     Bleicher, N.  "Preoperative and Postoperative Care of the
Laboratory Dog", Proc An Care Panel, March, 1960.

     Blue, J.T. and Short, C.E. "Preanesthetic Evaluation and
Clinical  Pathology",  Principles and Practice of Veterinary
Anesthesia, C.E. Short (ed,), Williams and Wilkins, Baltimore, MD;

     Chaffee, V.W. "Surgery of Laboratory Animals", Handbook of
Laboratory Animal Science,  E.C. Melby, Jr., and N.H. Altman
(eds.), CRC Press, Cleveland, OH; Vol. 1, 1974.

     Haskins, S.C.,  "Postoperative Care", Methods of Animal
Experimentation, W.I. Gay and J.E. Heavner (eds.), Academic
Press, Inc., Vol. 111, Part A, 1986.

     Hoffer, R.E., "Preoperative and Postoperative Care, and
Asceptic Surgery",  Atlas of Small Animal Surgery, Thoracic,
Abdominal, and Soft Tissue Techniques, 2nd Ed., The C.V. Mosby
Company, 1977.

     Hofmann, L.S., "Preoperative and Operative Patient
Management",  Small Animal Surgery: An Atlas of Operative
Techniques, W.E. Wingfield and C.A. Rawlings (eds.), W.B.
Saunders, Philadelphia, PA; 1979.

     University of California - Davis, Animal Use and Care
Adminstrative Advisory Committee, Guidelines for Post-Surgical
Monitoring, Davis, CA; 1986.

     Webb, A.I., "Postoperative Care and Oxygen Therapy",
Principles and Practice of Veterinary Anesthesia, C.E. Short
(ed.), Williams and Wilkins, Baltimore, MD; 1987.

                            Chapter 7

               B. Taylor Bennett, D.V.M., Ph.D.


     A chapter on euthanasia was included in this manual for
several reasons.  The first was to remind investigators of their
responsibilities in assuring institutional compliance with the
regulations and requirements of the various regulatory and
accrediting agencies as they relate to euthanatizing laboratory
animals.  The second was to make them aware of two documents that
will serve as excellent references when evaluating various
euthanasia techniques and when training staff to carry out these

     1)   The Report of the American Veterinary Medical
Association Panel on Euthanasia is recognized by all regulatory
agencies as the accepted published guidelines for selecting and
evaluating euthanasia techniques.

     2)   A more concise discussion of the issue appears in the
chapter entitled "Euthanasia: Providing A Good Death"  In: The
Biomedical Investigator's Handbook.

(Both of these sources are referenced at the end of this
chapter.)  The final reason was to provide the investigator with
a summarized version of the AVMA document for quick reference and
easy reading.

     The term euthanasia is now included in the Definition of
Terms (9 CFR Part 1) of the Animal Welfare Regulations:

   "Euthanasia means the humane destruction of an animal
accomplished by a method which produces rapid unconsciousness and
subsequent death without evidence of pain or distress, or a
method that utilizes anesthesia produced by an agent that causes
painless loss of consciousness and subsequent death."

     The AVMA Panel gives a much briefer definition with an
explanatory sentence:

   "Euthanasia is the act of inducing painless death.  Criteria
to be considered for a painless death are: rapidly occurring
unconsciousness and unconsciousness followed by cardiac and
respiratory arrest."

     The Guide for the Care and Use of Laboratory Animals is the
basis for complying with the Public Health Science Policy.  The
Guide defines euthanasia as: "the procedure of killing animals
rapidly and painlessly."

     In The Biomedical Investigator's Handbook the author states
that: "The word can serve as its own guideline."  He then refers
to its original Greek derivation from the term "eu" meaning
"good" and "thanotos" meaning "death" to define euthanasia simply
as "good death."  He then defines a good death as one: "in which
the animal experiences no pain, no fear, and no other significant
stress before dying."

     When selecting a euthanasia technique, remember that death
should be accompanied by no pain, no fear and no significant

     The key issue then in providing this "good death" is to
minimize the pain and stress experienced by the animal.  The
issue f pain is specifically addressed in some depth in the AVMA
Panel report which indicates that for pain to be perceived the
nerve impulses stimulated by various noxious stimuli must reach a
functional cerebral cortex.  A method which causes rapid loss of
consciousness would then, by definition, produce a painless
death.  Fear and stress in the animals to be euthanatized can be
minimized or eliminated entirely when they are handled in a
humane manner and the individuals charged with this task are well
trained in handling the species involved and cognizant of the
importance of their role in providing the animal a "good death."


     The regulations promulgated to implement the amended Animal
Welfare Act require that the euthanasia methods used be in
accordance with the definition of the term as detailed above,
except when scientifically justified in writing by the principle
investigator.  In addition, the program of adequate veterinary
care must contain a mechanism whereby investigators or other
personnel receive guidance concerning the euthanasia of the
animals they care for and use.

     The Guide requires that personnel performing euthanasia be
trained to use acceptable techniques which should follow the
guidelines established by the AVMA.  When methods recommended in
these guidelines cannot be used, the Guide indicates they be
reviewed and approved by the institutional veterinarian.

     The Public Health Service Policy on Humane Care and Use of
Laboratory Animals generally indicates that the recommendations
contained in the Guide should be those used to establish
acceptable animal care and use programs.  The use of euthanasia
techniques is an exception to this general rule.  The
Institutional Animal Care and Use Committee is specifically
charged with reviewing the methods of euthanasia to assure
compliance with the recommendations of the AVMA.  Methods
deviating from these recommendations must be "justified for
scientific reasons in writing by the investigator."

     In addition to the requirements contained in the PHS Policy,
the PHS Grant Application Form PHS 398 requires the investigator
to address the method of euthanasia in Section F.  The fifth
point in this section is: "Describe any euthanasia method to be
used and the reasons for its selection.  State whether this
method is consistent with the recommendations of the Panel on
Euthanasia of the American Veterinary Medical Association.  If
not, present a justification for not following the

     Regardless of which set of regulations and/or requirements
the use of animals falls under, the key issue in assuring
compliance, as it relates to euthanasia of the animals, is
adherence to the recommendations of the AVMA Panel on Euthanasia.
Responsibility for this compliance begins with the Principal
Investigator in designing the project, continues with the
Institutional Animal Care and Use Committee in reviewing the
project and with the veterinarian in monitoring the program.  All
those involved should have a working knowledge of the fundamental
principles contained in the AVMA document.  The remainder of this
chapter is designed to provide this knowledge by summarizing the
document and providing an easy-to-follow table applicable in most



     In the introduction emphasis is placed upon the need to
define and recognize pain in animals and be able to separate what
may be a response to pain from a reflex response.  For pain to be
experienced, the cerebral cortex and subcortical areas must be
functional and any technique which renders these areas
nonfunctional would eliminate an animal's ability to feel pain.
Emphasis is also placed on the importance of proper restraint in
euthanatizing animals to minimize stress to the animals and
prevent injuries to the personnel involved.

     Criteria for selection of an appropriate euthanasia method
are listed and include: species involved, need for restraint,
skill of personnel, numbers to be euthanatized and the cost of
the procedure iself.  While not discussed in this section, the
importance of considering the effect of the euthanasia technique
on the experimental data must also be of primary concern.
Techniques which potentially compromise results could result in
more animals being used.

Behavioral Considerations

     In this section the need to understand the behavior of the
animals in order to accurately evaluate the presence of pain
and/or distress is reemphasized.  The need to consider the effect
that performing euthanasia can have on staff involved with these
procedures is also discussed.  This factor is one that should be
considered by all those who supervise animal care and use
personnel.  Performing euthanasia can represent a significant
stress for many individuals and can result in job dissatisfaction
and/or failure to correctly perform the technique.  This is
particularly true when physical methods of euthanasia are being
used or large numbers of animals are routinely euthanatized.

Modes of Action

     Euthanatizing agents terminate life by three basic
mechanisms: (1) hypoxia, direct or indirect; (2) direct
depression of neurons for vital life functions; and (3) physical
damage to brain tissue.

     Euthanatizing agents which produce death by hypoxia can act
at various sites and the time of onset of unconsciousness can be
variable.  In some cases, unconsciousness may occur prior to
cessation of motor activity.  Hence, even if animals demonstrate
muscular contractions, they are not perceiving pain.

     Euthanatizing agents acting by direct neuronal depression
depress nerve cells first, blocking apprehension and pain
perception; this is followed by unconsciousness and death.

     Physical methods for euthanatizing animals place an added
responsibility on the principal investigator to insure that those
who perform euthanasia be knowledgeable, well-trained
individuals, because appropriate application of these methods is
essential to produce painless death.

Inhalant Agents

     In this section the use of anesthetic and nonanesthetic
gases which either produce hypoxemia or directly depress the CNS
is discussed.  Of key importance in the use of these agents is
properly operating equipment which assures that the appropriate
concentration of gas is obtained thus minimizing the potential
stress on the animals and the time necessary to produce
unconsciousness.  Of equal importance is the need to protect
personnel from these gases.  Many gases such as chloroform or
hydrogen cyanide are so toxic to personnel that their use cannot
be justified, while others such as ether and cyclopropane must be
used in designated areas.

     When using gases to euthanatize animals, it important that
the stress to the animal be minimized.  Stress can result when
the animal comes into contact with the liquid forms of these
agents, when the animal is placed into a chamber devoid of enough
oxygen to create a suffocating environment or when the gas is
forced into the chamber under pressure in a manner which upsets
the animals.

     Whereas many of the gaseous agents require highly
sophisticated equipment and are expensive or difficult to obtain
or use in an institution, CO2 is inexpensive to use, poses little
risk to personnel, is quite effective and does not interfere with
most types of research.  If CO2 is not available in your
institution, ask the veterinarian about the possibility of
acquiring the necessary equipment for use as a centralized

Noninhalant Pharmacological Agents

     The majority of agents included in this group are barbituric
acid derivatives which have the advantage of producing a rapid
loss of consciousness but have the disadvantage of being
controlled drugs for which a Drug Enforcement Agency (DEA) number
must be provided at purchase and special records of usage must be
maintained.  These drugs should be administered intravenously
except in rodents in which the intraperitoneal route is

     T-61 is an injectable noncontrolled drug which is often used
as a substitute for the barbituric acid derivatives.  It must be
administered intravenously and in accordance with the labeled
instructions.  For this reason the use of this drug is
discouraged except in the hands of highly skilled personnel.

     Another class of drugs included in this section are the
curariforms which induce death by immobilizing the respiratory
muscles resulting in the suffocation of a fully conscious animal.
Use of these drugs for euthanasia is absolutely condemned by the
AVMA.  The use of these drugs is also specifically addressed in
the amended AWA, which prohibits their use in the absence of
appropriate anesthesia.

Physical Methods

     The methods included in this section produce unconsciousness
by direct  damage to the brain and when performed correctly are
acceptable means of euthanasia.  The key to their use is that
they must be performed correctly to produce the "good death"
described earlier in this chapter.  Since these techniques
require the most skill to perform, they are most likely to be
affected by human error.  To minimize the chance of human error,
the personnel performing these techniques must be properly
trained and the responsibility for this training lies with the
principal investigator.  For those techniques commonly employed
in research, the AVMA Panel charges the IACUC with reviewing
those protocols using physical techniques to assure that those
performing the procedures are appropriately trained.

     When the 1986 Panel report was released, the recommendations
concerning the use of cervical dislocation and decapitation were
changed significantly from past reports.  These changes have
created a great deal of discussion within the biomedical
community and have required many investigators to reevaluate the
use of these techniques in terms of the scientific requirements
of their studies.

     The use of cervical dislocation in rabbits and rodents is
only recommended for animals of specified size; the use of
sedation or light anesthesia prior to euthanasia is recommended.
The recommendations concerning the use of a guillotine are more
restrictive and indicate that sedation or light anesthesia should
be used.  Since the publication of these recommendations, there
has been much discussion of their implication for research
projects where the use of concurrent drugs could affect the data
generated.  There has also been considerable discussion of the
study which the panel cited in making their recommendations on
decapitation. At this time it is clear to this author that
additional studies need to be performed to clarify these issues.
Until such studies are evaluated, those investigators who must
use these techniques should adequately justify their use
scientifically to their IACUC and the various funding agencies
and insure that those performing these techniques are adequately

Criteria for Judging Methods of Euthanasia

     Once a method of euthanasia has been selected and approved
by he IACUC, it should be evaluated by the principal investigator
on n ongoing basis to assure that it is indeed meeting the goal
of producing a "good death," by rapid loss of consciousness and a
painless death.  The procedures should also minimize the
potential psychological stress to the animals and personnel
involved.  The cost of the procedure, the compatibility with the
research goals and the safety of the personnel performing the
techniques should also be monitored.  Where controlled drugs are
used the potential for abuse must be considered, but the use of
commercially available euthanasia solutions would almost
eliminate this concern.

Summary of Recommendations for Euthanasia

     The table included with this chapter is an attempt to
summarize the 1986 Report of the AVMA Panel on Euthanasia.  It is
intended to be an easy to use reference source.  When questions
arise concerning the method of euthanasia to be used, the
institutional veterinarian should be consulted for additional


     The use of animals in biomedical research is a privilege.
That privilege places a great deal of responsibility with the
supervising scientist to assure compliance with the highest
scientific, regulatory and societal values.  At no time is this
compliance more subject to review and scrutiny than when it
becomes necessary to kill the animals that have been involved in
a study.  The importance of this final step is emphasized by the
prominence of the issue of euthanasia in the regulations,
policies and guidelines of the various regulatory, accrediting
and funding agencies.  If the "good death" definition is employed
as the standard for technique evaluation, then one should be able
to proceed with the confidence of carrying out the responsibility
that comes with the privilege of using animals in research,
teaching and testing.



Inhalant Agents:      Because in the liquid state most inhalant
Anesthetics           anesthetics act as topical irritants,
                      animals should be exposed to the vapors of
                      the anesthetic only.  Air or oxygen must
                      be provided during the induction period.

Ether                  Cats, young dogs,       Should be used in
                       birds, rodents and      an approved hood
                       other small species     according to

Chloroform               Cats, young dogs,     Potential health
                         rodents and other     hazards outweigh
                         small species         all other

Halothane                Cats, young dogs,        Acceptable
                         birds, rodents and
                         other small species

Methoxyflurane           Cats, young dogs,        Acceptable
                         birds, rodents and
                         other small species

Isoflurane               Cats, young dogs,        Acceptable
                         birds, rodents and
                         other small species

Nitrous oxide (NO2)      Cats, young dogs,         Acceptable in
                         birds, rodents and        100%          

                         other small species       concentration

Enflurane                Cats, young dogs,        Acceptable, but
                         birds, rodents and       not recommended
                         other small species

Inhalant Agents:          Most agents in this category require   

                          the use of special equipment.

Nitrogen (N2)             Dogs, cats, rodents,      Acceptable,
                          lagomorphs over           requires     

                          4-months old              special

Hydrogen Cyanide Gas      Dogs, cats, rodents,      Acceptable,  

                          rabbits                   but not



Carbon Monoxide (CO)     Dogs, cats, rodents,       Acceptable,
                         rabbits                    requires

Carbon Dioxide (CO2)     Dogs, cats, rodents,       Acceptable;
                         rabbits                    larger

Non-Inhalant          Uses of these agents requires adequate
                      restraint and mastery of appropriate
                      injection techniques.


Barbituric Acid         Mammalian species        Acceptable,
                        and birds                should be
                                                 administered IV
                                                 except in
                                                 where IP is an

T-61                     Mammalian species       Acceptable,
                         and birds               must be
                                                 administered IV
                                                 at recommended
                                                 dosages and

Chloral hydrate                                  Not recommended

Mixture Chloral          Horses and cattle       Acceptable
hydrate, pentobarbital

Strychnine                                       Absolutely

Magnesium Sulfate/KCL                            Not to be used
                                                 as sole agent

Nicotine                                         Absolutely

Curariform Drugs                                 Absolutely

Physical Methods:     These methods require that the user have
                      complete mastery of the techniques to be

Electrocution         Mammalian species          Acceptable,
                                                 requires special

Gun Shot: Captive      Large animals             Acceptable,
Bolt Pistol                                      requires special


Rapid Decompression      Dogs and smaller        Not recommended;
                         animals                 other preferable
                                                 Requires special

Stunning                 Small mammals          Acceptable, must
                                                produce immediate
                                                loss of
                                                consciousness and
                                                be followed by
                                                method which
                                                insures death.
                                                Must be
                                                reviewed on a
                                                on a case-by-case
                                                basis by IACUC

Cervical Dislocation     Small mammals,          Acceptable,
                         birds rats (200gm       proper technique
                         or less) and             essential
                         rabbits under 1 Kg.

Guillotine               Small mammals           Should only be
                                                 used when the
                                                 animal has been
                                                 sedated or

Microwave                Small rodents          Acceptable,
                                                special restraint
                                                and focusing
                                                Microwave ovens
                                                are absolutely
                                                for use.

Rapid Freezing           Small rodents          Acceptable,
                                                over 40g must be

Exsanguination           All species            Acceptable when
                                                animal is first

Air Embolism            Rabbits and other       Acceptable only
                        small species           in fully


     Application for Public Health Service, Grant PHS 398.
Revised 10/88 OMB No. 0925-0001.

     Euthanasia: Its History, Chemical and Physical Methods.
1982. Lab Animal, Vol. ll, No.4:l7-4l.

     Guide for the Care and Use of Laboratory Animals, NIH
Publication No. 86-23.

     Report of the AVMA Panel on Euthanasia, l986. JAVMA, Vol.
188, No. 3, 252-268.

     Public Health Service Policy on Humane Care and Use of
Laboratory Animals.  Department of Health and Human Services,
Bethesda, MD; 1986

     Public Law  99-198. Code of Federal Regulations, Title 9,
Subchapter A, Animal Welfare, 1986.

     The Biomedical Investigator's Handbook.  Foundation for
Biomedical Research, Washington, DC; 1987.

     Yoxall, A.T.  Pain in small animals - its recognition and
control. l98l.  ILAR News  Vol. XXV, NO. 1:16-25.

                               Chapter 8
                The Animal Welfare Information Center

                      Jean A. Larson, M.A.
                      Kevin P. Engler, M.S.
                 B. Taylor Bennett, D.V.M., Ph.D.


     The Animal Welfare Information Center (AWIC) was established
at the National Agricultural Library (NAL) in 1986 as a result of
language contained in the amended Animal Welfare Act (99-189).
In the Act, Congress mandated that:

     "The Secretary shall establish an information service at the
National Agricultural Library.  Such service shall, in
cooperation with the National Library of Medicine, provide

     (1)   pertinent to employee training;

     (2)   which could prevent unintended duplication of animal
experimentation as determined by the research facility;

     (3)   on improved methods of animal experimentation,
including methods which could--

          (A)   reduce or replace animal use; and

          (B)   minimize pain and distress to animals, such as
anesthetic and analgesic procedures."

     With appropriations of $750,000 per year for the fiscal year
1987 and 1988 directed to the Library through the Animal and
Plant Health Inspection Service (APHIS), the AWIC was established
as the thirteenth information center within the NAL.  Beginning
with the FY '89 budget year, the AWIC was incorporated with the
base budget of NAL.  This funding has been used to provide
services to patrons, develop information products, purchase
reference materials and hire staff.  Presently, the staff
includes a coordinator and four technical information

                         THROUGH AWIC

     The NAL currently houses over 2 million volumes including
books,  journals, newsletters, proceedings, reports, maps,
microforms, slides, video recordings, films and computer
software.  It also coordinates a national information delivery
network of state land-grant universities and USDA field
libraries.  The substantial resources of the Library enable the
Center's staff to supply information on a broad array of
subjects.  Materials that are commonly accessed for AWIC's
clientele cover important technical, ethical, political and legal
issues related to the welfare of animals.  The publication Animal
Welfare Information Center Scope Notes for Indexers outlines the
animals and subject areas considered to be within the scope of
the Center's collection.  Subjects that are indexed include:
anesthesia, analgesia, euthanasia, training and education of
technicians and investigators, transportation and acquisition of
animals, species husbandry, animal behavior, environmental
factors affecting animals, laboratory animal management,
Institutional Animal Care and Use Committees, regulations and
legislation concerning the humane treatment of animals,
philosophies of animal welfare/rights and alternatives to the use
of animals in research, testing, and ducation.

     Since a significant portion of the present NAL collection
deals with welfare of farm animals and wild animals, the AWIC
staff emphasizes the acquisition of new materials related to the
welfare of laboratory animals.  Literature that involves the use
of esearch animals as experimental subjects, but does not address
the elfare of the animals is generally not indexed.  This type of
information is collected by the National Library of Medicine. 
Since the Primate Information Center of the University of
Washington has an extensive collection of literature involving
the use of laboratory primates, these materials are not generally
collected or indexed by NAL.

     To access its extensive information resources the Library
provides computerized bibliographic retrieval services through
its in-house database Agricultural On-Line Access, or AGRICOLA.
This database and others enable the staff to develop customized
bibliographies tailored to the patron's specific information
needs.  Established in 1970, AGRICOLA contains over 2.6 million
citations covering aspects of agriculture and related subjects
such as plant and animal production, food and nutrition,
forestry, entomology, biotechnology and rural development.  While
there is currently no database specifically for animal welfare,
approximately one-fifth of the AGRICOLA database is devoted to
citations on animal production, laboratory animal science,
veterinary medicine and animal welfare.  AGRICOLA is currently
available through the database vendors DIALOG Information
Retrieval Service (in files 10 and 110) and the Bibliographic
Retrieval Service (BRS) (in file CAIN), or commercially on
compact disc.  AGRICOLA/CAIN may be accessed from the above
commercial vendors using standard dial-up computer terminals.
The publication Searching AGRICOLA for Animal Welfare details
strategies and techniques for efficiently searching the database
for animal welfare topics on both DIALOG and BRS.  Other
databases commonly utilized by the AWIC staff include the
DIALOG files CRIS (60), MEDLINE (154, 155), EMBASE (72, 172,
173), BIOSIS PREVIEWS (5, 55) and CAB ABSTRACTS (50, 53).

     The staff also maintains vertical files on subjects and
organizations related to animal care and use.  These provide a
source of contact persons and information about related
organizations, as well as quick reference to current events and
popular animal-related topics.  The files contain records of
acquisitions and clippings from current newspapers and magazines.
They also include information about the history of animal
welfare, legislation and guidelines pertaining to animal care and
use, and organizations involved in animal welfare or animal
research.  Other files are devoted to specific subject-related
topics such as laboratory ferrets, computer simulations,
guidelines for animal care in the United Kingdom, the Draize
test, laboratory animal identification and technician training.

     The staff has developed an extensive network of subject
experts and organizations active in the area of animal care and
use.  Referrals to individuals and groups may be provided upon

     A table-top exhibit describing the purpose and functions of
the Center is available for loan to interested groups.  The
display is sent via overnight express mail and copies of AWIC
publications may be included with the exhibit.  Return shipment
must be arranged and paid for by the requestor.

     A 12-minute tape entitled "Resources Today for the Research
of Tomorrow" has been developed for distribution to the
registered esearch facilities.  Accompanying the tape is a
brochure on the Animal Welfare Information Center and a Request
for Information form.  The tape provides a brief overview of the
organization and resources of the AWIC.  It can be used as part
of an institution's available training resources as an
introduction to the AWIC for faculty and staff.

     The services of AWIC are available to USDA employees,
federal, state and local government staff, academic and private
institutions, industry, students and the general public.  Under
some circumstances non-USDA personnel may be billed for services.
Materials held in the collection may be obtained on interlibrary
loan through institutional, business, academic, or public
libraries.  The information sheet Document Delivery Services to
Individuals details the photo duplication and loan services to
patrons for requested information.  Requests for information can
be placed by phone or mail, or by visiting the Center in person.


     To fill patron requests as quickly and thoroughly as
possible, a number of bibliographic reference publications on
specific topics defined as important animal welfare issues have
been developed and are continually being updated.  For example,
bibliographies are now available on the Draize and LD50 tests,
alternatives to the use of live animals for research and
education, euthanasia, legislation, training materials for
technicians and investigators, ethical and moral issues,
transgenic animals, and Institutional Animal Care and
Use Committees.  A list of current AWIC publications appears at
the end of this chapter.  Efforts will continue to be directed
toward developing new reference publications.  All AWIC
bibliographies are distributed from the Center without charge.

                       PROJECTS SUPPORTED BY AWIC

     From its inception the AWIC has supported projects that
promote the mandates of the Animal Welfare Act.  Support for
these projects has been provided financially and/or through
active participation by the Center.

     The following projects have been funded with grant monies
provided by AWIC:

   *   An annotated bibliography of important literature relating
to animal welfare entitled Laboratory Animal Welfare Bibliography
compiled by the Scientists Center for Animal Welfare (SCAW).
*(Completed and available from SCAW and AWIC)

   *   A handbook, partially funded by AWIC, produced by the
National Research Council, Institute for Laboratory Animal
Resources entitled Guidelines for the Recognition and Alleviation
of Pain and Distress in Laboratory Animals.

   *   An educational videotape program, Alternatives in Animal
Research, produced by Texas University Health Sciences Center,
which will survey past and present ethical issues relating to
animal research and discuss the concepts of reduction, refinement
and replacement in the context of experimental design and

   *   Proceedings of a conference held June 22-25, 1988, by the
Scientists Center for Animal Welfare (SCAW) entitled  Science and
Animals: Addressing Contemporary Issues, covering various aspects
of animal experimentation.  *(Completed and available for
purchase from SCAW @ $25.00)

   *   Two updated guidelines, Laboratory Animal Management:
Rodents and Laboratory Animal Management: Dogs, produced by the
National Research Council, Institute of Laboratory Animal

   *   Two publications regarding alternative animal toxicology
testing entitled  Benchmarks: Alternative Methods in Toxicology
and A Predictive Model for Estimating Rat Oral LD50 Values
produced by the Princeton Scientific Publishing Company.
*(Completed and available for purchase from Princeton Scientific)

                UPDATES REGARDING AWIC and NAL

     Patrons are welcome to visit AWIC and other NAL offices on
weekdays from 8:00 a.m. to 4:30 p.m.  A tour of the Library's
facilities is available by appointment.  For current updates
regarding AWIC and NAL, the Library's newsletter Agricultural
Libraries Information Notes is available on a monthly basis
free-of-charge.  The Agricultural Library Forum (ALF) renewal, an
electronic bulletin board system, also provides current
information about new and existing products and services of AWIC
and NAL, and serves as a forum for the exchange of agricultural
information between libraries, information centers, and other
users. A "Brief Guide" to ALF has been prepared to introduce the
major features of the system and to help callers get started.

     For additional information please contact the AWIC staff by
mail or by telephone at:

               Animal Welfare Information Center
               National Agricultural Library
               10301 Baltimore Blvd., Room 205
               Beltsville, MD  20705
               (301) 504-6212


Quick Bibliographies:

Animal Models of Disease QB 92-61

Animal Welfare Legislation and Regulation QB 92-35

BST - Bovine Somatotropin/Growth Hormone QB 92-30

Ethical and Moral Issues Relating to Animals QB 92-51

Housing, Stress and Welfare of Sheep and Goats QB 92-59

Laboratory Animal Facilities and Management QB 92-58

PST - Porcine Growth Hormone QB 92-31

Raising Quail, Partridge, Pheasant, Bobwhites, and Ostriches
QB 92-20

Stress in Cattle QB 91-18

Stress in Horses QB 91-06

Stress in Poultry QB 91-01

Stress in Swine QB 91-16

Training Materials for Animal Facility Personnel QB 91-07

Transport and Handling of Livestock QB 92-57

Welfare of Experimental Animals QB 91-83

Veal Calves QB 92-67

Annotated Bibliographies:

Laboratory Animal Welfare Bibliography (Scientist Center for
Animal Welfare/National Agricultural Library, October 1991)

Search Tip Series:

Searching AGRICOLA for Animal Welfare STS-03

Special Reference Briefs:

Animal Care and Use Committees SRB 92-16

Animal Euthanasia SRB 91-02

The Draize Eye-Irritancy Test 1979-1988  SRB 91-03

The LD50 (Median Lethal Dose) Toxicity Test 1980-1988 SRB 92-12

Animal Models in Biomedical Research: Poultry SRB 92-17

Animal Models in Biomedical Research: Swine SRB 91-06

Reference Materials for Members of Animal Care and Use Committees
AWIC Series #10


ALF (Agricultural Library Forum): The National Agricultural
Library's Electronic Bulletin Board System: A Brief Guide

The Animal Welfare Information Center Brochure

Animal Welfare Information Center Scope Notes for Indexers, AWIC
Series #6

Animal Welfare Legislation: Bills and Public Laws 1980 - October
1988, AWIC Series 8

Animal Welfare Legislation: Bills and Public Laws 1989, AWIC
Series #2

Animal Welfare Legislation: Bills and Public Laws 1990, AWIC
Series #4

Animal Welfare Legislation: Bills and Public Laws 1991, AWIC
Series #9

Audio-Visuals Relating to Animal Care, Use and Welfare, AWIC
Series 7

Environmental Enrichment Information Resources for Nonhuman
Primates: 1987 -1992

Essentials for Animal Research: A Primer for Research Personnel
(NAL/Univ. of Illinois)

Selected Readings on the History and Use of Old Livestock Breeds
(AWIC Jan. 1992)

Sterilization of Marine Mammal Pool Waters: Theoretical and
Health Considerations (APHIS - Tech. Bull. No. 1797)

The Well-being of Agricultural Animals in Biomedical and
Agricultural Research (SCAW Feb. 1992)


     Animal Welfare Act - Title 7 U.S.C. 2131 - 2156 as amended
by PL-99-198 

                            Chapter 9
             Organizations, Associations and Societies

                Marilyn J. Brown, D.V.M., M.S.
            John C. Schofield, B.V.Sc., M.R.C.V.S.
                B. Taylor Bennett, D.V.M., Ph.D.


American Association for Accreditation of Laboratory Animal Care
   Voluntary accrediting body for demonstrating achievement of
certain standards for an animal care and use program.
    Albert E. New, Executive Director, 9650 Rockville Pike,
    Bethesda, MD  20814. (301) 571-1850.

American Association for Laboratory Animal Science
   A professional association for veterinarians, animal care
workers, managers and manufacturers involved in laboratory animal
science.  Publisher of Laboratory Animal Science.
      Donald W. Keene, Executive Director, 70 Timber Creek Drive,
      Suite 5, Cordova, TN  38018. (901) 754-8620.

Association of American Medical Colleges
   Through its ad Hoc Group for Medical Research Funding
published recommendations and guidelines on the use of animals in
      1 Dupont Circle, Suite 200, Washington, DC 20036.
      (312) 828-0470.

American College of Laboratory Animal Medicine
   Certifies veterinarians (Diplomates) who achieve certain
standards in Laboratory Animal Medicine.
      C. Max Lang, Secretary-Treasurer, Dept. Comparative
      Medicine, The Milton Hershey Medical Center, The
      Pennsylvania State University, P.O. Box 850,  Hershey, PA
      17033.  (717) 531-8462.

American Medical Association
   A professional association of physicians.  Published a White
Paper on the Use of Animals in Biomedical Research.
      535 North Dearborn St., Chicago, IL  60610. (312) 645-5000.

American Psychological Association
   An association founded to advance the understanding of basic
behavioral principles.  Publishes a detailed statement on the
care and use of animals entitled Guidelines for Ethical Conduct
in the Care and Use of Animals.
      1200 17th Street, N.W., Washington, DC  20036.
      (202) 955-7653.

Animal Plant and Health Inspection Service
   That division of the U.S. Department of Agriculture that
administers the federal Animal Welfare Act.
      U.S. Department of Agriculture, Animal and Plant Health
      Inspection Service, REAC, 6505 Belcrest Rd., Room 268-FB,
      Hyattsville, MD  20782.  (301) 436-7833.

American Physiological Society
   First scientific society to adopt a written statement on the
prevention of cruelty to research animals.  Distributes the
Guiding Principles in the Care and Use of Animals to members for
signing and posting.
      9650 Rockville Pike, Bethesda, MD  20814. (301) 530-7164.

American Society of Laboratory Animal Practitioners
   An organization of veterinarians engaged or interested in the
practice of laboratory animal medicine.
      Farol N. Tomson, Secretary-Treasurer. 182 Grinter Hall,
University of Florida, Gainesville, FL  32611. (904) 392-9917.

American Veterinary Medical Association
   A professional association of veterinarians. In 1986, the AVMA
published recommended standards for euthanasia procedures which
are accepted as national guidelines.
      930 N. Meacham Rd., Schaumburg, IL  60196-1074.
     (800) 248-2862

Association for Veterinary Technicians and Educators, Inc.
   The original name of the group was the Association of Animal
Technicians Educators. Its purposes is to stimulate improvement
of instruction in animal technology education.

Animal Welfare Institute
   A national organization active in laboratory animal welfare
issues.  Its sister organization, the Society for Animal
Protective Legislation, is a major lobbying force.  The AWI
encourages lay persons to serve on IACUC's and has a number of
publications pertinent to laboratory animal welfare.
      Mrs. Christine Stevens, 1686-34th Street, N.W., Washington,
      DC  20007.

Animal Welfare Information Center
   The information center of the National Agricultural Library
established as result of the 1985 amendment to the Animal Welfare
Act.  See Chapter 8.
      Animal Welfare Information Center, National Agricultural
      Library, Room 205, Beltsville, MD  20705. (301) 504-6212.

Center for Alternatives to Animal Testing
   Established in 1981 to encourage and support the development
of non-animal testing methods.  The center supports grants,
sponsors symposia and publishes a variety of materials.
      Johns Hopkins School of Public Health, 615 North Wolfe St.,
      Baltimore, MD  21205. (301) 955-3343.

Canadian Association of Laboratory Animal Science
   A professional association for veterinarians and technicians
involved with laboratory animal science.
      Donald G. McKay, Executive Director, BioScience Animal
      Services, M524 Biological Sciences Building, The University

      of Alberta, T6G 2E9 Canada.  (403) 432-5193.

Canadian Council on Animal Care
   The national body that establishes policy on the care and use
of laboratory animals in Canada.  Has many useful publications.
      1000-151 Slater Street, Ottawa, Ontario, K1P 5H3. (613)

Drug Enforcement Administration - United States Department of
     The regulatory agency responsible for the enforcement of
laws pertaining to controlled substances.  Licenses to use
controlled substance are obtained from this agency.
      P.O. Box 28083, Central Station, Washington, DC  20005.
      (202) 724-1013.

Federation of American Societies of Experimental Biology.
   A federation of leading professional associations, including,
physiologists and pharmacologists and other major disciplines
involved with animal experiments.
      9650 Rockville Pike, Bethesda, MD  20814. (301) 530-7000.

Foundation for Biomedical Research
   A nonprofit educational organization established to inform the
American public about the proper and necessary role of animal
models in biomedical research and testing.
      818 Connecticut Ave., N.W., Suite 303, Washington, DC
      20006. (202) 457-0654.

U.S. Food and Drug Administration
   The federal agency responsible for enforcement of the Good
Laboratory Practices (GLP) regulations.
      5600 Fishers Lane, Rockville, MD  20857.  (301) 443-1544.

International Association for the Study of Pain
   Publishes the journal Pain and has developed "Ethical
Standards for Investigators of Experimental Pain in Animals."
      909 NE, 43rd St., Suite 306, Seattle, WA  98105.
      (206) 547-6409.

Institute of Laboratory Animal Resources
   That part of the National Academy of Sciences which has
responsibility for laboratory animal issues.  ILAR is responsible
for preparing part of the Public Health Service Policy entitled
Guide for the Care and Use of Laboratory Animals.
      2101 Constitution Ave., NW,  Washington, DC  20418.
      (202) 334-2590.

International Council for Laboratory Animal Science
      Osmo Hanninen, University of Kuopio, P.O. Box 140,
      SF-70101, Kuopio 10, Finland.

National Association for Biomedical Research
   An association of biomedical facilities concerned with
legislation on laboratory animal welfare and with presenting
information about the benefits to human health resulting from
animal experiments.
      Frankie Trull, Executive Director, 818 Connecticut Ave.,
      NW, Suite 303, Washington, DC  20006. (202) 857-0540.

National Agricultural Library
   (See AWIC)

National Academy of Sciences
   Established the National Research Council in 1916 for the
purpose of furthering knowledge and advising the federal
government.  The Guide for the Care and Use of Laboratory Animals
was reviewed and approved by the Governing Board of the National
Research Council.  See ILAR.

National Institutes of Health
   A federal agency which disburses funds for biomedical research
and sets policy on laboratory animal welfare, (Public Health
Service Policy).
      Office of Animal Care and Use, 9000 Rockville Pike,
      Bethesda, MD  20892. (301) 496-5793.

National Science Foundation
   A federal agency responsible for disbursement of funds in
support of non-biomedical research, i.e., zoological and wildlife
      1800 G. Street N.W., Washington, DC 20550.  (202) 357-9854.

Office for Protection From Research Risks, National Institutes of
   The office which oversees compliance with the Public Health
Service Policy on Humane Care and Use of Laboratory Animals.
      9000 Rockville Pike, Building 31, Room 4B09, Bethesda, MD
      20892.  (301) 496-7005.

Public Health Service
   Comprises several federal agencies that are involved with
either medical research or provision of medical health services.
The National Institutes of Health is the major agency within the
PHS relevant to laboratory animal issues.

Scientists Center for Animal Welfare
   A nonprofit educational organization of scientists that
upholds justifiable animal research and conducts programs to help
ensure compliance with federal policies, introduction of
alternatives where feasible, and sensitivity to humane issues
among scientists.
      4805 St. Elmo Ave., Bethesda, MD  20814. (301) 654-6390.

United States Department of Agriculture
   The federal agency responsible for enforcement of the federal
Animal Welfare Act, see also APHIS.

                           Chapter 10
                       General References

            John C. Schofield, B.V.Sc., M.R.C.V.S.
                Marilyn J. Brown, D.V.M., M.S.
               B. Taylor Bennett, D.V.M., Ph.D.


ILAR News (quarterly). Washington, DC; Institute of Laboratory
Animal Resources, National Research Council. Mailing address:
2101 Constitution Ave.  NW, Washington, DC  20077-5576.

Laboratory Animal Science (bi-monthly). Joliet, Illinois:
American ssociation for Laboratory Animal Science.  Mailing
address: 70 Timber Creek Drive, Suite 5, Cordova, TN  38018.

Laboratory Animals (quarterly). Journal of the Laboratory Animal
Science Association  Laboratory Animals Ltd., London.  Mailing
address: The Registered Office, Laboratory Animals Ltd., 1
Thrifts Mead, Theydon Bois, Essex, CM16 7NF, United Kingdom.


Biology Data Book  2nd ed. P.L. Altman and D.S. Dittmer. Vol. 1,
1971, 606 pp.; Vol. 2, 1973, 1432 pp.; Vol. 3, 1974, 2123 pp.,
Federation of American Societies for Experimental Biology,
Bethesda, MD.

The Biology and Medicine of Rabbits and Rodents  J.E. Harkness
and J. E. Wagner.  Lea and Febiger, Philadelphia, PA; 1983, 210

Clinical Laboratory Animal Medicine  D.D. Holmes, Iowa State
University Press, Ames, IA; 1984, 138 pp.

Environmental and genetic factors affecting laboratory animals:
Impact on biomedical research. Introduction. C.M. Lang and E.S.
Vesell. Fed. Proc. 35:1123-1124 (1976).

The Future of Animals, Cells, Models, and Systems in Research,
Development, Education, and Testing.  ILAR (Institute of
Laboratory Animal Resources). Proceedings of a symposium
organized by an ILAR committee. National Academy Press,
Washington, DC; 1977.

Guide for the Care and Use of Laboratory Animals.  NIH No. 86-23.
U.S. Government Printing Office, Washington, DC.

Guide for the Care and Use of Agricultural Animals in
Agricultural Research and Teaching.  Consortium for Developing a
Guide for the Care and Use of Agricultural Animals in
Agricultural Research and Teaching.  309 West Clark Street,
Champaign, IL  61820.

Guide to the Care and Use of Experimental Animals.  Canadian
Council on Animal Care (CCAC). Canadian Council on Animal Care,
Ottawa, Ontario; Vol.1, 1980, 112 pp.; Vol. 2, 1984, 208 pp.
(Available from CCAC, 1105-151 Slater Street, Ottawa, Ontario K1P
5H3, Canada)

Handbook of Laboratory Animal Science. E.C. Melby, Jr. and N.H.
Altman, (eds.), Vol. 1, 1974, 451 pp.; Vol. 2, 1974, 523 pp.;
Vol. 3, 1976, 943 pp.; CRC Press, Cleveland, OH.

Inbred Strains in Biomedical Research  M.F.W. Festing. Macmillian
Pub., London; 1979, 483 pp.

The Importance of Laboratory Animal Genetics, Health, and the
Environment in Biomedical Research. E.C. Melby, Jr. and M.W. Balk
(eds.), Academic Press, Orlando, FL; 1983, 284 pp.

Laboratory Animal Medicine. J.G. Fox; B.J. Cohen and F.M. Loew
(eds.), Academic Press, New York, NY; 1984, 750 pp.

Laboratory Animal Welfare. National Library of Medicine (NLM)
Specialized Bibliography Series.  Compiled by F.P. Gluckstein.
SBS No. 1984-1. U.S. Department of Health and Human Services,
Washington, DC; 85 citations, 1984, 18 pp. (Available from
Reference Services Division, NLM, Bethesda, MD  20209).

Laboratory Animal Welfare: Supplement 1.  National Library of
Medicine (NLM) Specialized Bibliography Series.  Compiled by F.P.
Gluckstein.  SBS No. 1985-1.  U.S. Department of Health and Human
Services, Washington, DC; 31 citations; 1985, 6 pp. (Available
from Reference Services Division, NLM, Bethesda, MD 20209).
Methods of Animal Experimentation.  W.I. Gay (ed.), Vol. 1, 1965,
382 pp.;  Vol. 2, 1965, 608 pp.; Vol. 3, 1968, 469 pp.; Vol. 4,
1973, 384 pp.; Vol. 5, 1974, 400 pp.; Vol. 6, 1981, 365 pp.,
Academic Press, New York, NY.

Of Mice, Models, and Men: A Critical Evaluation of Animal Research.
A.N. Rowan. State University of New York Press, Albany, NY; 1984,
323 pp.

Practical Guide to Laboratory Animals.  C.S.F. Williams.  C.V.
Mosby, Co., St. Louis, MO; 1976, 207 pp.

Reproduction and Breeding Techniques for Laboratory Animals.
E.S.E. Hafez (ed.), Lea and Febiger, Philadelphia, PA; 1970, 375

Restraint of Animals.  J.R. Leahy and P. Barrow. Cornell Campus
Store, Ithaca, NY; 2nd ed., 1953, 269 pp.

Scientific Perspectives on Animal Welfare.  W.J. Dodds and F.B.
Orlans (eds.), Academic Press, New York, NY; 1982, 131 pp.

The UFAW Handbook on the Care and Management of Laboratory
Animals. UFAW (Universities Federation for Animal Welfare) (ed.),
Churchill Livingstone, New York, NY; 6th ed., 1987, 635 pp.


Holders of Inbred and Mutant Mice in the United States Including
the Rules for Standardized Nomenclature of Inbred Strains, Gene
Loci, and Biochemical Variants.  D.D. Greenhouse (ed.), ILAR News
27(2):1A-30A (1984).

Inbred and Genetically Defined Strains of Laboratory Animals.
P.L. Altman and D.D. Katz (eds.), 1979. Part 1,  Mouse and Rat,
418 pp.; Part 2, Hamster,  Guinea Pig, Rabbit, and Chicken, 319
pp., Federation of American Societies for Experimental Biology,
Bethesda, MD.

International Standardized Nomenclature for Outbred Stocks of
Laboratory Animals.  Issued by the International Committee on
Laboratory Animals.  M. Festing; K. Kondo; R. Loosli; S.M. Poiley
and A. Spiegel. ICLA Bulletin 30:4-17 (March 1972). (Available
from the Institute of Laboratory Animal Resources,  National
Research Council, 2101 Constitution Avenue, N.W., Washington, DC

Laboratory Animal Management: Genetics.  ILAR (Institute of
Laboratory Animal Resources). ILAR News 23(1):A1-A16 (1979).


Complications of Viral and Mycoplasmal Infections in Rodents to
Research and Testing.  T.E. Hamm, McGraw-Hill, Washington, DC;
1986, 191 pp.

Outline of Veterinary Clinical Pathology.  M.M. Benjamin. Iowa
State  University Press, Ames, IA; 3rd ed., 1978, 352 pp.

Viral and Mycoplasmal Infections of Laboratory Rodent-Effects on
Biomedical Research.  P.N. Bhatt; R.O. Jacoby; H.C. Morse and
A.E. New (eds.), Academic Press, Orlando, FL; 1986, 844 pp.


Animal Anesthesia.  C.J. Green. Laboratory Animals Ltd., London;
1979, 300 pp.

Animal Pain. Perception and Alleviation.  R.L. Kitchell; H.H.
Erickson; E. Carstens and L.E. Davis.  American Physiological
Society, Bethesda, MD; 1983, 221 pp.

Animal Physiologic Surgery. C.M. Lang (ed.), Springer-Verlag, New
York, NY; 2nd ed., 1982, 180 pp.

Basic Surgical Exercises Using Swine. M.M. Swindle. Praeger, New
York, NY;  1983, 237 pp.

Experimental Surgery: Including Surgical Physiology.  J.
Markowitz; J.  Archibald and H.G. Downie.  Williams and Wilkens,
Baltimore, MD; 5th ed.,  1964, 659 pp.

Experimental and Surgical Technique in the Rat.  H.B. Waynforth.
Academic Press, New York, NY; 1980, 269 pp.

Laboratory Animal Anesthesia - An Introduction for Research
Workers and Technicians. P.A. Flecknell. Academic Press, London;
1987, 151 pp.

Large Animal Anesthesia: Principles and Techniques.  T.W.
Riebold; D.O. Goble and D.R. Geiser.  State University Press,
Ames, IA; 1982, 154 pp.

The relief of pain in laboratory animals.  P.A. Flecknell.
Laboratory Animal 18:147-160 (1984).

Textbook of Veterinary Anesthesia.  L.R. Soma (ed.),  Williams
and Wilkins, Baltimore, MD; 1971, 621 pp.

Veterinary Anesthesia.  W.V. Lumb and E.W. Jones.  Lea and
Febiger, Philadelphia, PA; 2nd ed., 1984, 693 pp.

Veterinary Anesthesia. C.E. Short. Williams and Wilkins, Baltimore,
MD; 1987, 669 pp.

Veterinary Anesthesia. L.W. Hall and K.W. Clarke. Bailliere
Tindall, East Sussex, England; 1983, 417 pp.


Control of Diets in Laboratory Animal Experimentation.  ILAR
(Institute of Laboratory Animal Resources), Committee on
Laboratory Animal Diets. ILAR News 21(2):A1-A12 (1978).

Nutrient Requirements of Laboratory Animals.  BARR (Board on
Agriculture and Renewable Resources) Subcommittee on Laboratory
Animal Nutrition, Committee on Animal Nutrition.  Nutrient
Requirements of Domestic Animals Series  National Academy of
Sciences, Washington, DC; 3rd rev. ed., 1978, 96 pp.


Laboratory Animal Housing. ILAR (Institute of Laboratory Animal
Resources), Committee on Laboratory Animal Housing. National
Academy of Sciences, Washington, DC; 1978, 220 pp.


Clinical Textbook for Veterinary Technicians.  D.M. McCurnin.
W.B. Saunders, Philadelphia, PA; 1985, 511 pp.

The Education and Training of Laboratory Animal Technicians.  S.
Erichsen; W.J.I. van der Gulden; O. Hanninen; G.J.R. Hovell; L.
Kallai and M. Khemmani. Prepared for the International Committee
on Laboratory Animals.  World Health Organization, Geneva; 1976,
42 pp.

Laboratory Animal Medicine: Guidelines for Education and
Training. ILAR (Institute of Laboratory Animal Resources),
Committee on Education. ILAR News 22(2):M1-M26 (1979).

Manual for Assistant Laboratory Animal Technicians.  AALAS
(American Association for Laboratory Animal Science). AALAS Pub.
No. 84-1  American Association for Laboratory Animal Science,
Joliet, IL; 1984, 454 pp.

Manual for Assistant Laboratory Animal Technicians.  AALAS
(American Association for Laboratory Animal Science). AALAS Pub.
No. 84-2  American Association for Laboratory Animal Science,

Joliet, IL; 1984, 248 pp.

Syllabus of the Basic Principles of Laboratory Animal Science. Ad
Hoc Committee on Education of the Canadian Council on Animal Care
(CCAC). Canadian Council on Animal Care, Ottawa, Ontario; 1984,
46 pp. (Available from CCAC, 1105-151 Slater Street, Ottawa,
Ontario K1P 5H3, Canada)

Syllabus for the Laboratory Animal Technologist.  AALAS (American
Association for Laboratory Animal Science). AALAS Pub. No. 72-2 
American Association for Laboratory Animal Science, Joliet, IL;
1972, 462 pp. (Available from AALAS, 70 Timber Creek Drive, Suite
5, Cordova, TN  38018.


Biohazards and Zoonotic Problems of Primate Procurement,
Quarantine and Research.  M.L. Simmons (ed.), Cancer Research
Safety Monograph Series, Vol. 2. DHEW Pub. No. (NIH) 76-890  U.S.
Department of Health, Education, and Welfare, Washington, DC;
1975, 137 pp.

Biosafety in Microbiological and Biomedical Laboratories.  HHS
Publication No. (NIH) 88-839  U.S. Department of Health and Human
Services, Washington, DC; 139 pp.

Code of Federal Regulations. Title 40; Part 260, Hazardous Waste
Management System: General; Part 261, Identification and Listing
of Hazardous Waste; Part 262, Standards Applicable to Generators
of Hazardous Waste; Part 263, Standards Applicable to
Transporters of Hazardous Waste; Part 264, Standards for Owners
and Operators of Hazardous Waste Treatment, Storage, and Disposal
Facilities; Part 265, Interim Status Standards for Owners and
Operators of Hazardous Waste Treatment, Storage, and Disposal
Facilities; and Part 270, EPA Administered Permit Programs: The
Hazardous Waste Permit Program. Office of the Federal Register,
Washington, DC; 1984.

Guidelines for Prevention of Herpesvirus Simiae (B-Virus)
Infection in Monkey Handlers.  J.E. Kaplan, et al. Laboratory
Animal Science 37(6): 709-712 (1987).

NIH Guidelines for the Laboratory Use of Chemical Carcinogens.
National Institutes of Health. NIH Pub. No. 81-2385. U.S.
Department of Health and Human Services, Washington, DC; 1981, 15


Environments and genetic factors affecting the response of
laboratory animals to drugs.  E.S. Vesell; C.M. Lang; W.J. White;
G.T. Passananti; R.N. Hill; T.L.  Clemens; D.K. Liu and W.D.
Johnson.  Federal Proceedings 35:1125-1132

Influence on pharmacological experiments of chemicals and other
factors in diets of laboratory animals. P.M. Newberne. Federal
Proceedings 34:209-218 (1975).


Alternatives to Animals Use in Research, Testing and Education 
U.S. Congress,  Office of Technology Assessment. U.S. Government
Printing Office, Washington, DC; OTA-BA-273, 1986.


          Animal Welfare Information Center
          National Agricultural Library
          10301 Baltimore Blvd., Room 205
          Beltsville, MD  20705