GUIDELABORATORYANIMALS

FOR THE CARE AND USE OF

Eighth Edition

Committee for the Update of the Guide for the Careand Use of Laboratory Animals
Institute for Laboratory Animal Research
Division on Earth and Life Studies

THE NATIONAL ACADEMIES PRESS

500 Fifth Street, NW

Washington, DC 20001

NOTICE: The project that is the subject of this report was approved by the Governing Board of the National Research Council, whose members are drawn from thecouncils of the National Academy of Sciences, the National Academy of Engineering, and the Institute of Medicine. The members of the Committee responsible forthe report were chosen for their special competences and with regard for appropriatebalance.
This study was supported by the Office of Extramural Research, Office of the Director, National Institutes of Health/Department of Health and Human Services underContract Number N01-OD-4-2139 Task Order #188; the Office of Research Integrity,
Department of Health and Human Services; the Animal and Plant Health Inspection
Service, U.S. Department of Agriculture; Association for Assessment and Accreditationof Laboratory Animal Care International; American Association for Laboratory Animal
Science; Abbott Fund; Pfizer; American College of Laboratory Animal Medicine; American Society of Laboratory Animal Practitioners; Association of Primate Veternarians.
Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of theorganizations or agencies that provided support for the project. The content of
this publication does not necessarily reflect the views or policies of the National
Institutes of Health, nor does mention of trade names, commercial products, ororganizations imply endorsement by the US government.
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The National Academy of Sciences is a private, nonprofit, self-perpetuating societyof distinguished scholars engaged in scientific and engineering research, dedicatedto the furtherance of science and technology and to their use for the general welfare.
Upon the authority of the charter granted to it by the Congress in 1863, the Academy has a mandate that requires it to advise the federal government on scientificand technical matters. Dr. Ralph J. Cicerone is president of the National Academyof Sciences.
The National Academy of Engineering was established in 1964, under the charterof the National Academy of Sciences, as a parallel organization of outstanding
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sharing with the National Academy of Sciences the responsibility for advising thefederal government. The National Academy of Engineering also sponsors engineering programs aimed at meeting national needs, encourages education and research,and recognizes the superior achievements of engineers. Dr. Charles Vest is presidentof the National Academy of Engineering.
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Fineberg is president of the Institute of Medicine.
The National Research Council was organized by the National Academy of Sciences in 1916 to associate the broad community of science and technology with the
Academy’s purposes of furthering knowledge and advising the federal government.
Functioning in accordance with general policies determined by the Academy, theCouncil has become the principal operating agency of both the National Academyof Sciences and the National Academy of Engineering in providing services to
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Dr. Ralph J. Cicerone and Dr. Charles M. Vest are chair and vice chair, respectively,of the National Research Council.
www.national-academies.org

COMMITTEE FOR THE UPDATE OF THE GUIDE FOR THE CARE
AND USE OF LAbORATORy ANIMALSMembers
Janet C. Garber (Chair), Garber Consulting
R. Wayne barbee, Virginia Commonwealth University
Joseph T. bielitzki, University of Central Florida
Leigh Ann Clayton, National Aquarium, Baltimore
John C. Donovan, BioResources, Inc.
Coenraad F. M. Hendriksen, Netherlands Vaccine Institute, Bilthoven,
The Netherlands (until March 2009)
Dennis F. Kohn, Columbia University (retired)
Neil S. Lipman, Memorial Sloan-Kettering Cancer Center and Weill
Cornell Medical College
Paul A. Locke, Johns Hopkins Bloomberg School of Public HealthJohn Melcher, U.S. Senate (retired)Fred W. Quimby, Rockefeller University (retired)
Patricia V. Turner, University of Guelph, Canada
Geoffrey A. Wood, University of Guelph, Canada
Hanno Würbel, Justus Liebig University of Giessen, Germany
Staff
Lida Anestidou, Study Director
Frances Sharples, Acting Director
Kathleen beil, Administrative Coordinator
Cameron H. Fletcher, Senior Editor
Ruth Crossgrove, Senior Editor
Radiah Rose, Manager of Editorial Projects
Rhonda Haycraft, Senior Project Assistant
Joanne Zurlo, Director (until April 2010)

INSTITUTE FOR LAbORATORy ANIMAL RESEARCH COUNCIL
Members
Stephen W. barthold (Chair), Center for Comparative Medicine,
University of California-Davis
Kathryn A. bayne, Association for Assessment and Accreditation ofLaboratory Animal Care International, Frederick, Maryland
Myrtle A. Davis, National Cancer Institute, National Institutes of Health,Bethesda, Maryland
Jeffrey I. Everitt, Comparative Medicine and Investigator Support,
GlaxoSmithKline Research and Development, Research Triangle Park,
North Carolina (until June 2010)
James G. Fox, Division of Comparative Medicine, Massachusetts Instituteof Technology, Cambridge
Nelson L. Garnett, Laboratory Animal Care and Use Programs,
Dickerson, MD
Estelle b. Gauda, Johns Hopkins University School of Medicine, Johns
Hopkins Hospital, Baltimore, Maryland (until June 2010)
Joseph W. Kemnitz, Institute for Clinical and Translational Research andDepartment of Physiology, University of Wisconsin-Madison
Judy A. MacArthur Clark, Animals in Scientific Procedures Inspectorate,
Home Office, London, United Kingdom
Martha K. McClintock, Institute for Mind and Biology, University of
Chicago, Illinois
Leticia V. Medina, Animal Welfare and Compliance, Abbott Laboratories,
Abbott Park, Illinois
Timo Olavi Nevalainen, National Laboratory Animal Center, University ofEastern Finland, Kuopio, Finland
bernard E. Rollin, Department of Animal Sciences, Colorado State
University, Fort Collins
Abigail L. Smith, School of Veterinary Medicine, University ofPennsylvania, Philadelphia (until June 2010)
Stephen A. Smith, Department of Biomedical Sciences and Pathobiology,
Virginia Polytechnic Institute and State University, Blacksburg
James E. Womack, Department of Veterinary Pathology, Texas A&M
University, College Station (until June 2010)

i

StaffFrances Sharples, Acting Director
Lida Anestidou, Senior Program Officer
Kathleen beil, Administrative Coordinator
Cameron H. Fletcher, Managing Editor, ILAR Journal
Rhonda Haycraft, Program Associate
Joanne Zurlo, Director (until April 2010)

ii

INSTITUTE FOR LAbORATORy ANIMAL RESEARCH PUbLICATIONS
Recognition and Alleviation of Pain in Laboratory Animals (2009)
Scientific and Humane Issues in the Use of Random Source Dogs andCats for Research (2009)
Recognition and Alleviation of Distress in Laboratory Animals (2008)
Toxicity Testing in the 21st Century: A Vision and a Strategy (2007)
Overcoming Challenges to Develop Countermeasures Against Aerosolized
Bioterrorism Agents: Appropriate Use of Animal Models (2006)
Guidelines for the Humane Transportation of Research Animals (2006)
Science, Medicine, and Animals: Teacher’s Guide (2005)
Animal Care and Management at the National Zoo: Final Report (2005)
Science, Medicine, and Animals (2004)
The Development of Science-based Guidelines for Laboratory Animal
Care: Proceedings of the November 2003 International Workshop(2004)
Animal Care and Management at the National Zoo: Interim Report (2004)
National Need and Priorities for Veterinarians in Biomedical Research(2004)
Guidelines for the Care and Use of Mammals in Neuroscience andBehavioral Research (2003)
International Perspectives: The Future of Nonhuman Primate Resources,
Proceedings of the Workshop Held April 17-19, 2002 (2003)
Occupational Health and Safety in the Care and Use of NonhumanPrimates (2003)
Definition of Pain and Distress and Reporting Requirements for Laboratory
Animals: Proceedings of the Workshop Held June 22, 2000 (2000)
Strategies That Influence Cost Containment in Animal Research Facilities(2000)
Microbial Status and Genetic Evaluation of Mice and Rats: Proceedings ofthe 1999 US/Japan Conference (2000)
Microbial and Phenotypic Definition of Rats and Mice: Proceedings of the1998 US/Japan Conference (1999)
Monoclonal Antibody Production (1999)
The Psychological Well-Being of Nonhuman Primates (1998)
Biomedical Models and Resources: Current Needs and Future
Opportunities (1998)
Approaches to Cost Recovery for Animal Research: Implications for
Science, Animals, Research Competitiveness and Regulatory
Compliance (1998)
Chimpanzees in Research: Strategies for Their Ethical Care, Management,
and Use (1997)

iii

Occupational Health and Safety in the Care and Use of Research Animals(1997)
Guide for the Care and Use of Laboratory Animals (1996)
Rodents (1996)
Nutrient Requirements of Laboratory Animals, Fourth Revised Edition(1995)
Laboratory Animal Management: Dogs (1994)
Recognition and Alleviation of Pain and Distress in Laboratory Animals(1992)
Education and Training in the Care and Use of Laboratory Animals: A
Guide for Developing Institutional Programs (1991)
Companion Guide to Infectious Diseases of Mice and Rats (1991)
Infectious Diseases of Mice and Rats (1991)
Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry,and Use (1989)
Use of Laboratory Animals in Biomedical and Behavioral Research (1988)
Animals for Research: A Directory of Sources, Tenth Edition and
Supplement (1979)
Amphibians: Guidelines for the Breeding, Care and Management ofLaboratory Animals (1974)
Copies of these reports may be ordered from the National Academies Press(800) 624-6242 or (202) 334-3313
www.nap.edu

ixReviewersT

his eighth edition of the Guide for the Care and Use of Laboratory
Animals has been reviewed in draft form by individuals chosen for
their diverse perspectives and expertise, in accordance with procedures approved by the Report Review Committee of the National ResearchCouncil. The purpose of this independent review is to provide candid andcritical comments that will assist the Committee in making its publishedreport as sound as possible, and to ensure that the report meets institutional
standards for objectivity, evidence, and responsiveness to the study charge.
The review comments and draft manuscript remain confidential to protect
the integrity of the deliberation process. The Committee thanks the following individuals for their review of the draft report:
Michael B. Ballinger, Amgen
Philippe J.R. Baneux, PreLabs
Stephen W. Barthold, University of California-Davis
Linda C. Cork, Stanford University
Jann Hau, University of Copenhagen, Denmark
Michael J. Huerkamp, Emory University
Michael D. Kastello, sanofi-aventis
Arthur L. Lage, Harvard Medical School
Christian Lawrence, Children’s Hospital Boston
Randall J. Nelson, University of Tennessee College of MedicineMemphis
Steven M. Niemi, Massachusetts General Hospital
Melinda A. Novak, University of Massachusetts-Amherst
xi

xii

REVIEWERS

Gemma Perretta, National Research Council, Italy
Marky E. Pitts, IACUC Consultant
George E. Sanders, University of Washington
Allen W. Singer, Battelle Memorial InstituteWilliam J. White, Charles River Laboratories
Although the reviewers listed above have provided many constructive
comments and suggestions, they were not asked to endorse the conclusions
or recommendations nor did they see the final draft of the report before
its release. The review of this report was overseen by John Dowling, Harvard University, and John Vandenbergh, North Carolina State University.
Appointed by the National Research Council, they were responsible formaking certain that an independent examination of this report was carriedout in accordance with institutional procedures and that all review comments were carefully considered. Responsibility for the final content of thisreport rests entirely with the authoring committee and the institution.

PrefaceT

he purpose of the Guide for the Care and Use of Laboratory Animals(the Guide), as expressed in the charge to the Committee for theUpdate of the Guide, is to assist institutions in caring for and using
animals in ways judged to be scientifically, technically, and humanely
appropriate. The Guide is also intended to assist investigators in fulfilling
their obligation to plan and conduct animal experiments in accord with thehighest scientific, humane, and ethical principles. Recommendations in the
Guide are based on published data, scientific principles, expert opinion,and experience with methods and practices that have proved to be consistent with both high-quality research and humane animal care and use.
These recommendations should be used as a foundation for the development of a comprehensive animal care and use program, recognizing thatthe concept and application of performance standards, in accordance withgoals, outcomes, and considerations defined in the Guide, is essential tothis process.
The Guide is an internationally accepted primary reference on animalcare and use, and its use is required in the United States by the Public
Health Service Policy. It was first published in 1963, under the title Guide
for Laboratory Animal Facilities and Care, and was revised in 1965, 1968,
1972, 1978, 1985, and 1996. More than 550,000 copies have been printed
since its first publication.
In 2006 an ad hoc committee appointed by the Institute for Laboratory
Animal Research recommended that the Guide be updated. The Committee
for the Update of the Guide for the Care and Use of Laboratory Animals
was appointed in 2008 by the National Research Council; its 13 members
xiii

xi

PREFACE

included research scientists, veterinarians, and nonscientists representing
biomedical ethics and the public’s interest in animal welfare. The Committee widely solicited written and oral comments on the update of the Guide
from the scientific community and the general public; comments at open
meetings (on September 26, 2008, in Washington, DC; October 16, 2008,
in Irvine, California; and November 14, 2008, in Chicago) as well as writtencomments submitted to or requested by the Committee were considered. In
addition, the Committee studied the materials submitted to NIH in response
to its 2005 Request for Information (NOT-OD-06-011). All comments contributed substantially to this eighth edition of the Guide.
In approaching its task, the Committee carried forward the balance
between ethical and science-based practice that has always been the basisof the Guide, and fulfilled its role to provide an updated resource thatenables the research community to proceed responsibly and in a self-regulatory manner with animal experimentation. The Guide is predicated on theunderstanding that the exercise of professional judgment both upholds thecentral notion of performance standards and obviates the need for more
stringent regulations.
Laboratory animal science is a rapidly evolving field and the Committee identified a number of areas in which current available scientificinformation is insufficient; additional objective information and assessment
are needed to provide a scientific basis for recommendations in future
editions of the Guide. Although pursuing these concepts was beyond thisCommittee’s charge, the following two topics merit further study: (1) spaceand housing needs of laboratory species and (2) the need and best methods
for providing enrichment, exercise, and human contact.
The need for continual updating of the Guide is implicit in its objective“to provide information that will enhance animal well-being, the quality of
research, and the advancement of scientific knowledge that is relevant toboth humans and animals” (Chapter 1). The irregular and increasing intervals between updates, reaching a 14-year gap between the seventh edition
and this eighth edition, mean that important new research findings might
wait more than a decade before being reflected in recommended practice.
Addressing this concern was beyond the charge of this Committee; wenoted, however, that regular and more frequent updates of the informationin the Guide will promote laboratory animal welfare and support high-quality scientific data. A formal process for revising the information in the Guide,
including the updating of practice standards, could meet this need.
In undertaking this update, the Committee acknowledged the contributions of William I. Gay and Bennett J. Cohen in the development of the original Guide. In 1959, Animal Care Panel (ACP) President Cohen appointed
the Committee on Ethical Considerations in the Care of Laboratory Animals
to evaluate animal care and use. That Committee was chaired by Dr. Gay,

PREFACEx

who soon recognized that the Committee could not evaluate animal careprograms objectively without appropriate criteria on which to base its
evaluations—that is, standards were needed. The ACP Executive Committee agreed, and the Professional Standards Committee was appointed. NIH
later awarded the ACP a contract to “determine and establish a professional
standard for laboratory animal care and facilities.” Dr. Cohen chaired the
ACP Animal Facilities Standards Committee, which prepared the first Guide
for Laboratory Animal Facilities and Care.
This edition of the Guide was financially supported by the National
Institutes of Health; the Office of Research Integrity, Department of Healthand Human Services; the US Department of Agriculture (USDA); the Association for Assessment and Accreditation of Laboratory Animal Care International; the American Association for Laboratory Animal Science; Abbott
Fund; Pfizer, Inc.; the American College of Laboratory Animal Medicine; theAmerican Society of Laboratory Animal Practitioners; and the Associationof Primate Veterinarians.
The Committee for the Update of the Guide for the Care and Use ofLaboratory Animals expresses its appreciation to the Animal Welfare Information Center, National Agricultural Library, USDA, for its assistance incompiling bibliographies and references. This task would have been formidable without the help of the Center’s staff. Appreciation is also extended to
the reviewers of this volume, to Rhonda Haycraft for providing exemplary
administrative and logistical assistance, and especially to Lida Anestidou,
Study Director, who, through extraordinary patience, persistence, and scientific insight, managed the process from beginning to end.
Readers who detect errors of omission or commission are invited tosend corrections and suggestions to the Institute for Laboratory Animal
Research, National Research Council, 500 Fifth Street NW, Washington,
DC 20001.
Janet C. Garber, Chair
Committee for the Update of the Guide for theCare and Use of Laboratory Animals

Overview

T

his eighth edition of the Guide is divided into five chapters and four
appendices.
Chapter 1 presents the goals and intended audiences of the
Guide as well as key concepts and terminology essential to its premiseand use. Incorporating some of the material from the Introduction to thelast edition, the chapter highlights a commitment to the concepts of theThree Rs—Replacement, Reduction, and Refinement—and provides anenhanced discussion of the ethics of animal use and investigator/institutional obligations.
Chapter 2 focuses on the overall institutional animal care and use
program (Program), in addition to many of the topics previously covered in
Chapter 1 of the seventh edition. It defines the evolved concept of Programand provides a framework for its intra-institutional integration, taking into
account institutional policies and responsibilities, regulatory considerations,
Program and personnel management (including training and occupational
health and safety), and Program oversight. Discussions of the latter include
institutional animal care and use committee (IACUC) functions, protocol
and Program review, postapproval monitoring (a new section), and considerations such as humane endpoints and multiple survival surgical procedures. The Committee endorses the American College of Laboratory Animal
Medicine’s “Guidelines for Adequate Veterinary Care.”
Chapter 3 focuses on the animals themselves and, unlike previous editions, addresses terrestrial and aquatic species in separate sections, reflect-xii

xiiiOVERVIEW

ing the growing role of aquatic animals in biomedical research. The chapter
provides recommendations for housing and environment, discusses theimportance of social housing, and includes enhanced sections on environmental enrichment, animal well-being, and scientific validity.
Space recommendations were nominally expanded based on the Committee’s professional and expert opinion and on current housing methods.
Cage sizes have historically been interpreted as minimum space needs byusers of the Guide, and were labeled as such (“recommended minimum
space”) in this edition. The use of the word “minimum” does not further
restrict users of the Guide because, although the space requirements arenumbers (i.e., engineering standards), they are used in a performance standards framework. The Committee recommends minimum space for femalerodents with litter and an increase of the cage height for rabbits to 16”.
Further, in light of many comments submitted to the Committee requesting
more information on performance goals and how to achieve them, rodent
breeding recommendations are accompanied by substantial guidance.
With respect to nonhuman primates (NHPs), the Committee endorses
social housing as the default and has provided some species-specific guidance. An additional group has been added for monkeys, and chimpanzees
are separated in a new category. These changes were motivated by theCommittee’s recognition (affirmed in comments solicited from NHP experts)
that these animals need more floor and vertical space, at least in somegroups, to exercise their natural habits.
Chapter 4 discusses veterinary care and the responsibilities of theattending veterinarian. It introduces the concept of animal biosecurity andupholds its central role in ensuring the health of laboratory animals. The
chapter includes recommendations relative to animal procurement, transportation, and preventive medicine, and expands the sections on clinicalcare and management, surgery (with a new section on intraoperative monitoring), pain and distress, and euthanasia.
Chapter 5 discusses physical plant–related topics and includes updated
and new material on vibration control; physical security and access control; hazardous agent containment; and special facilities for imaging andwhole body irradiation, barrier housing, behavioral studies, and aquatic species housing. The chapter provides detailed discussion of centralized versus
decentralized animal facilities and introduces the concept of variable-volume
HVAC systems with a nod toward energy conservation and efficiency.
Appendix A is the updated bibliography; Appendix B presents the U.S.
Government Principles for the Utilization and Care of Vertebrate Animals
Used in Testing, Research, and Training; Appendix C presents the Statement

OVERVIEW

xix

of Task; and Appendix D provides the biographical sketches of the Committee members.
In accordance with the Statement of Task (“In addition to the publishedreport, the updated Guide will be posted on the Internet in a pdf or equivalent format such that users will be able to search the entire document at
one time”), the Guide is available in searchable pdf format on the National
Academies Press website, www.nap.edu.

Contents

1

KEy CONCEPTS
Applicability and Goals, 2Intended Audiences and Uses of the Guide, 3
Ethics and Animal Use, 4
The Three Rs, 4
Key Terms Used in the Guide, 5
Humane Care, 6
Animal Care and Use Program, 6Engineering, Performance, and Practice Standards, 6
Policies, Principles, and Procedures, 7Must, Should, and May, 8References, 8

1

2

ANIMAL CARE AND USE PROGRAM
Regulations, Policies, and Principles, 12Program Management, 13
Program Management Responsibility, 13
The Institutional Official, 13
The Attending Veterinarian, 14
The Institutional Animal Care and Use Committee, 14
Collaborations, 15
Personnel Management, 15
Training and Education, 15
Occupational Health and Safety of Personnel, 17

11

xxi

xxii

CONTENTS

Personnel Security, 23
Investigating and Reporting Animal Welfare Concerns, 23
Program Oversight, 24
The Role of the IACUC, 24
IACUC Constitution and Function, 24
Protocol Review, 25
Special Considerations for IACUC Review, 27
Postapproval Monitoring, 33
Disaster Planning and Emergency Preparedness, 35
References, 35
3

ENVIRONMENT, HOUSING, AND MANAGEMENT
Terrestrial Animals, 42
Terrestrial Environment, 42
Microenvironment and Macroenvironment, 42
Temperature and Humidity, 43
Ventilation and Air Quality, 45
Illumination, 47
Noise and Vibration, 49
Terrestrial Housing, 50
Microenvironment (Primary Enclosure), 50
Environmental Enrichment, 52
Sheltered or Outdoor Housing, 54
Naturalistic Environments, 55
Space, 55
Terrestrial Management, 63
Behavioral and Social Management, 63
Husbandry, 65
Population Management, 75
Aquatic Animals, 77
Aquatic Environment, 77
Microenvironment and Macroenvironment, 77
Water Quality, 78
Life Support System, 79
Temperature, Humidity, and Ventilation, 80
Illumination, 81
Noise and Vibration, 81
Aquatic Housing, 82
Microenvironment (Primary Enclosure), 82
Environmental Enrichment and Social Housing, 82
Sheltered, Outdoor, and Naturalistic Housing, 83
Space, 83

41

CONTENTS

xxiii

Aquatic Management, 84
Behavior and Social Management, 84
Husbandry, 84
Population Management, 87
References, 88
4

VETERINARy CARE
105
Animal Procurement and Transportation, 106
Animal Procurement, 106
Transportation of Animals, 107
Preventive Medicine, 109
Animal Biosecurity, 109
Quarantine and Stabilization, 110
Separation by Health Status and Species, 111
Surveillance, Diagnosis, Treatment, and Control of Disease, 112
Clinical Care and Management, 113
Medical Management, 114
Emergency Care, 114
Recordkeeping, 115
Surgery, 115
Training, 115
Presurgical Planning, 116
Surgical Facilities, 116
Surgical Procedures, 117
Aseptic Technique, 118
Intraoperative Monitoring, 119
Postoperative Care, 119
Pain and Distress, 120
Anesthesia and Analgesia, 121
Euthanasia, 123
References, 124

5

PHySICAL PLANT
General Considerations, 133
Location, 134
Centralization Versus Decentralization, 134
Functional Areas, 135
Construction Guidelines, 136
Corridors, 136
Animal Room Doors, 137
Exterior Windows, 137
Floors, 137

133

xxi

CONTENTS

Drainage, 138
Walls and Ceilings, 138
Heating, Ventilation, and Air Conditioning (HVAC), 139
Power and Lighting, 141
Storage Areas, 141
Noise Control, 142
Vibration Control, 142
Facilities for Sanitizing Materials, 143
Environmental Monitoring, 143
Special Facilities, 144
Surgery, 144
Barrier Facilities, 145
Imaging, 146
Whole Body Irradiation, 147
Hazardous Agent Containment, 148
Behavioral Studies, 149
Aquatic Species Housing, 150
Security and Access Control, 151
References, 151
ADDENDUM

155

APPENDICESAADDITIONAL SELECTED REFERENCES
Subject Matter, 161
Use of Laboratory Animals, 162
Alternatives, 162
Ethics and Welfare, 163
Experimental Design and Statistics, 164
Research and Testing Methodology, 165
Program Management, 167
General References, 167
Laws, Regulations, and Policies, 168
Education, 169
Monitoring the Care and Use of Animals, 169
Occupational Health and Safety, 170
Environment, Housing, and Management, 172
General References, 172
Environmental Enrichment, 173
Genetics and Genetically Modified Animals, 175

161

CONTENTS

xx

Species-Specific References—Environment, Housing, andManagement, 176
Agricultural Animals, 176
Amphibians, Reptiles, and Fish, 178
Birds, 179
Cats and Dogs, 180
Exotic, Wild, and Zoo Animals, 181
Nonhuman Primates, 182
Rodents and Rabbits, 184
Other Animals, 187
Veterinary Care, 188
Transportation, 188
Anesthesia, Pain, and Surgery, 188
Disease Surveillance, Diagnosis, and Treatment, 190
Pathology, Clinical Pathology, and Parasitology, 190
Species-Specific References—Veterinary Care, 191
Agricultural Animals, 191
Amphibians, Reptiles, and Fish, 192
Birds, 193
Cats and Dogs, 193
Exotic, Wild, and Zoo Animals, 193
Nonhuman Primates, 194
Rodents and Rabbits, 194
Design and Construction of Animal Facilities, 196bU.S. GOVERNMENT PRINCIPLES FOR THE UTILIZATIONAND CARE OF VERTEbRATE ANIMALS USED IN TESTING,RESEARCH, AND TRAINING

199

C

STATEMENT OF TASK201

D

AbOUT THE AUTHORS

203

INDEx

209

1
Key ConceptsThis edition of the Guide for the Care and Use of Laboratory Animals(the Guide) strongly affirms the principle that all who care for, use,or produce animals for research, testing, or teaching must assume
responsibility for their well-being. The Guide is created by scientists andveterinarians for scientists and veterinarians to uphold the scientific rigor
and integrity of biomedical research with laboratory animals as expectedby their colleagues and society at large.
The Guide plays an important role in decision making regarding theuse of vertebrate laboratory animals because it establishes the minimum
ethical, practice, and care standards for researchers and their institutions.
The use of laboratory animals in research, teaching, testing, and productionis also governed or affected by various federal and local laws, regulations,and standards; for example, in the United States the Animal Welfare Act(AWA 1990) and Regulations (PL 89-544; USDA 1985) and/or Public HealthService (PHS) Policy (PHS 2002) may apply. Compliance with these laws,
regulations, policies, and standards (or subsequent revised versions) in theestablishment and implementation of a program of animal care and use isdiscussed in Chapter 2.
Taken together, the practical effect of these laws, regulations, and policies is to establish a system of self-regulation and regulatory oversight thatbinds researchers and institutions using animals. Both researchers and institutions have affirmative duties of humane care and use that are supported
by practical, ethical, and scientific principles. This system of self-regulation
establishes a rigorous program of animal care and use and provides flexibility in fulfilling the responsibility to provide humane care. The specific12

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

scope and nature of this responsibility can vary based on the scientificdiscipline, nature of the animal use, and species involved, but because it
affects animal care and use in every situation this responsibility requiresthat producers, teachers, researchers, and institutions carry out purposeful
analyses of proposed uses of laboratory animals. The Guide is central tothese analyses and to the development of a program in which humane careis incorporated into all aspects of laboratory animal care and use.
APPLICAbILITy AND GOALSIn the Guide, laboratory animals (also referred to as animals) are generally defined as any vertebrate animal (i.e., traditional laboratory animals,
agricultural animals, wildlife, and aquatic species) produced for or usedin research, testing, or teaching. Animal use is defined as the proper care,use, and humane treatment of laboratory animals produced for or used inresearch, testing, or teaching.
When appropriate, considerationsor specific emphases for agricultural
Laboratory animals or animals:
animals and nontraditional species
Any vertebrate animal (e.g.,
are presented. The Guide does not
traditional laboratory animals,
address in detail agricultural aniagricultural animals, wildlife, andmals used in production, agriculaquatic species) produced fortural research or teaching, wildlife
or used in research, testing, orteaching.
and aquatic species studied in natural settings, or invertebrate animals(e.g., cephalopods) used in research,but establishes general principles and ethical considerations that are also
applicable to these species and situations. References provide the reader
with additional resources, and supplemental information on breeding, care,management, and use of selectedlaboratory animal species is availAnimal use: The proper care,able in other publications prepareduse, and humane treatment ofby the Institute for Laboratory Animallaboratory animals produced for
Research (ILAR) and other organizaor used in research, testing, ortions (Appendix A).
teaching.
The goal of the Guide is to promote the humane care and use oflaboratory animals by providing information that will enhance animal wellbeing, the quality of research, and the advancement of scientific knowledge
that is relevant to both humans and animals. The Committee recognizes thatthe use of different species in research is expanding and that researchers
and institutions will face new and unique challenges in determining how
to apply the Guide in these situations. In making such determinations, it isKEy CONCEPTS

3

important to keep in mind that the Guide is intended to provide information
to assist researchers, institutional animal care and use committees (IACUCs),
veterinarians, and other stakeholders in ensuring the implementation ofeffective and appropriate animal care and use programs that are based onhumane care. Throughout the Guide, scientists and institutions are encouraged to give careful and deliberate thought to the decision to use animals,
taking into consideration the contribution that such use will make to newknowledge, ethical concerns, and the availability of alternatives to animaluse (NRC 1992). A practical strategy for decision making, the “Three Rs”(Replacement, Reduction, and Refinement) approach, is discussed in moredetail below. Institutions should use the recommendations in the Guide asa foundation for the development of a comprehensive animal care and use
program and a process for continually improving this program.
INTENDED AUDIENCES AND USES OF THE Guide
The Guide is intended for a wide and diverse audience, including
•
•
•
•
•
•
•
•
•

the scientific community
administrators
IACUCsveterinarians
educators and trainers
producers of laboratory animals
accreditation bodies
regulators
the public.

The Guide is meant to be read by the user in its entirety, as there aremany concepts throughout that may be helpful. Individual sections will
be particularly relevant to certain users, and it is expected that the reader
will explore in more detail the references provided (including those inAppendix A) on topics of interest.
Members of the scientific community (investigators and other animal
users) will find Chapters 1 and 2 (and portions of Chapter 4) of the Guide
useful for their interactions with the IACUC, attending veterinarian, andadministrators regarding animal care as well as the preparation of animalcare and use protocols. Scientific review committees and journal editors
may choose to refer to multiple sections of the Guide to determine whether
scientists contributing proposals and manuscripts have met the appropriate
standards in their planned use of animals. The Guide can assist IACUCs
and administrators in protocol review, assessment, and oversight of an animal care and use program. Veterinarians should find Chapters 3 through 5

4

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

valuable for their oversight and support of animal care and use. Educatorsand trainers can use the Guide as a document to assess both the scope andadequacy of training programs supported by the institution. Accreditation
bodies will find the Guide useful for evaluating many areas of animal careand use programs not subject to strict engineering standards (see definition
below). Finally, members of the public should feel assured that adherence
to the Guide will ensure humane care and use of laboratory animals.
Readers are reminded that the Guide is used by a diverse group ofnational and international institutions and organizations, many of which arecovered by neither the Animal Welfare Act nor the PHS Policy. The Guide
uses some terminology that is both defined by US statute and denotes a
general concept (e.g., “attending veterinarian,” “adequate veterinary care,”and “institutional official”). Even if these terms are not consistent with thoseused by non-US institutions, the underlying principles can still be applied.
In all instances where Guide recommendations are different from applicablelegal or policy requirements, the higher standard should apply.
ETHICS AND ANIMAL USE
The decision to use animals in research requires critical thought, judgment, and analysis. Using animals in research is a privilege granted by society
to the research community with the expectation that such use will provide
either significant new knowledge or lead to improvement in human and/or
animal well-being (McCarthy 1999; Perry 2007). It is a trust that mandates
responsible and humane care and use of these animals. The Guide endorses
the responsibilities of investigators as stated in the U.S. Goernment Principles
for Utilization and Care of Vertebrate Animals Used in Testing, Research, andTraining (IRAC 1985; see Appendix B). These principles direct the researchcommunity to accept responsibility for the care and use of animals during allphases of the research effort. Other government agencies and professionalorganizations have published similar principles (NASA 2008; NCB 2005; NIH
2006, 2007; for additional references see Appendix A). Ethical considerations
discussed here and in other sections of the Guide should serve as a starting
point; readers are encouraged to go beyond these provisions. In certain situations, special considerations will arise during protocol review and planning;several of these situations are discussed in more detail in Chapter 2.
THE THREE Rs
The Three Rs represent a practical method for implementation of the principles described above. In 1959, W.M.S. Russell and R.L. Burch publisheda practical strategy of replacement, refinement, and reduction—referred toas the Three Rs—for researchers to apply when considering experimental

5

KEy CONCEPTS

design in laboratory animal research (Russell and Burch 1959). Over theyears, the Three Rs have become an internationally accepted approachfor researchers to apply when deciding to use animals in research and indesigning humane animal research studies.
Replacement refers to methods that avoid using animals. The term
includes absolute replacements (i.e., replacing animals with inanimate
systems such as computer programs) as well as relative replacements (i.e.,
replacing animals such as vertebrates with animals that are lower on thephylogenetic scale).
Refinement refers to modifications of husbandry or experimental procedures to enhance animal well-being and minimize or eliminate painand distress. While institutions and investigators should take all reasonablemeasures to eliminate pain and distress through refinement, IACUCs should
understand that with some types of studies there may be either unforeseen
or intended experimental outcomes that produce pain. These outcomes may
or may not be eliminated based on the goals of the study.
Reduction involves strategies for obtaining comparable levels of information from the use of fewer animals or for maximizing the information
obtained from a given number of animals (without increasing pain or distress) so that in the long run fewer animals are needed to acquire the samescientific information. This approach relies on an analysis of experimental
design, applications of newer technologies, the use of appropriate statistical methods, and control of environmentally related variability in animal
housing and study areas (see Appendix A).
Refinement and reduction goals should be balanced on a case-by-casebasis. Principal investigators are strongly discouraged from advocating animal reuse as a reduction strategy, and reduction should not be a rationale
for reusing an animal or animals that have already undergone experimental
procedures especially if the well-being of the animals would be compromised. Studies that may result in severe or chronic pain or significant alterations in the animals’ ability to maintain normal physiology, or adequately
respond to stressors, should include descriptions of appropriate humane
endpoints or provide science-based justification for not using a particular,
commonly accepted humane endpoint. Veterinary consultation must occur
when pain or distress is beyond the level anticipated in the protocol description or when interventional control is not possible.
KEy TERMS USED IN THE Guide
The Committee for the Update of the Guide believes that the terms set
out below are important for a full understanding of the Guide. Accordingly,
we have defined these terms and concepts to provide users of the Guide
with additional assistance in implementing their responsibilities.

6

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

Humane Carehumane care means those actions taken to ensure that laboratory
animals are treated according to high ethical and scientific standards.
Implementation of a humane care program, and creation of a laboratoryenvironment in which humane care and respect for animals are valued andencouraged, underlies the core requirements of the Guide and the systemof self-regulation it supports (Klein and Bayne 2007).
Animal Care and Use Program
The animal care and use program (the Program) means the policies,
procedures, standards, organizational structure, staffing, facilities, and practices put into place by an institution to achieve the humane care and useof animals in the laboratory and throughout the institution. It includes theestablishment and support of an IACUC or equivalent ethical oversight committee and the maintenance of an environment in which the IACUC canfunction successfully to carry out its responsibilities under the Guide andapplicable laws and policies. Chapter 2 provides a more expansive discussion of the importance of the Guide and its application to animal care anduse programs.
Engineering, Performance, and Practice Standards
Engineering standard means a standard or guideline that specifies in detail amethod, technology, or technique for achieving a desired outcome; it does
not provide for modification in the event that acceptable alternative methods are available or unusual circumstances arise. Engineering standards areprescriptive and provide limited flexibility for implementation. However, anengineering standard can be useful to establish a baseline and is relatively
easy to use in evaluating compliance.
Performance standard means a standard or guideline that, while describing adesired outcome, provides flexibility in achieving this outcome by granting
discretion to those responsible for managing the animal care and use program, the researcher, and the IACUC. The performance approach requires
professional input, sound judgment, and a team approach to achieve specific goals. It is essential that the desired outcomes and/or goals be clearly
defined and appropriate performance measures regularly monitored in orderto verify the success of the process. Performance standards can be advantageous because they accommodate the consideration of many variables(such as the species and previous history of the animals, facilities, staff

KEy CONCEPTS

expertise, and research goals) so that implementation can be best tailored
to meet the recommendations in the Guide.
Ideally, engineering and performance standards are balanced, setting atarget for optimal practices, management, and operations while encouraging flexibility and judgment, if appropriate, based on individual situations(Gonder et al. 2001).
Scientists, veterinarians, technicians, and others have extensive experience and information covering many of the topics discussed in the Guide.
For topics on which information is insufficient or incomplete, sustained
research into improved methods of laboratory animal management, care,and use is needed for the continued evaluation and improvement of performance and engineering standards.
Practice standard means the application of professional judgment by qualified, experienced individuals to a task or process over time, an approachthat has been demonstrated to benefit or enhance animal care and use. Professional judgment comes from information in the peer-reviewed scientific
literature and textbooks and, as in many other disciplines, from time-provenexperiences in the field (for additional information see Chapter 2). In theabsence of published scientific literature or other definitive sources, whereexperience has demonstrated that a particular practice improves animalcare and use, practice standards have been used in determining appropriate
recommendations in the Guide. In most situations, the Guide is intended toprovide flexibility so that institutions can modify practices and procedureswith changing conditions and new information.

POLICIES, PRINCIPLES, AND PROCEDURES
Policies commonly derive from a public agency or private entity. They
are generally practical statements of collective wisdom, convention, ormanagement direction that are internal to the entity. However, policies may
assume broader force when they become the means by which an implementing agency interprets existing statutes (e.g., PHS Policy). Principles
are broader in their scope and intended application, and are accepted
generalizations about a topic that are frequently endorsed by many anddiverse organizations (e.g., the U.S. Government Principles). Procedures(often called “operating procedures” or “standard operating procedures”)
are typically detailed, step-by-step processes meant to ensure the consistent
application of institutional practices. Establishing standard operating procedures can assist an institution in complying with regulations, policies, andprinciples as well as with day-to-day operations and management.

8

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

MUST, SHOULD, AND MAy
Must indicates actions that the Committee for the Update of the Guide
considers imperative and mandatory duty or requirement for providing
humane animal care and use. Should indicates a strong recommendation
for achieving a goal; however, the Committee recognizes that individual
circumstances might justify an alternative strategy. May indicates a suggestion to be considered.
The Guide is written in general terms so that its recommendations canbe applied in diverse institutions and settings that produce or use animals forresearch, teaching, and testing. This approach requires that users, IACUCs,
veterinarians, and producers apply professional judgment in making specificdecisions regarding animal care and use. Because the Guide is written ingeneral terms, IACUCs have a key role in interpretation, implementation,
oversight, and evaluation of institutional animal care and use programs.
REFERENCES
AWA [Animal Welfare Act]. 1990. Animal Welfare Act. PL (Public Law) 89-544. Available atwww.nal.usda.gov/awic/legislat/awa.htm; accessed January 14, 2010.
Gonder JC, Smeby RR, Wolfle TL. 2001. Performance Standards and Animal Welfare: Definition, Application and Assessment, Parts I and II. Greenbelt MD: Scientists Center for
Animal Welfare.
IRAC [Interagency Research Animal Committee]. 1985. U.S. Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training. Federal
Register, May 20, 1985. Washington: Office of Science and Technology Policy. Available
at http://oacu.od.nih.gov/regs/USGovtPrncpl.htm; accessed May 10, 2010.
Klein HJ, Bayne KA. 2007. Establishing a culture of care, conscience, and responsibility:
Addressing the improvement of scientific discovery and animal welfare through sciencebased performance standards. ILAR J 48:3-11.
McCarthy CR. 1999. Bioethics of laboratory animal research. ILAR J 40:1-37.
NASA [National Aeronautics and Space Administration]. 2008. NASA Principles for the EthicalCare and Use of Animals. NPR 8910.1B-Appendix A. May 28. Available at http://nodis3.
gsfc.nasa.gov/displayDir.cfm?t=NPDandc=8910ands=1B; accessed May 10, 2010.
NCB [Nuffield Council on Bioethics]. 2005. The Ethics of Research Using Animals. London:
NCB.
NIH [National Institutes of Health]. 2007. Memorandum of Understanding Between the Officeof Laboratory Animal Welfare, National Institutes of Health, US Department of Health andHuman Services and the Office of Research Oversight and the Office of Research and Development, Veterans Health Administration, US Department of Veterans Affairs Concerning
Laboratory Animal Welfare. November 2007. Bethesda: Office of Extramural Research, NIH.
Available at http://grants.nih.gov/grants/olaw/references/mou_olaw_va_2007_11.htm.
NIH. 2006. Memorandum of Understanding Among the Animal and Plant Health Inspection
Service USDA and the Food and Drug Administration, US Department of Health andHuman Services, and the National Institutes of Health Concerning Laboratory Animal
Welfare. March 1, 2006. Bethesda: Office of Extramural Research, NIH. Available at
http://grants.nih.gov/grants/olaw/references/finalmou.htm.

KEy CONCEPTS



NRC [National Research Council]. 1992. Report on Responsible Science. Washington: National Academy Press.
Perry P. 2007. The ethics of animal research: A UK perspective. ILAR J 48:42-46.
PHS [Public Health Service]. 2002. Public Health Service Policy on Humane Care and Use ofLaboratory Animals. Publication of the Department of Health and Human Services, National Institutes of Health, Office of Laboratory Animal Welfare. Available at http://grants.
nih.gov/grants/olaw/references/phspol.htm; accessed June 9, 2010.
Russell WMS, Burch RL. 1959. The Principles of Humane Experimental Technique. London:
Methuen and Co. [Reissued: 1992, Universities Federation for Animal Welfare, Herts,
UK].
USDA [US Department of Agriculture]. 1985. 9 CFR 1A. (Title 9, Chapter 1, Subchapter A):
Animal Welfare. Available at http://ecfr.gpoaccess.gov/cgi/t/text/text-idx?sid=8314313bd
7adf2c9f1964e2d82a88d92andc=ecfrandtpl=/ecfrbrowse/Title09/9cfrv1_02.tpl; accessed
January 14, 2010.

2
Animal Care and Use ProgramT

he proper care and use of laboratory animals in research, testing,teaching, and production (animal use) require scientific and professional judgment based on the animals’ needs and their intended
use. An animal care and use program(hereafter referred to as the Program)comprises all activities conducted by
Program: The activities conand at an institution that have a direct
ducted by and at an institutionthat have a direct impact on theimpact on the well-being of animals,
well-being of animals, including
including animal and veterinary care,animal and veterinary care, polipolicies and procedures, personnel
cies and procedures, personnel
and program management and overand program management andsight, occupational health and safety,
oversight, occupational healthand safety, IACUC functions,institutional animal care and use comand animal facility design andmittee (IACUC) functions, and animal
management.
facility design and management.
This chapter defines the overall
Program and key oversight responsibilities and provides guidelines to aid in developing an effective Program.
Chapters 3, 4, and 5 cover the details of Program components: environment,
housing, and management; veterinary care; and physical plant, respectively.
Each institution should establish and provide sufficient resources for a Program that is managed in accord with the Guide and in compliance withapplicable regulations, policies, and guidelines.

11

12

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALSREGULATIONS, POLICIES, AND PRINCIPLES
The use of laboratory animals is governed by an interrelated, dynamic
system of regulations, policies, guidelines, and procedures. The Guide
takes into consideration regulatory requirements relevant to many US-based
activities, including the Animal Welfare Regulations (USDA 1985; US Code,
42 USC § 289d) and the Public Health Service Policy on Humane Care andUse of Laboratory Animals (PHS 2002). The use of the Guide by non-US
entities also presumes adherence to all regulations relevant to the humane
care and use of laboratory animals applicable in those locations. The Guide
also takes into account the U.S. Government Principles for Utilization andCare of Vertebrate Animals Used in Testing, Research, and Training (IRAC
1985; see Appendix B) and endorses the following principles:
•
•
•
•
•
•
•
•
•

consideration of alternatives (in vitro systems, computer simulations, and/or mathematical models) to reduce or replace the use ofanimals
design and performance of procedures on the basis of relevance tohuman or animal health, advancement of knowledge, or the goodof society
use of appropriate species, quality, and number of animals
avoidance or minimization of discomfort, distress, and painuse of appropriate sedation, analgesia, and anesthesia
establishment of humane endpoints
provision of adequate veterinary careprovision of appropriate animal transportation and husbandry
directed and performed by qualified persons
conduct of experimentation on living animals exclusively by
and/or under the close supervision of qualified and experienced
personnel.

Interpretation and application of these principles and the Guide require
knowledge, expertise, experience, and professional judgment. Programs
should be operated in accord with the Guide and relevant regulations,policies, and principles. Also, institutions are encouraged to establish andperiodically review written procedures to ensure consistent application of
Guide standards. Supplemental information on various aspects of animalcare and use is available in other publications prepared by the Institute forLaboratory Animal Research (ILAR) and other organizations (Appendix A).
References in the Guide provide the reader with additional informationthat supports statements made in the Guide. In the absence of publishedliterature, some information in the Guide is derived from currently accepted
practice standards in laboratory animal science (see Chapter 1). The bodyANIMAL CARE ANd USE PROGRAM

13

of literature related to animal science and use of animals is constantly
evolving, requiring Programs to remain current with the information andbest practices.
PROGRAM MANAGEMENT
An effective Program requires clearly defined roles that align responsibility with regulatory and management authority. US federal law createsa statutory basis for the institutional official (IO), the attending eterinarian(AV), and the institutional animal care and use committee (IACUC). The
Guide endorses these concepts as important operating principles for all US
and non-US animal care and use programs. Effective leadership in and collaboration among these three components, which not only oversee but also
support animal users, are necessary (Lowman 2008; Van Sluyters 2008). In
addition, interactions with regulatory and funding agencies and accreditation organizations are an integral part of the Program.
As summarized here and discussed throughout the Guide, the primary
oversight responsibilities in the Program rest with the IO, the AV, and the
IACUC. Their roles fit in a defined organizational structure where thereporting relationships, authorities, and responsibilities of each are clearly
defined and transparent. Together they establish policies and procedures,
ensure regulatory compliance, monitor Program performance, and support
high-quality science and humane animal use. A Program that includes theseelements and establishes a balance among them has the best chance of efficiently using resources while attaining the highest standards of animal wellbeing and scientific quality (Bayne and Garnett 2008; Van Sluyters 2008).
Program Management Responsibility
The Institutional Official
The institutional official (IO) bears ultimate responsibility for the Program, although overall Program direction should be a shared responsibility
among the IO, AV, and IACUC. The
IO has the authority to allocate theInstitutional official: The indiresources needed to ensure the Providual who, as a representative
gram’s overall effectiveness. Programof senior administration, bears
needs should be clearly and regularly
ultimate responsibility for thecommunicated to the IO by the AV,
Program and is responsible for
the IACUC, and others associatedresource planning and ensuring
alignment of Program goals with
with the Program (e.g., facilities manthe institution’s mission.
agement staff, occupational healthand safety personnel, scientists). As a

14

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

representative of senior administration, the IO is responsible for resource
planning and ensuring the alignment of Program goals of quality animalcare and use with the institution’s mission.
The Attending Veterinarian
The attending eterinarian (AV) is responsible for the health and wellbeing of all laboratory animals used at the institution. The institution must
provide the AV with sufficient authority, including access to all animals,
and resources to manage the programof veterinary care. The AV should
Attending veterinarian: The
oversee other aspects of animal careveterinarian responsible forand use (e.g., husbandry, housing)
the health and well-being of all
to ensure that the Program complies
laboratory animals used at thewith the Guide.
institution.
Institutional mission, programmatic goals, including the nature ofanimal use at the institution, and Program size will determine whether fulltime, part-time, or consultative veterinary services are needed. If a full-time
veterinarian is not available on site, a consulting or part-time veterinarian
should be available in visits at intervals appropriate to programmatic needs.
In such instances, there must be an individual with assigned responsibility
for daily animal care and use and facility management. While institutions
with large animal care and use programs may employ multiple veterinarians, the management of veterinary medicine, animal care, and facility
operations by a single administrative unit is often an efficient mechanism
to administer all aspects of the Program.
The Guide endorses the American College of Laboratory Animal Medicine’s (ACLAM) “Guidelines for Adequate Veterinary Care” (ACLAM 1996).
These guidelines include veterinary access to all animals and their medical
records, regular veterinary visits to facilities where animals are or may behoused or used, provisions for appropriate and competent clinical, preventive, and emergency veterinary care, and a system for legal animal procurement and transportation. Other responsibilities of the AV are outlined in the
Program Oversight section below and in later chapters. For a Program towork effectively, there should be clear and regular communication between
the AV and the IACUC.
The Institutional Animal Care and Use Committee
The IACUC (or institutional equivalent) is responsible for assessmentand oversight of the institution’s Program components and facilities. It
should have sufficient authority and resources (e.g., staff, training, comput-

ANIMAL CARE ANd USE PROGRAM

15

ers and related equipment) to fulfill this responsibility. Detailed informationon the role and function of the IACUC is provided later in this chapter.
Collaborations
Interinstitutional collaboration has the potential to create ambiguities
about responsibility for animal care and use. In cases of such collaboration
involving animal use (beyond animal transport), the participating institutions
should have a formal written understanding (e.g., a contract, memorandumof understanding, or agreement) that addresses the responsibility for offsite
animal care and use, animal ownership, and IACUC review and oversight(AAALAC 2003). In addition, IACUCs from the participating institutions may
choose to review protocols for the work being conducted.
Personnel Management
Training and Education
All personnel involved with the care and use of animals must be adequately educated, trained, and/or qualified in basic principles of laboratory
animal science to help ensure high-quality science and animal well-being.
The number and qualifications of personnel required to conduct and support a Program depend on several factors, including the type and size ofthe institution, the administrative structure for providing adequate animalcare, the characteristics of the physical plant, the number and species ofanimals maintained, and the nature of the research, testing, teaching, andproduction activities. Institutions are responsible for providing appropriate
resources to support personnel training (Anderson 2007), and the IACUC
is responsible for providing oversight and for evaluating the effectivenessof the training program (Foshay and Tinkey 2007). All Program personnel
training should be documented.
Veterinary and Other Professional Staff Veterinarians providing clinicaland/or Program oversight and support must have the experience, training,and expertise necessary to appropriately evaluate the health and wellbeing of the species used in the context of the animal use at the institution. Veterinarians providing broad Program direction should be trained orhave relevant experience in laboratory animal facility administration andmanagement. Depending on the scope of the Program, professionals withexpertise in other specific areas may be needed—in, for example, facility
design and renovation, human resource management, pathology of laboratory animals, comparative genomics, facility and equipment maintenance,
diagnostic laboratory operations, and behavioral management. Laboratory

16

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

animal science and medicine are rapidly changing and evolving disciplines.
The institution should provide opportunities and support for regular professional development and continuing education to ensure both that professional staff are knowledgeable about the latest practices and procedures andthat laboratory animals receive high-quality care (Colby et al. 2007).
Animal Care Personnel Personnel caring for animals should be appropriately trained (see Appendix A, Education), and the institution should provide
for formal and/or on-the-job training to facilitate effective implementation of
the Program and the humane care and use of animals. Staff should receive
training and/or have the experience to complete the tasks for which they
are responsible. According to the Program scope, personnel with expertisein various disciplines (e.g., animal husbandry, administration, veterinary
medical technology) may be required.
There are a number of options for training animal care personnel andtechnicians (Pritt and Duffee 2007). Many colleges have accredited programs in veterinary technology (AVMA 2010); most are 2-year programs
that award Associate of Science degrees, some are 4-year programs thataward Bachelor of Science degrees. Nondegree training, via certification
programs for laboratory animal technicians and technologists, is available
from the American Association for Laboratory Animal Science (AALAS), andthere are various commercially available training materials appropriate forself-guided study (Appendix A).
Personnel caring for laboratory animals should also regularly engage incontinuing education activities and should be encouraged to participate inlocal and national laboratory animal science meetings and in other relevant
professional organizations. On-the-job training, supplemented with institution-sponsored discussion and training programs and reference materials
applicable to their jobs and the species in their care, should be provided toeach employee responsible for animal care (Kreger 1995).
Coordinators of institutional training programs can seek assistance fromthe Animal Welfare Information Center (AWIC), the Laboratory Animal
Welfare and Training Exchange (LAWTE), AALAS, and ILAR (NRC 1991).
The Guide to the Care and Use of Experimental Animals by the CanadianCouncil on Animal Care (CCAC 1993) and guidelines from other countries are valuable additions to the libraries of laboratory animal scientists(Appendix A).
The Research Team The institution should provide appropriate educationand training to members of research teams—including principal investigators, study directors, research technicians, postdoctoral fellows, students,and visiting scientists—to ensure that they have the necessary knowledge
and expertise for the specific animal procedures proposed and the species

ANIMAL CARE ANd USE PROGRAM

1

used (Conarello and Shepard 2007). Training should be tailored to theparticular needs of research groups; however, all research groups should
receive training in animal care and use legislation, IACUC function, ethics of animal use and the concepts of the Three Rs, methods for reporting
concerns about animal use, occupational health and safety issues pertaining
to animal use, animal handling, aseptic surgical technique, anesthesia andanalgesia, euthanasia, and other subjects, as required by statute. Continuing education programs should be offered to reinforce training and provide
updates that reflect changes in technology, legislation, and other relevant
areas. Frequency of training opportunities should ensure that all animal
users have adequate training before beginning animal work.
The IACUC It is the institution’s responsibility to ensure that IACUC members are provided with training opportunities to understand their workand role. Such training should include formal orientation to introduce
new members to the institution’s Program; relevant legislation, regulations,guidelines, and policies; animal facilities and laboratories where animal use
occurs; and the processes of animal protocol and program review (Greene
et al. 2007). Ongoing opportunities to enhance their understanding of animal care and use in science should also be provided. For example, IACUC
members may meet with animal care personnel and research teams; beprovided access to relevant journals, materials, and web-based training; andbe given opportunities to attend meetings or workshops.
Occupational health and Safety of Personnel
Each institution must establish and maintain an occupational health andsafety program (OHSP) as an essential part of the overall Program of animalcare and use (CFR 1984a,b,c; DHHS 2009; PHS 2002). The OHSP must
be consistent with federal, state, and local regulations and should focus on
maintaining a safe and healthy workplace (Gonder 2002; Newcomer 2002;
OSHA 1998a). The nature of the OHSP will depend on the facility, research
activities, hazards, and animal species involved. The National ResearchCouncil’s publication Occupational health and Safety in the Care andUse of Research Animals (NRC 1997) contains guidelines and references
for establishing and maintaining an effective, comprehensive OHSP (alsosee Appendix A). An effective OHSP requires coordination between the
research program (as represented by the investigator), the animal care anduse Program (as represented by the AV, IO, and IACUC), the environmental
health and safety program, occupational health services, and administration(e.g., human resources, finance, and facility maintenance personnel). Establishment of a safety committee may facilitate communication and promote
ongoing evaluation of health and safety in the workplace. In some cases

18

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

there is a regulatory requirement for such a committee. Operational andday-to-day responsibility for safety in the workplace resides with the laboratory or facility supervisor (e.g., principal investigator, facility director, or astaff veterinarian) and depends on safe work practices by all employees.
Control and Preention Strategies A comprehensive OHSP should include ahierarchy of control and prevention strategies that begins with the identification of hazards and the assessment of risk associated with those hazards.
Managing risk involves the following steps: first, the appropriate design andoperation of facilities and use of appropriate safety equipment (engineering
controls); second, the development of processes and standard operatingprocedures (SOPs; administrative controls); and finally, the provision of
appropriate personal protective equipment (PPE) for employees. Special
safety equipment should be used in combination with appropriate management and safety practices (NIH 2002; OSHA 1998a,b). Managing risk using
these strategies requires that personnel be trained, maintain good personal
hygiene, be knowledgeable about the hazards in their work environment,
understand the proper selection and use of equipment, follow establishedprocedures, and use the PPE provided.
hazard Identification and Risk Assessment The institutional OHSP should
identify potential hazards in the work environment and conduct a criticalassessment of the associated risks. An effective OHSP ensures that the risks
associated with the experimental use of animals are identified and reduced tominimal and acceptable levels. Hazard identification and risk assessment are
ongoing processes that involve individuals qualified to assess dangers associated with the Program and implement commensurate safeguards. Health andsafety specialists with knowledge in relevant disciplines should be involved inrisk assessment and the development of procedures to manage such risks.
Potential hazards include experimental hazards such as biologic agents(e.g., infectious agents or toxins), chemical agents (e.g., carcinogens andmutagens), radiation (e.g., radionuclides, X-rays, lasers), and physical hazards (e.g., needles and syringes). The risks associated with unusual experimental conditions such as those encountered in field studies or wildlife
research should also be addressed. Other potential hazards—such as animal
bites, exposure to allergens, chemical cleaning agents, wet floors, cage
washers and other equipment, lifting, ladder use, and zoonoses—that are
inherent in or intrinsic to animal use should be identified and evaluated.
Once potential hazards have been identified, a critical ongoing assessmentof the associated risks should be conducted to determine appropriate strategies to minimize or manage the risks.
The extent and level of participation of personnel in the OHSP should
be based on the hazards posed by the animals and materials used (theANIMAL CARE ANd USE PROGRAM

1

severity or seriousness of the hazard); the exposure intensity, duration, andfrequency (prevalence of the hazard); to some extent, the susceptibility (e.g.,
immune status) of the personnel; and the history of occupational illness andinjury in the particular workplace (Newcomer 2002; NRC 1997). Ongoing
identification and evaluation of hazards call for periodic inspections andreporting of potential hazardous conditions or “near miss” incidents.
Facilities, Equipment, and Monitoring The facilities required to support
the OHSP will vary depending on the scope and activities of the Program.
Their design should preferentially use engineering controls and equipment
to minimize exposure to anticipated hazards (also see Chapter 5). Because
a high standard of personal cleanliness is essential, changing, washing,and showering facilities and supplies appropriate to the Program should
be available.
Where biologic agents are used, the Centers for Disease Control andPrevention (CDC) and National Institutes of Health (NIH) publication biosafety in Microbiological and biomedical Laboratories (BMBL; DHHS 2009)
and the USDA standards (USDA 2002) should be consulted for appropriate
facility design and safety procedures. These design and safety features are
based on the level of risk posed by the agents used. Special facilities andsafety equipment may be needed to protect the animal care and investigative staff, other occupants of the facility, the public, animals, and theenvironment from exposure to hazardous biologic, chemical, and physicalagents used in animal experimentation (DHHS 2009; Frasier and Talka
2005; NIH 2002). When necessary, these facilities should be separated fromother animal housing and support areas, research and clinical laboratories,
and patient care facilities. They should be appropriately identified andaccess to them limited to authorized personnel.
Facilities, equipment, and procedures should also be designed, selected,
and developed to reduce the possibility of physical injury or health risk
to personnel (NIOSH 1997a,b). Engineering controls and equipment that
address the risk of ergonomic injury in activities such as the lifting of heavyequipment or animals should be considered (AVMA 2008). Those are alsofrequently used to limit or control personnel exposure to animal allergens(Harrison 2001; Huerkamp et al. 2009). The potential for repetitive motion
injuries in animal facilities (e.g., maintenance of large rodent populations
and other husbandry activities) should also be assessed.
The selection of appropriate animal housing systems requires professional knowledge and judgment and depends on the nature of the hazards
in question, the types of animals used, the limitations or capabilities of the
facilities, and the design of the experiments. Experimental animals should
be housed so that possibly contaminated food and bedding, feces, and urine
can be handled in a controlled manner. Appropriate facilities, equipment,

20

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

and procedures should be used for bedding disposal. Safety equipment
should be properly maintained and its function periodically validated.
Appropriate methods should be used for assessing and monitoring exposure
to potentially hazardous biologic, chemical, and physical agents whererequired (e.g., ionizing radiation) or where the possibility of exceeding
permissible exposure limits exists (CFR 1984b).
Personnel Training As a general rule, safety depends on trained personnel
who rigorously follow safe practices. Personnel at risk should be provided
with clearly defined procedures and, in specific situations, personal protective equipment to safely conduct their duties, understand the hazards
involved, and be proficient in implementing the required safeguards. They
should be trained regarding zoonoses, chemical, biologic, and physicalhazards (e.g., radiation and allergies), unusual conditions or agents thatmight be part of experimental procedures (e.g., the use of human tissue
in immunocompromised animals), handling of waste materials, personal
hygiene, the appropriate use of PPE, and other considerations (e.g., precautions to be taken during pregnancy, illness, or immunosuppression) as
appropriate to the risk imposed by their workplace.
Personal hygiene The use of good personal hygiene will often reduce thepossibility of occupational injury and cross contamination. Appropriate
policies should be established and enforced, and the institution should
supply suitable attire and PPE (e.g., gloves, masks, face shields, head covers,
coats, coveralls, shoes or shoe covers) for use in the animal facility andlaboratories in which animals are used. Soiled attire should be disposed of,laundered, or decontaminated by the institution as appropriate, and may
require that special provisions be implemented if outside vendors are used.
Personnel should wash and/or disinfect their hands and change clothing asoften as necessary to maintain good personal hygiene. Outer garments wornin the animal rooms should not be worn outside the animal facility unlesscovered (NRC 1997). Personnel should not be permitted to eat, drink, use
tobacco products, apply cosmetics, or handle or apply contact lenses inrooms and laboratories where animals are housed or used (DHHS 2009;
NRC 1997; OSHA 1998a).
Animal Experimentation Inoling hazards When selecting specific safeguards for animal experimentation with hazardous agents, careful attention
should be given to procedures for animal care and housing, storage anddistribution of the agents, dose preparation and administration, body fluid
and tissue handling, waste and carcass disposal, items that might be used
temporarily and removed from the site (e.g., written records, experimental
devices, sample vials), and personal protection.

ANIMAL CARE ANd USE PROGRAM

21

Institutions should have written policies and procedures governing
experimentation with hazardous biologic, chemical, and physical agents.
An oversight process (such as the use of a safety committee) should be
developed to involve persons who are knowledgeable in the evaluation andsafe use of hazardous materials or procedures and should include review ofthe procedures and facilities to be used for specific safety concerns. Formal
safety programs should be established to assess hazards, determine thesafeguards needed for their control, and ensure that staff have the necessary
training and skills and that facilities are adequate for the safe conduct of
the research. Technical support should be provided to monitor and ensurecompliance with institutional safety policies. A collaborative approach
involving the investigator and research team, attending veterinarian, animal care technician, and occupational health and safety professionals may
enhance compliance.
The BMBL (DHHS 2009) and NRC (1997) recommend practices andprocedures, safety equipment, and facility requirements for working withhazardous biologic agents and materials. Facilities that handle agents of
unknown risk should consult with appropriate CDC personnel about hazard control and medical surveillance. The use of highly pathogenic “selectagents and toxins” in research requires that institutions develop a programand procedures for procuring, maintaining, and disposing of these agents(CFR 1998, 2002a,b; NRC 2004; PL 107-56; PL 107-188; Richmond et al.
2003). The use of immunodeficient or genetically modified animals (GMAs)
susceptible to or shedding human pathogens, the use of human tissues andcell lines, or any infectious disease model can lead to an increased risk to
the health and safety of personnel working with the animals (Lassnig et al.
2005; NIH 2002).
Hazardous agents should be contained in the study environment, forexample through the use of airflow control during the handling and administering of hazardous agents, necropsies on contaminated animals (CDC
and NIH 2000), and work with chemical hazards (Thomann 2003). Waste
anesthetic gases should be scavenged to limit exposure.
Personal Protection While engineering and administrative controls are thefirst considerations for the protection of personnel, PPE appropriate for thework environment, including clean institution-issued protective clothing,should be provided as often as necessary. Protective clothing and equipment
should not be worn beyond the boundary of the hazardous agent work areaor the animal facility (DHHS 2009). If appropriate, personnel should shower
when they leave the animal care, procedure, or dose preparation areas.
Personnel with potential exposure to hazardous agents or certain species
should be provided with PPE appropriate to the situation (CFR 1984c); forexample, personnel exposed to nonhuman primates should have PPE such

22

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

as gloves, arm protectors, suitable face masks, face shields, and goggles(NRC 2003a). Hearing protection should be available in high-noise areas(OSHA 1998c). Personnel working in areas where they might be exposed tocontaminated airborne particulate material or vapors should have suitable
respiratory protection (Fechter 1995; McCullough 2000; OSHA 1998d),
with respirator fit testing and training in the proper use and maintenance ofthe respirator (OSHA 1998d; Sargent and Gallo 2003).
Medical Ealuation and Preentie Medicine for Personnel Development
and implementation of a program of medical evaluation and preventive
medicine should involve input from trained health professionals, such as
occupational health physicians and nurses. Confidentiality and other medical and legal factors must be considered in the context of appropriate federal, state, and local regulations (e.g., PL 104-191).
A preemployment health evaluation and/or a health history evaluation
before work assignment is advisable to assess potential risks for individual
employees. Periodic medical evaluations are advisable for personnel in
specific risk categories. For example, personnel required to use respiratory protection may also require medical evaluation to ensure that they
are physically and psychologically able to use the respirator properly (Sargent and Gallo 2003). An appropriate immunization schedule should be
adopted. It is important to immunize animal care personnel against tetanus(NRC 1997), and preexposure immunization should be offered to people atrisk of infection or exposure to specific agents such as rabies virus (e.g., if
working with species at risk for infection) or hepatitis B virus (e.g., if working with human blood or human tissues, cell lines, or stocks). Vaccinationis recommended if research is to be conducted on infectious diseases forwhich effective vaccines are available. More specific recommendations areavailable in the BMBL (DHHS 2009). Preemployment or preexposure serum
collection is advisable only in specific circumstances as determined by an
occupational health and safety professional (NRC 1997). In such cases,
identification, traceability, retention, and storage conditions of samples
should be considered, and the purpose for which the serum samples will be
used must be consistent with applicable federal and state laws.
Laboratory animal allergy has become a significant issue for individuals
in contact with laboratory animals (Bush and Stave 2003; Gordon 2001;
Wolfle and Bush 2001; Wood 2001). The medical surveillance program
should promote the early diagnosis of allergies (Bush 2001; Bush and Stave
2003; Seward 2001) and include evaluation of an individual’s medical
history for preexisting allergies. Personnel training should include information about laboratory animal allergies, preventive control measures, earlyrecognition and reporting of allergy symptoms, and proper techniques forworking with animals (Gordon et at. 1997; Schweitzer et al. 2003; Thulin

ANIMAL CARE ANd USE PROGRAM

23

et al. 2002). PPE should be used to supplement, not replace, engineeringor process controls (Harrison 2001; Reeb-Whitaker et al. 1999). If PPE for
respiratory protection is necessary, appropriate fit testing and training should
be provided.
Zoonosis surveillance should be a part of an OHSP (DHHS 2009; NRC
1997). Personnel should be instructed to notify their supervisors of potential
or known exposures and of suspected health hazards and illnesses. Nonhuman primate diseases that are transmissible to humans can be serious hazards (NRC 2003a). Animal technicians, veterinarians, investigators, students,
research technicians, maintenance workers, and others who have contact
with nonhuman primates or their tissues and body fluids or who have duties
in nonhuman primate housing areas should be routinely screened for tuberculosis. Because of the potential for exposure to Macacine herpesirus 1 (formerly Cercopithecine herpesirus 1 or Herpes B virus), personnel who workwith or handle biologic samples (blood and tissues) from macaques shouldhave access to and be instructed in the use of bite and scratch emergency carestations (Cohen et al. 2002). Injuries associated with macaques, their tissues
or body fluids, or caging and equipment with which the animals have had
direct contact, should be carefully evaluated and appropriate postexposure
treatment and follow-up implemented (ibid.; NRC 2003a).
Clear procedures should be established for reporting all accidents, bites,
scratches, and allergic reactions (NRC 1997), and medical care for such
incidents should be readily available (Cohen et al. 2002; DHHS 2009).
Personnel Security
While contingency plans normally address natural disasters, they should
also take into account the threats that criminal activities such as personnel
harassment and assault, facility trespassing, arson, and vandalism poseto laboratory animals, research personnel, equipment and facilities, andbiomedical research at the institution. Preventive measures should be considered, including preemployment screening and physical and information
technology security (Miller 2007).
Inestigating and Reporting Animal Welfare Concerns
Safeguarding animal welfare is the responsibility of every individual
associated with the Program. The institution must develop methods forreporting and investigating animal welfare concerns, and employees should
be aware of the importance of and mechanisms for reporting animal welfare concerns. In the United States, responsibility for review and investigation of these concerns rests with the IO and the IACUC. Response tosuch reports should include communication of findings to the concerned

24

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

employee(s), unless such concerns are reported anonymously; corrective
actions if deemed necessary; and a report to the IO of the issue, findings,
and actions taken. Reported concerns and any corrective actions taken
should be documented.
Mechanisms for reporting concerns should be posted in prominent
locations in the facility and on applicable institutional website(s) with
instructions on how to report the concern and to whom. Multiple pointsof contact, including senior management, the IO, IACUC Chair, and AV,
are recommended. The process should include a mechanism for anonymity, compliance with applicable whistleblower policies, nondiscrimination
against the concerned/reporting party, and protection from reprisals.
Training and regular communication with employees (including personnel such as custodial, maintenance, and administrative staff, who are farther
removed from the animal use) about the institution’s animal use activities
may reduce potential concerns.
PROGRAM OVERSIGHT
The Role of the IACUC
IACUC Constitution and Function
The responsibility of the IACUC is to oversee and routinely evaluate theProgram. It is the institution’s responsibility to provide suitable orientation,
background materials, access to appropriate resources, and, if necessary,
specific training to assist IACUC members in understanding their roles andresponsibilities and evaluating issues brought before the committee.
Committee membership includes the following:
•

•
•
•

a Doctor of Veterinary Medicine either certified (e.g., by ACLAM,ECLAM, JCLAM, KCLAM) or with training and experience in laboratory animal science and medicine or in the use of the species at
the institution
at least one practicing scientist experienced in research involving
animals
at least one member from a nonscientific background, drawn frominside or outside the institution
at least one public member to represent general community interests in the proper care and use of animals.

Public members should not be laboratory animal users, affiliated inany way with the institution, or members of the immediate family of a person who is affiliated with the institution. The public member may receiveANIMAL CARE ANd USE PROGRAM

25

compensation for participation and ancillary expenses (e.g., meals, parking, travel), but the amount should be sufficiently modest that it does not
become a substantial source of income and thus risk compromising themember’s association with the community and public at large.
For large institutions with many administrative units or departments,
no more than three voting members should be associated with a single
administrative unit (USDA 1985). The size of the institution and the natureand extent of the Program will determine the number of members of thecommittee and their terms of appointment. Institutions with broad research
programs may need to choose scientists from a number of disciplines andexperience to properly evaluate animal use protocols.
The committee is responsible for oversight and evaluation of the entire
Program and its components as described in other sections of the Guide.
Its oversight functions include review and approval of proposed animaluse (protocol review) and of proposed significant changes to animal use;regular inspection of facilities and animal use areas; regular review of theProgram; ongoing assessment of animal care and use; and establishment ofa mechanism for receipt and review of concerns involving the care and useof animals at the institution. The committee must meet as often as necessary to fulfill its responsibilities, and records of committee meetings andresults of deliberations should be maintained. Program review and facilities
inspections should occur at least annually or more often as required (e.g.,by the Animal Welfare Act and PHS Policy). After review and inspection, awritten report (including any minority views) should be provided to the IO
about the status of the Program.
Protocol Reiew
The animal use protocol is a detailed description of the proposed use oflaboratory animals. The following topics should be considered in the preparation of the protocol by the researcher and its review by the IACUC:••••

rationale and purpose of the proposed use of animals
a clear and concise sequential description of the procedures involving the use of animals that is easily understood by all members of
the committee
availability or appropriateness of the use of less invasive procedures, other species, isolated organ preparation, cell or tissue culture, or computer simulation (see Appendix A, Alternatives)
justification of the species and number of animals proposed; whenever possible, the number of animals and experimental group sizes
should be statistically justified (e.g., provision of a power analysis;
see Appendix A, Experimental Design and Statistics)

26

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

•
•
•
•
•
•
•
•
•
•
•

unnecessary duplication of experiments
nonstandard housing and husbandry requirements
impact of the proposed procedures on the animals’ well-being
appropriate sedation, analgesia, and anesthesia (indices of pain or
invasiveness might aid in the preparation and review of protocols;see Appendix A, Anesthesia, Pain, and Surgery)conduct of surgical procedures, including multiple operative
procedures
postprocedural care and observation (e.g., inclusion of post-treatment or postsurgical animal assessment forms)
description and rationale for anticipated or selected endpoints
criteria and process for timely intervention, removal of animals
from a study, or euthanasia if painful or stressful outcomes are
anticipatedmethod of euthanasia or disposition of animals, including planningfor care of long-lived species after study completion
adequacy of training and experience of personnel in the procedures
used, and roles and responsibilities of the personnel involved
use of hazardous materials and provision of a safe working
environment.

While the responsibility for scientific merit review normally lies outside
the IACUC, the committee members should evaluate scientific elementsof the protocol as they relate to the welfare and use of the animals. For
example, hypothesis testing, sample size, group numbers, and adequacyof controls can relate directly to the prevention of unnecessary animaluse or duplication of experiments. For some IACUC questions, input fromoutside experts may be advisable or necessary. In the absence of evidenceof a formal scientific merit review, the IACUC may consider conducting
or requesting such a review (Mann and Prentice 2004). IACUC members
named in protocols or who have other conflicts must recuse themselvesfrom decisions concerning these protocols.
At times, protocols include procedures that have not been previously
encountered or that have the potential to cause pain or distress that cannot
be reliably predicted or controlled. Relevant objective information about
the procedures and the purpose of the study should be sought from theliterature, veterinarians, investigators, and others knowledgeable about theeffects on animals. If little is known about a specific procedure, limited
pilot studies, designed to assess both the procedure’s effects on the animals
and the skills of the research team and conducted under IACUC oversight,
are appropriate. General guidelines for protocol or method evaluation forsome of these situations are provided below, but they may not apply in all
instances.

ANIMAL CARE ANd USE PROGRAM

2

Special Considerations for IACUC Reiew
Certain animal use protocols include procedures or approaches that
require special consideration during the IACUC review process due to theirpotential for unrelieved pain or distress or other animal welfare concerns.
The topics below are some of the most common requiring special IACUC
consideration. For these and other areas the IACUC is obliged to weighthe objectives of the study against potential animal welfare concerns. By
considering opportunities for refinement, the use of appropriate nonanimalalternatives, and the use of fewer animals, both the institution and theprincipal investigator (PI) can begin to address their shared obligations forhumane animal care and use.
Experimental and humane Endpoints The experimental endpoint of a study
occurs when the scientific aims and objectives have been reached. The
humane endpoint is the point at which pain or distress in an experimental
animal is prevented, terminated, or relieved. The use of humane endpoints
contributes to refinement by providing an alternative to experimental endpoints that result in unrelieved or severe animal pain and distress, including
death. The humane endpoint should be relevant and reliable (Hendriksen
and Steen 2000; Olfert and Godson 2000; Sass 2000; Stokes 2002). For
many invasive experiments, the experimental and humane endpoints areclosely linked (Wallace 2000) and should be carefully considered during
IACUC protocol review. While all studies should employ endpoints thatare humane, studies that commonly require special consideration include
those that involve tumor models, infectious diseases, vaccine challenge,
pain modeling, trauma, production of monoclonal antibodies, assessment of toxicologic effects, organ or system failure, and models of cardiovascular shock.
The PI, who has precise knowledge of both the objectives of the study
and the proposed model, should identify, explain, and include in the animal
use protocol a study endpoint that is both humane and scientifically sound.
The identification of humane endpoints is often challenging, however,
because multiple factors must be weighed, including the model, species(and sometimes strain or stock), animal health status, study objectives,
institutional policy, regulatory requirements, and occasionally conflicting
scientific literature. Determination of humane endpoints should involve thePI, the veterinarian, and the IACUC, and should be defined when possible
before the start of the study (Olfert and Godson 2000; Stokes 2000).
Information that is critical to the IACUC’s assessment of appropriate endpoint consideration in a protocol includes precise definition of the humane
endpoint (including assessment criteria), the frequency of animal observation, training of personnel responsible for assessment and recognition of the

28

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

humane endpoint, and the response required upon reaching the humane
endpoint. An understanding of preemptive euthanasia (Toth 2000), behavioral
or physiologic definitions of the moribund state (ibid.), and the use of studyspecific animal assessment records (Morton 2000; Paster et al. 2009) can
aid the PI and IACUC when considering or developing proposed endpoints.
When novel studies are proposed or information for an alternative endpointis lacking, the use of pilot studies is an effective method for identifying anddefining humane endpoints and reaching consensus among the PI, IACUC,
and veterinarian. A system for communication with the IACUC should bein place both during and after such studies. Numerous publications address
specific proposals for the application and use of humane endpoints (e.g.,
CCAC 1998; ILAR 2000; OECD 1999; Toth 1997; UKCCCR 1997).
Unexpected Outcomes Fundamental to scientific inquiry is the investigation of novel experimental variables. Because of the potential for unexpected outcomes that may affect animal well-being when highly novel
variables are introduced, more frequent monitoring of animals may berequired. With their inherent potential for unanticipated phenotypes, GMAs
are an example of models for which increased monitoring for unexpected
outcomes could be implemented (Dennis 1999).
GMAs, particularly mice and fish, are important animal models, andnew methods and combinations of genetic manipulation are constantly
being developed (Gondo 2008). Regardless of whether genetic manipulation is targeted or random, the phenotype that initially results is often unpredictable and may lead to expected or unexpected outcomes that affect the
animal’s well-being or survival at any stage of life. For example, in someinstances genetic modification has led to unforeseen immunodeficiency,
requiring the GMA offspring to be held under specialized bioexclusion
conditions (Mumphrey et al. 2007); and the promoter sequences used todirect expression of transgenes to specific tissues have varying degrees of
specificity (“leakiness”) that can lead to unanticipated phenotypes (Moorehead et al. 2003). These examples illustrate the diversity of unanticipatedoutcomes and emphasize the need for diligent monitoring and professional
judgment to ensure the animals’ well-being (Dennis 2000). The first offspring of a newly generated GMA line should be carefully observed frombirth into early adulthood for signs of disease, pain, or distress. Investigators
may find that the phenotype precludes breeding of particular genotypes orthat unexpected infertility occurs, situations that could lead to increases in
the numbers of animals used and revision of the animal use protocol. Whenthe initial characterization of a GMA reveals a condition that negatively
affects animal well-being, this should be reported to the IACUC, and more
extensive analysis may be required to better define the phenotype (Brown etal. 2000; Crawley 1999; Dennis 2000). Such monitoring and reporting may

ANIMAL CARE ANd USE PROGRAM

2

help to determine whether proactive measures can circumvent or alleviate
the impact of the genetic modification on the animal’s well-being and toestablish humane endpoints specific to the GMA line.
Physical Restraint Physical restraint is the use of manual or mechanicalmeans to limit some or all of an animal’s normal movement for the purposeof examination, collection of samples, drug administration, therapy, or
experimental manipulation. Animals are restrained for brief periods, usually
minutes, in many research applications.
Restraint devices should be suitable in size, design, and operation
to minimize discomfort, pain, distress, and the potential for injury to theanimal and the research staff. Dogs, nonhuman primates, and many otheranimals can be trained, through use of positive reinforcement techniques,to cooperate with research procedures or remain immobile for brief periods (Boissy et al. 2007; Laule et al. 2003; Meunier 2006; Prescott andBuchanan-Smith 2003; Reinhardt 1991, 1995; Sauceda and Schmidt 2000;
Yeates and Main 2009).
Prolonged restraint, including chairing of nonhuman primates, should
be avoided unless it is essential for achieving research objectives and isspecifically approved by the IACUC (NRC 2003b). Systems that do notlimit an animal’s ability to make normal postural adjustments (e.g., subcutaneous implantation of osmotic minipumps in rodents, backpack-fitted
infusion pumps in dogs and nonhuman primates, and free-stall housing forfarm animals) should be used when compatible with protocol objectives.
Animals that do not adapt to necessary restraint systems should be removed
from the study. When restraint devices are used, they should be specificallydesigned to accomplish research goals that are impossible or impractical toaccomplish by other means or to prevent injury to animals or personnel.
The following are important guidelines for restraint:
•
•
•
•
•
•
•

Restraint devices should not be considered a normal method of
housing, and must be justified in the animal use protocol.
Restraint devices should not be used simply as a convenience inhandling or managing animals.
Alternatives to physical restraint should be considered.
The period of restraint should be the minimum required to accomplish the research objectives.
Animals to be placed in restraint devices should be given training (with positive reinforcement) to adapt to the equipment andpersonnel.
Animals that fail to adapt should be removed from the study.
Provision should be made for observation of the animal at appropriate intervals, as determined by the IACUC.

30

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS••

Veterinary care must be provided if lesions or illnesses associatedwith restraint are observed. The presence of lesions, illness, orsevere behavioral change often necessitates the temporary or permanent removal of the animal from restraint.
The purpose of the restraint and its duration should be clearly
explained to personnel involved with the study.

Multiple Surial Surgical Procedures Surgical procedures in the laboratory
setting may be categorized as major or minor (USDA 1985). Whether aprocedure is major or minor should be evaluated on a case-by-case basis,
as determined by the veterinarian and IACUC (NRC 2003b; Silverman et al.
2007; for additional discussion see Chapter 4, Surgical Procedures).
Regardless of classification, multiple surgical procedures on a single
animal should be evaluated to determine their impact on the animal’s wellbeing. Multiple major surgical procedures on a single animal are acceptable only if they are (1) included in and essential components of a single
research project or protocol, (2) scientifically justified by the investigator, or(3) necessary for clinical reasons. Conservation of scarce animal resources
may justify the conduct of multiple major surgeries on a single animal,
but the application of such a practice on a single animal used in separate
protocols is discouraged and should be reviewed critically by the IACUC.
When applicable, the IO must submit a request to the USDA/APHIS andreceive approval in order to allow a regulated animal to undergo multiple
major survival surgical procedures in separate unrelated research protocols(USDA 1985, 1997a). Justifications for allowing animals not regulated by
the USDA to undergo multiple survival procedures that meet the above
criteria should conform to those required for regulated species. If multiple
survival surgery is approved, the IACUC should pay particular attention toanimal well-being through continuing evaluation of outcomes. Cost savings alone is not an adequate reason for performing multiple major survival
surgical procedures.
Some procedures characterized as minor may induce substantial postprocedural pain or impairment and should similarly be scientifically justified if performed more than once in a single animal.
Food and Fluid Regulation Regulation of food or fluid intake may berequired for the conduct of some physiological, neuroscience, and behavioral research protocols. The regulation process may entail scheduled
access to food or fluid sources, so an animal consumes as much as desired
at regular intervals, or restriction, in which the total volume of food or fluid
consumed is strictly monitored and controlled (NRC 2003b). The objective
when these studies are being planned and executed should be to use theANIMAL CARE ANd USE PROGRAM

31

least restriction necessary to achieve the scientific objective while maintaining animal well-being.
The development of animal protocols that involve the use of food or
fluid regulation requires the evaluation of three factors: the necessary levelof regulation, potential adverse consequences of regulation, and methods
for assessing the health and well-being of the animals (NRC 2003b). In addition, the following factors influence the amount of food or fluid restrictionthat can be safely used in a specific protocol: the species, strain, or stock,gender, and age of the animals; thermoregulatory demand; type of housing;time of feeding, nutritive value, and fiber content of the diet (Heiderstadt etal. 2000; Rowland 2007); and prior experimental manipulation. The degreeof food or fluid restriction necessary for consistent behavioral performanceis influenced by the difficulty of the task, the individual animal, the motivation required of the animal, and the effectiveness of animal training for a
specific protocol-related task.
The animals should be closely monitored to ensure that food andfluid intake meets their nutritional needs (Toth and Gardiner 2000). Body
weights should be recorded at least weekly and more often for animals
requiring greater restrictions (NRC 2003b). Written records should be maintained for each animal to document daily food and fluid consumption,
hydration status, and any behavioral and clinical changes used as criteria
for temporary or permanent removal of an animal from a protocol (Morton
2000; NRC 2003b). In the case of conditioned-response research protocols,use of a highly preferred food or fluid as positive reinforcement, instead ofrestriction, is recommended. Caloric restriction, as a husbandry techniqueand means of weight control, is discussed in Chapter 3.
Use of Non-Pharmaceutical-Grade Chemicals and Other Substances The
use of pharmaceutical-grade chemicals and other substances ensures that
toxic or unwanted side effects are not introduced into studies conductedwith experimental animals. They should therefore be used, when available,
for all animal-related procedures (USDA 1997b). The use of non-pharmaceutical-grade chemicals or substances should be described and justified in
the animal use protocol and be approved by the IACUC (Wolff et al. 2003);
for example, the use of a non-pharmaceutical-grade chemical or substance
may be necessary to meet the scientific goals of a project or when a veterinary or human pharmaceutical-grade product is unavailable. In such
instances, consideration should be given to the grade, purity, sterility, pH,
pyrogenicity, osmolality, stability, site and route of administration, formulation, compatibility, and pharmacokinetics of the chemical or substance tobe administered, as well as animal welfare and scientific issues relating to
its use (NIH 2008).

32

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

Field Inestigations Investigations may involve the observation or use of
nondomesticated vertebrate species under field conditions. Many field
investigations require international, federal, state, and/or local permits,which may call for an evaluation of the scientific merit of the proposed
study and a determination of the potential impact on the population or
species to be studied.
Additionally, occupational health and safety issues, including zoonoses, should be reviewed by the institution’s health and safety committee or office, with assurances to the IACUC that the field study does notcompromise the health and safety of either animals or persons in the field.
Principal investigators conducting field research should be knowledgeable
about relevant zoonotic diseases, associated safety issues, and any laws or
regulations that apply. Exceptions to the above should be clearly definedand evaluated by the IACUC.
In preparing the design of a field study, investigators are encouraged
to consult with relevant professional societies and available guidelines (seeAppendix A). Veterinary input may be needed for projects involving capture,individual identification, sedation, anesthesia, surgery, recovery, holding,transportation, release, or euthanasia. Issues associated with these activities
are similar if not identical to those for species maintained and used in thelaboratory. When species are removed from the wild, the protocol should
include plans for either a return to their habitat or their final disposition, as
appropriate.
The Guide does not purport to be a compendium of all information
regarding field biology and methods used in wildlife investigations, but thebasic principles of humane care and use apply to animals living under natural conditions. IACUCs engaged in the review of field studies are encouraged to consult with a qualified wildlife biologist.
Agricultural Animals The use of agricultural animals in research is subject to the same ethical considerations as for other animals in research,
although it is often categorized as either biomedical or agricultural becauseof government regulations and policies, institutional policies, administrative structure, funding sources, and/or user goals (Stricklin et al. 1990). This
categorization has led to a dual system with different criteria for evaluating
protocols and standards of housing and care for animals of the same specieson the basis of stated biomedical or agricultural research objectives (Stricklin and Mench 1994). With some studies, differences in research goals maylead to a clear distinction between biomedical and agricultural research.
For example, animal models of human diseases, organ transplantation, andmajor surgery are considered biomedical uses; and studies on food and fiber
production, such as feeding trials, are usually considered agricultural uses.
But when the distinction is unclear, as in the case of some nutrition andANIMAL CARE ANd USE PROGRAM

33

disease studies, administrators, regulators, and IACUCs face a dilemma in
deciding how to handle such studies (Stricklin et al. 1990). Decisions on
categorizing research uses of agricultural animals and defining standards
for their care and use should be made by the IACUC based on both theresearcher’s goals and concern for animal well-being. Regardless of thecategory of research, institutions are expected to provide oversight of all
research animals and ensure that pain and distress are minimized.
The protocol, rather than the category of research, should determine thesetting (farm or laboratory). Housing systems for agricultural animals used inbiomedical research may or may not differ from those used in agriculturalresearch; animals used in either type of research can be housed in cages,
stalls, paddocks, or pastures (Tillman 1994). Some agricultural studies need
uniform conditions to minimize environmental variability, and some biomedical studies are conducted in farm settings. Agricultural research often
necessitates that animals be managed according to contemporary farm
production practices (Stricklin and Mench 1994), and natural environmental conditions might be desirable for agricultural research, whereas control
of environmental conditions to minimize variation might be desirable inbiomedical research (Tillman 1994).
The Guide applies to agricultural animals used in biomedical research,
including those maintained in typical farm settings. For animals maintainedin a farm setting, the Guide for the Care and Use of Agricultural Animals in
Research and Teaching (FASS 2010) is a useful resource. Information about
environmental enrichment, transport, and handling may be helpful in both
agricultural and biomedical research settings. Additional information about
facilities and management of farm animals in an agricultural setting is available from the Midwest Plan Service (1987) and from agricultural engineersor animal science experts.
Postapproval Monitoring
Continuing IACUC oversight of animal activities is required by federallaws, regulations, and policies. A variety of mechanisms can be used tofacilitate ongoing protocol assessment and regulatory compliance. Postapproval monitoring (PAM) is considered here in the broadest sense, consisting of all types of protocol monitoring after the IACUC’s initial protocol
approval.
PAM helps ensure the well-being of the animals and may also provide
opportunities to refine research procedures. Methods include continuing
protocol review; laboratory inspections (conducted either during regularfacilities inspections or separately); veterinary or IACUC observation of
selected procedures; observation of animals by animal care, veterinary, and
IACUC staff and members; and external regulatory inspections and assess-

34

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

ments. The IACUC, veterinary, animal care, and compliance staff may all
conduct PAM, which may also serve as an educational tool.
Continuing protocol review typically consists of an annual update orreview as well as the triennial review required by the PHS. The depth ofsuch reviews varies from a cursory update to a full committee review of theentire protocol. Some institutions use the annual review as an opportunity
for the investigator to submit proposed amendments for future procedures,to provide a description of any adverse or unanticipated events, and to provide updates on work progress. For the triennial review, many institutions
require a complete new protocol submission and may request a progress
report on the use of animals during the previous 3 years.
Both the Health Research Extension Act and the AWA require the
IACUC to inspect animal care and use facilities, including sites used foranimal surgeries, every 6 months. As part of a formal PAM program someinstitutions combine inspection of animal study sites with concurrent review
of animal protocols. Based on risks to animals and their handlers, otherstudy areas may require more or less frequent inspections. Examples ofeffective monitoring strategies include
•
•
•
•
•

examination of surgical areas, including anesthetic equipment, useof appropriate aseptic technique, and handling and use of controlled substances
review of protocol-related health and safety issues
review of anesthetic and surgical records
regular review of adverse or unexpected experimental outcomesaffecting the animals
observation of laboratory practices and procedures and comparison
with approved protocols.

Institutions may also consider the use of veterinary staff and/or animal
health technicians to observe increased risk procedures for adverse events(e.g., novel survival surgeries, pain studies, tumor growth studies) and reporttheir findings for review by the IACUC. The level of formality and intensityof PAM should be tailored to institutional size and complexity, and in allcases should support a culture of care focusing on the animals’ well-being(Klein and Bayne 2007). Regardless of the methods used or who conducts
and coordinates the monitoring, PAM programs are more likely to succeed
when the institution encourages an educational partnership with investigators (Banks and Norton 2008; Collins 2008; Dale 2008; Lowman 2008;
Plante and James 2008; Van Sluyters 2008).

35

ANIMAL CARE ANd USE PROGRAMDISASTER PLANNING AND EMERGENCy PREPAREDNESSAnimal facilities may be subject to unexpected conditions that result in
the catastrophic failure of critical systems or significant personnel absenteeism, or other unexpected events that severely compromise ongoing animalcare and well-being (ILAR 2010). Facilities must therefore have a disasterplan. The plan should define the actions necessary to prevent animal pain,
distress, and deaths due to loss of systems such as those that control ventilation, cooling, heating, or provision of potable water. If possible the planshould describe how the facility will preserve animals that are necessary
for critical research activities or are irreplaceable. Knowledge of the geographic locale may provide guidance as to the probability of a particular
type of disaster.
Disaster plans should be established in conjunction with the responsible investigator(s), taking into consideration both the priorities for triaging
animal populations and the institutional needs and resources. Animals thatcannot be relocated or protected from the consequences of the disaster mustbe humanely euthanized. The disaster plan should identify essential personnel who should be trained in advance in its implementation. Efforts should
be taken to ensure personnel safety and provide access to essential personnel during or immediately after a disaster. Such plans should be approvedby the institution and be part of the overall institutional disaster response
plan that is coordinated by the IO or another senior-level administrator. Law
enforcement and emergency personnel should be provided with a copyof the plan for comment and integration into broader, areawide planning(Vogelweid 1998).
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GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALSMann MD, Prentice ED. 2004. Should IACUCs review scientific merit of animal research
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3
Environment, Housing,and ManagementT

his chapter provides guidelines for the environment, housing, andmanagement of laboratory animals used or produced for research,
testing, and teaching. These guidelines are applicable across species
and are relatively general; additional information should be sought about
how to apply them to meet the specific needs of any species, strain, or use(see Appendix A for references). The chapter is divided into recommendations for terrestrial (page 42) and aquatic animals (page 77), as there arefundamental differences in their environmental requirements as well as animal husbandry, housing, and care needs. Although formulated specificallyfor vertebrate species, the general principles of humane animal care as set
out in the Guide may also apply to invertebrate species.
The design of animal facilities combined with appropriate animal housing and management are essential contributors to animal well-being, thequality of animal research and production, teaching or testing programs
involving animals, and the health and safety of personnel. An appropriate
Program (see Chapter 2) provides environments, housing, and management that are well suited for the species or strains of animals maintained
and takes into account their physical, physiologic, and behavioral needs,
allowing them to grow, mature, and reproduce normally while providing
for their health and well-being.
Fish, amphibians, and reptiles are poikilothermic animals: their coretemperature varies with environmental conditions and they have limited
ability (compared with birds and mammals) to metabolically maintain coretemperature. The majority of poikilothermic laboratory animals are aquatic
species—for example, fish and most amphibians—although some, such as
41

42

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

reptiles and certain amphibian species, are terrestrial. Personnel working
with aquatic animals should be familiar with management implications,e.g., the importance of providing appropriate temperature ranges for basic
physiologic function.
TERRESTRIAL ANIMALS
Terrestrial Environment
Microenironment and Macroenironment
The microenironment of a terrestrial animal is the physical environment immediately surrounding it; that is, the primary enclosure such as
the cage, pen, or stall. It contains all
the resources with which the animals
come directly in contact and also
Microenvironment: The immediprovides the limits of the animals’
ate physical environment surimmediate environment. The microrounding the animal (i.e., theenvironment in the primary enenvironment is characterized by manyclosure such as the cage, pen,factors, including illumination, noise,or stall).
vibration, temperature, humidity, andgaseous and particulate compositionof the air. The physical environmentof the secondary enclosure, such as a room, a barn, or an outdoor habitat,
constitutes the macroenironment.
Although the microenvironment and the macroenvironment are generally related, the microenvironment can be appreciably different and affectedby several factors, including the design of the primary enclosure and macroenvironmental conditions.
Evaluation of the microenvironment of small enclosures can be difficult. Available data indicate that
Macroenvironment: The physitemperature, humidity, and concencal environment of the secondary enclosure (e.g., a room, atrations of gases and particulate matbarn, or an outdoor habitat).
ter are often higher in the animal
microenvironment than in the macroenvironment (Besch 1980; Hasenau
et al. 1993; Perkins and Lipman 1995; E. Smith et al. 2004), while lightlevels are usually lower. Microenvironmental conditions can directly affectphysiologic processes and behavior and may alter disease susceptibility(Baer et al. 1997; Broderson et al. 1976; Memarzadeh et al. 2004; Schoeb
et al. 1982; Vesell et al. 1976).

ENVIRONMENT, hOUSING, ANd MANAGEMENT

43

Temperature and humidity
Maintenance of body temperature within normal circadian variationis necessary for animal well-being. Animals should be housed within temperature and humidity ranges appropriate for the species, to which they canadapt with minimal stress and physiologic alteration.
The ambient temperature range in which thermoregulation occurs without the need to increase metabolic heat production or activate evaporative
heat loss mechanisms is called the thermoneutral zone (TNZ) and is boundedby the lower and upper critical temperatures (LCTs and UCTs; Gordon 2005).
To maintain body temperature under a given environmental temperature
animals adjust physiologically (including their metabolism) and behaviorally (including their activity level and resource use). For example, the TNZ
of mice ranges between 26°C and 34°C (Gordon 1993); at lower temperatures, building nests and huddling for resting and sleeping allow them tothermoregulate by behaviorally controlling their microclimate. Althoughmice choose temperatures below their LCT of 26°C during activity periods,
they strongly prefer temperatures above their LCT for maintenance and resting behaviors (Gaskill et al. 2009; Gordon 2004; Gordon et al. 1998). Similar LCT values are found in the literature for other rodents, varying between
26-30°C for rats and 28-32°C for gerbils (Gordon 1993). The LCTs of rabbits(15-20°C; Gonzalez et al. 1971) and cats and dogs (20-25°C) are slightly
lower, while those of nonhuman primates and farm animals vary dependingon the species. In general, dry-bulb temperatures in animal rooms should
be set below the animals’ LCT to avoid heat stress. This, in turn, means thatanimals should be provided with adequate resources for thermoregulation(nesting material, shelter) to avoid cold stress. Adequate resources for thermoregulation are particularly important for newborn animals whose LCT isnormally considerably higher than that of their adult conspecifics.
Environmental temperature and relative humidity can be affected byhusbandry and housing design and can differ considerably between primaryand secondary enclosures as well as within primary enclosures. Factors that
contribute to variation in temperature and humidity between and within
enclosures include housing design; construction material; enrichment
devices such as shelters and nesting material; use of filter tops; number,age, type, and size of the animals in each enclosure; forced ventilation of
enclosures; and the type and frequency of contact bedding changes (Besch1980).
Exposure to wide temperature and humidity fluctuations or extremes
may result in behavioral, physiologic, and morphologic changes, whichmight negatively affect animal well-being and research performance aswell as outcomes of research protocols (Garrard et al. 1974; Gordon 1990,

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1993; Pennycuik 1967). These effects can be multigenerational (Barnett
1965, 1973).
The dry-bulb temperatures listed in Table 3.1 are broad and generally
reflect tolerable limits for common adult laboratory animal species, provided
they are housed with adequate resources for behavioral thermoregulation;
temperatures should normally be selected and maintained with minimal
fluctuation near the middle of these ranges. Depending on the specifichousing system employed, the selection of appropriate macro- and microenvironmental temperatures will differ based on a variety of factors, including but not limited to the species or strain, age, numbers of animals in theenclosure, size and construction of the primary enclosure, and husbandry
conditions (e.g., use/provision of contact bedding, nesting material and/or
shelter, individually ventilated cages). Poikilotherms and young birds ofsome species generally require a thermal gradient in their primary enclosure
to meet basic physiological processes. The temperature ranges shown may
not apply to captive wild animals, wild animals maintained in their naturalenvironment, or animals in outdoor enclosures that have the opportunity toadapt by being exposed to seasonal changes in ambient conditions.
Some conditions require increased environmental temperatures forhousing (e.g., postoperative recovery, neonatal animals, rodents with hairless phenotypes, reptiles and amphibians at certain stages of reproduction).
The magnitude of the temperature increase depends on housing details;
sometimes raising the temperature in the microenvironment alone (e.g.,
by using heating pads for postoperative recovery or radiant heat sources
for reptiles) rather than raising the temperature of the macroenvironment issufficient and preferable.
Relative humidity should also be controlled, but not nearly as narrowly
as temperature for many mammals; the acceptable range of relative humidity is considered to be 30% to 70% for most mammalian species. MicroTABLE 3.1 Recommended Dry-Bulb Macroenvironmental Temperatures
for Common Laboratory Animals
Dry-Bulb Temperature
Animal°C°F

Mouse, rat, hamster, gerbil, guinea piga
RabbitCat, dog, nonhuman primate
Farm animals, poultry

20-26
16-22
18-29
16-27

68-79
61-72
64-84
61-81

aDry-bulb room temperature settings for rodents are typically set below the animals’ LCT toavoid heat stress, and should reflect different species-specific LCT values. Animals should be
provided with adequate resources for thermoregulation (nesting material, shelter) to avoid
cold stress.

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45

environmental relative humidity may be of greater importance for animals
housed in a primary enclosure in which the environmental conditions differ
greatly from those of the macroenvironment (e.g., in static filter-top [isolator] cages).
Some species may require conditions with high relative humidity (e.g.,
selected species of nonhuman primates, tropical reptiles, and amphibians;
Olson and Palotay 1983). In mice, both abnormally high and low humidity may increase preweaning mortality (Clough 1982). In rats, low relative
humidity, especially in combination with temperature extremes, may lead to
ringtail, a condition involving ischemic necrosis of the tail and sometimes
toes (Crippa et al. 2000; Njaa et al. 1957; Totten 1958). For some species,
elevated relative humidity may affect an animal’s ability to cope with thermal extremes. Elevated microenvironmental relative humidity in rodent
isolator cages may also lead to high intracage ammonia concentrations(Corning and Lipman 1991; Hasenau et al. 1993), which can be irritating to
the nasal passages and alter some biologic responses (Gordon et al. 1980;
Manninen et al. 1998). In climates where it is difficult to provide a sufficientlevel of environmental relative humidity, animals should be closely monitored for negative effects such as excessively flaky skin, ecdysis (molting)difficulties in reptiles, and desiccation stress in semiaquatic amphibians.
Ventilation and Air Quality
The primary purpose of ventilation is to provide appropriate air quality
and a stable environment. Specifically, ventilation provides an adequate
oxygen supply; removes thermal loads caused by the animals, personnel,
lights, and equipment; dilutes gaseous and particulate contaminants including allergens and airborne pathogens; adjusts the moisture content andtemperature of room air; and, where appropriate, creates air pressure differentials (directional air flow) between adjoining spaces. Importantly, ventilating the room (i.e., the macroenvironment) does not necessarily ensure
adequate ventilation of an animal’s primary enclosure (i.e., the microenvironment), that is, the air to which the animal is actually exposed. The typeof primary enclosure may considerably influence the differences between
these two environments—for example, differences may be negligible when
animals are housed in open caging or pens, whereas they can be significant
when static isolator cages are used.
The volume and physical characteristics of the air supplied to a room
and its diffusion pattern influence the ventilation of an animal’s primary
enclosure and are important determinants of the animal’s microenvironment. The type and location of supply air diffusers and exhaust registers
in relation to the number, arrangement, location, and type of primary andsecondary enclosures affect how well the microenvironments are ventilated

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GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

and should therefore be considered. The use of computer modeling forassessing those factors in relation to heat loading, air diffusion patterns, andparticulate movement may be helpful in optimizing ventilation of microand macroenvironments (Hughes and Reynolds 1995).
Direct exposure of animals to air moving at high velocity (drafts) should
be avoided as the speed of air to which animals are exposed affects the rate
at which heat and moisture are removed from an animal. For example, air at
20°C moving at 60 linear feet per minute (18.3 m/min) has a cooling effectof approximately 7°C (Weihe 1971). Drafts can be particularly problematicfor neonatal homeotherms (which may be hairless and have poorly developed mechanisms for thermoregulatory control), for mutants lacking fur, andfor semiaquatic amphibians that can desiccate.
Provision of 10 to 15 fresh air changes per hour in animal housingrooms is an acceptable guideline to maintain macroenvironmental air quality by constant volume systems and may also ensure microenvironmental
air quality. Although this range is effective in many animal housing settings,
it does not take into account the range of possible heat loads; the species,
size, and number of animals involved; the type of primary enclosure andbedding; the frequency of cage changing; the room dimensions; or the efficiency of air distribution both in the macroenvironment and between themacro- and microenvironments. In some situations, the use of such a broad
guideline might overventilate a macroenvironment containing few animals,
thereby wasting energy, or underventilate a microenvironment containing
many animals, allowing heat, moisture, and pollutants to accumulate.
Modern heating, ventilation, and air conditioning (HVAC) systems (e.g.,variable air volume, or VAV, systems) allow ventilation rates to be set inaccordance with heat load and other variables. These systems offer considerable advantages with respect to flexibility and energy conservation,
but should always provide a minimum amount of air exchange, as recommended for general use laboratories (Bell 2008; DiBerardinis et al. 2009).
Individually ventilated cages (IVCs) and other types of specialized primary enclosures, that either directly ventilate the enclosure using filtered
room air or are ventilated independently of the room, can effectively address
animals’ ventilation requirements without the need to increase macroenvironmental ventilation. However, cautions mentioned above regarding highvelocity air should be considered (Baumans et al. 2002; Krohn et al. 2003).
Nevertheless, the macroenvironment should be ventilated sufficiently toaddress heat loads, particulates, odors, and waste gases released from primary enclosures (Lipman 1993).
If ventilated primary enclosures have adequate filtration to address contamination risks, air exhausted from the microenvironment may be returnedto the room in which animals are housed, although it is generally prefer-

ENVIRONMENT, hOUSING, ANd MANAGEMENT

4

able to exhaust these systems directly into the building’s exhaust system toreduce heat load and macroenvironmental contamination.
Static isolation caging (without forced ventilation), such as that used insome types of rodent housing, restricts ventilation (Keller et al. 1989). To
compensate, it may be necessary to adjust husbandry practices, including
sanitation and cage change frequency, selection of contact bedding, placement of cages in a secondary enclosure, animal densities in cages, and/or
decrease in macroenvironmental relative humidity to improve the microenvironment and heat dissipation.
The use of recycled air to ventilate animal rooms may save energy but
entails risks. Because many animal pathogens can be airborne or travel
on fomites (e.g., dust), exhaust air recycled into HVAC systems that serve
multiple rooms presents a risk of cross contamination. Recycling air from
nonanimal use areas (e.g., some human occupancy areas and food, bedding, and supply storage areas) may require less intensive filtration orconditioning and pose less risk of infection. The risks in some situations,however, might be too great to consider recycling (e.g., in the case of nonhuman primates and biohazard areas). The exhaust air to be recycled should
be filtered, at minimum, with 85-95% ASHRAE efficient filters to remove
airborne particles before it is recycled (NAFA 1996). Depending on the air
source, composition, and proportion of recycled air used (e.g., ammonia
and other gases emitted from excrement in recirculating air from animalrooms), consideration should also be given to filtering volatile substances.
In areas that require filtration to ensure personnel and/or animal safety (e.g.,hazardous containment holding), filter efficiency, loading, and integrity
should be assessed.
The successful operation of any HVAC system requires regular preventive maintenance and evaluation, including measurement of its function at
the level of the secondary enclosure. Such measurements should include
supply and exhaust air volumes, fluctuation in temperature and relative
humidity, and air pressure differentials between spaces as well as criticalmechanical operating parameters.
Illumination
Light can affect the physiology, morphology, and behavior of various
animals (Azar et al. 2008; Brainard et al. 1986; Erkert and Grober 1986;
Newbold et al. 1991; Tucker et al. 1984). Potential photostressors include
inappropriate photoperiod, photointensity, and spectral quality of the light(Stoskopf 1983).
Numerous factors can affect animals’ needs for light and should beconsidered when an appropriate illumination level is being established foran animal holding room. These include light intensity and wavelength as

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GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

well as the duration of the animal’s current and prior exposure to light, andthe animal’s pigmentation, circadian rhythm, body temperature, hormonal
status, age, species, sex, and stock or strain (Brainard 1989; Duncan and
O’Steen 1985; O’Steen 1980; Saltarelli and Coppola 1979; Semple-Rowland and Dawson 1987; Wax 1977). More recent studies in rodents andprimates have shown the importance of intrinsically photosensitive retinal
ganglion cells (distinct from rods and cones) for neuroendocrine, circadian,and neurobehavioral regulation (Berson et al. 2002; Hanifin and Brainard2007). These cells can respond to light wavelengths that may differ fromother photoreceptors and may influence the type of lighting, light intensity,and wavelength selected for certain types of research.
In general, lighting should be diffused throughout an animal holding area and provide sufficient illumination for the animals’ well-being
while permitting good housekeeping practices, adequate animal inspection
including for the bottom-most cages in racks, and safe working conditions for personnel. Light in animal holding rooms should provide for both
adequate vision and neuroendocrine regulation of diurnal and circadiancycles (Brainard 1989).
Photoperiod is a critical regulator of reproductive behavior in many animal species (Brainard et al. 1986; Cherry 1987), so inadvertent light exposure during the dark cycle should be minimized or avoided. Because somespecies, such as chickens (Apeldoorn et al. 1999), will not eat in low lightor darkness, such illumination schedules should be limited to a durationthat will not compromise their well-being. A time-controlled lighting systemshould be used to ensure a regular diurnal cycle, and system performance
should be checked regularly to ensure proper cycling.
Most commonly used laboratory rodents are nocturnal. Because albino
rodents are more susceptible to phototoxic retinopathy than other animals(Beaumont 2002), they have been used as a basis for establishing room
illumination levels (Lanum 1979). Data for room light intensities for otheranimals, based on scientific studies, are not available. Light levels of about
325 lux (30-ft candles) approximately 1 m (3.3 ft) above the floor appear tobe sufficient for animal care and do not cause clinical signs of phototoxic
retinopathy in albino rats (Bellhorn 1980). Levels up to 400 lux (37-ft
candles) as measured in an empty room 1 m from the floor have been foundto be satisfactory for rodents if management practices are used to prevent
retinal damage in albinos (Clough 1982). However, the light experienceof an individual animal can affect its sensitivity to phototoxicity; light of
130-270 lux above the light intensity under which it was raised has beenreported to be near the threshold of retinal damage in some individual
albino rats according to histologic, morphometric, and electrophysiologic
evidence (Semple-Rowland and Dawson 1987). Some guidelines recommend a light intensity as low as 40 lux at the position of the animal in

ENVIRONMENT, hOUSING, ANd MANAGEMENT

4

midcage (NASA 1988). Rats and mice generally prefer cages with low lightintensity (Blom et al. 1996), and albino rats prefer areas with a light intensityof less than 25 lux (Schlingmann et al. 1993a). Young mice prefer much
lower illumination than adults (Wax 1977). For animals that have beenshown to be susceptible to phototoxic retinopathy, light should be between
130 and 325 lux in the room at cage level.
Light intensity decreases with the square of the distance from its source.
Thus the location of a cage on a rack affects the intensity of light to which
the animals within are exposed. Light intensity may differ as much as 80fold in transparent cages from the top to the bottom of a rack, and differences up to 20-fold have been recorded within a cage (Schlingmann et al.
1993a,b). Management practices, such as rotating cage position relative
to the light source (Greenman et al. 1982) or providing animals with ways
to control their own light exposure by behavioral means (e.g., nesting or
bedding material adequate for tunneling), can reduce inappropriate lightstimulation. Variable-intensity lights are often used to accommodate theneeds of research protocols, certain animal species, and energy conservation. However, such a system should also provide for the observation andcare of the animals. Caution should be exercised as increases in daytime
room illumination for maintenance purposes have been shown to change
photoreceptor physiology and can alter circadian regulation (NRC 1996;
Reme et al. 1991; Terman et al. 1991).
Noise and Vibration
Noise produced by animals and animal care activities is inherent in theoperation of an animal facility (Pfaff and Stecker 1976) and noise control
should be considered in facility design and operation (Pekrul 1991). Assessment of the potential effects of noise on an animal warrants consideration of
the intensity, frequency, rapidity of onset, duration, and vibration potentialof the sound and the hearing range, noise exposure history, and sound effectsusceptibility of the species, stock, or strain. Similarly, occupational exposure
to animal or animal care practices that generate noise may be of concern for
personnel and, if of sufficient intensity, may warrant hearing protection.
Separation of human and animal areas minimizes disturbances to bothhuman and animal occupants of the facility. Noisy animals, such as dogs,
swine, goats, nonhuman primates, and some birds (e.g., zebra finches),should be housed away from quieter animals, such as rodents, rabbits, andcats. Environments should be designed to accommodate animals that makenoise rather than resorting to methods of noise reduction. Exposure to soundlouder than 85 dB can have both auditory and nonauditory effects (Fletcher
1976; Peterson 1980)—for example, eosinopenia, increased adrenal glandweights, and reduced fertility in rodents (Geber et al. 1966; Nayfield and50

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

Besch 1981; Rasmussen et al. 2009), and increased blood pressure in
nonhuman primates (Peterson et al. 1981)—and may necessitate hearing
protection for personnel (OSHA 1998). Many species can hear sound frequencies inaudible to humans (Brown and Pye 1975; Heffner and Heffner2007); rodents, for example, are very sensitive to ultrasound (Olivier et al.
1994). The potential effects of equipment (such as video display terminals;
Sales 1991; Sales et al. 1999) and materials that produce noise in the hearing range of nearby animals can thus become an uncontrolled variable forresearch experiments and should therefore be carefully considered (Turner
et al. 2007; Willott 2007). To the greatest extent possible, activities thatgenerate noise should be conducted in rooms or areas separate from thoseused for animal housing.
Because changes in patterns of sound exposure have different effects on
different animals (Armario et al. 1985; Clough 1982), personnel should try
to minimize the production of unnecessary noise. Excessive and intermittent
noise can be minimized by training personnel in alternatives to noisy practices, the use of cushioned casters and bumpers on carts, trucks, and racks,
and proper equipment maintenance (e.g., castor lubrication). Radios, alarms,
and other sound generators should not be used in animal rooms unless they
are part of an approved protocol or enrichment program. Any radios or soundgenerators used should be switched off at the end of the working day to minimize associated adverse physiologic changes (Baldwin 2007).
While some vibration is inherent to every facility and animal housing
condition, excessive vibration has been associated with biochemical andreproductive changes in laboratory animals (Briese et al. 1984; Carman et al.
2007) and can become an uncontrolled variable for research experiments.
The source of vibrations may be located within or outside the animal facility. In the latter case, groundborne vibration may affect both the structure
and its contents, including animal racks and cages. Housing systems withmoving components, such as ventilated caging system blowers, may create vibrations that could affect the animals housed within, especially if not
functioning properly. Like noise, vibration varies with intensity, frequency,
and duration. A variety of techniques may be used to isolate groundborne(see Chapter 5) and equipment-generated vibration (Carman et al. 2007).
Attempts should be made to minimize the generation of vibration, includingfrom humans, and excessive vibration should be avoided.
Terrestrial Housing
Microenironment (Primary Enclosure)
All animals should be housed under conditions that provide sufficientspace as well as supplementary structures and resources required to meet

ENVIRONMENT, hOUSING, ANd MANAGEMENT

51

physical, physiologic, and behavioral needs. Environments that fail to meet
the animals’ needs may result in abnormal brain development, physiologic
dysfunction, and behavioral disorders (Garner 2005; van Praag et al. 2000;
Würbel 2001) that may compromise both animal well-being and scientificvalidity. The primary enclosure or space may need to be enriched to prevent
such effects (see also section on Environmental Enrichment).
An appropriate housing space or enclosure should also account for
the animals’ social needs. Social animals should be housed in stable pairs
or groups of compatible individuals unless they must be housed alone forexperimental reasons or because of social incompatibility (see also section
on Behavioral and Social Management). Structural adjustments are frequently required for social housing (e.g., perches, visual barriers, refuges),and important resources (e.g., food, water, and shelter) should be provided
in such a way that they cannot be monopolized by dominant animals (seealso section on Environmental Enrichment).
The primary enclosure should provide a secure environment that does
not permit animal escape and should be made of durable, nontoxic materials that resist corrosion, withstand the rigors of cleaning and regular handling, and are not detrimental to the health and research use of the animals.
The enclosure should be designed and manufactured to prevent accidental
entrapment of animals or their appendages and should be free of sharpedges or projections that could cause injury to the animals or personnel.
It should have smooth, impervious surfaces with minimal ledges, angles,
corners, and overlapping surfaces so that accumulation of dirt, debris, andmoisture is minimized and cleaning and disinfecting are not impaired. All
enclosures should be kept in good repair to prevent escape of or injury to
animals, promote physical comfort, and facilitate sanitation and servicing. Rusting or oxidized equipment, which threatens the health or safetyof animals, needs to be repaired or replaced. Less durable materials, suchas wood, may be appropriate in select situations, such as outdoor corrals,
perches, climbing structures, resting areas, and perimeter fences for primary
enclosures. Wooden items may need to be replaced periodically becauseof damage or difficulties with sanitation. Painting or sealing wood surfaces
with nontoxic materials may improve durability in many instances.
Flooring should be solid, perforated, or slatted with a slip-resistant surface. In the case of perforated or slatted floors, the holes and slats shouldhave smooth edges. Their size and spacing need to be commensurate withthe size of the housed animal to minimize injury and the developmentof foot lesions. If wire-mesh flooring is used, a solid resting area may bebeneficial, as this floor type can induce foot lesions in rodents and rabbits(Drescher 1993; Fullerton and Gilliatt 1967; Rommers and Meijerhof 1996).
The size and weight of the animal as well as the duration of housing on
wire-mesh floors may also play a role in the development of this condi-

52

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

tion (Peace et al. 2001). When given the choice, rodents prefer solid floors(with bedding) to grid or wire-mesh flooring (Blom et al. 1996; Manser etal. 1995, 1996).
Animals should have adequate bedding substrate and/or structures
for resting and sleeping. For many animals (e.g., rodents) contact bedding
expands the opportunities for species-typical behavior such as foraging, digging, burrowing, and nest building (Armstrong et al. 1998; Ivy
et al. 2008). Moreover, it absorbs urine and feces to facilitate cleaningand sanitation. If provided in sufficient quantity to allow nest building
or burrowing, bedding also facilitates thermoregulation (Gordon 2004).
Breeding animals should have adequate nesting materials and/or substitutestructures based on species-specific requirements (mice: Sherwin 2002;
rats: Lawlor 2002; gerbils: Waiblinger 2002).
Specialized housing systems (e.g., isolation-type cages, IVCs, and gnotobiotic1 isolators) are available for rodents and certain species. Thesesystems, designed to minimize the spread of airborne particles between
cages or groups of cages, may require different husbandry practices, such asalterations in the frequency of bedding change, the use of aseptic handlingtechniques, and specialized cleaning, disinfecting, or sterilization regimens
to prevent microbial transmission by other than airborne routes.
Appropriate housing strategies for a particular species should be developed and implemented by the animal care management, in consultation
with the animal user and veterinarian, and reviewed by the IACUC. Housing should provide for the animals’ health and well-being while being
consistent with the intended objectives of animal use. Expert advice should
be sought when new species are housed or when there are special requirements associated with the animals or their intended use (e.g., geneticallymodified animals, invasive procedures, or hazardous agents). Objective
assessments should be made to substantiate the adequacy of the animal’senvironment, housing, and management. Whenever possible, routine procedures for maintaining animals should be documented to ensure consistency of management and care.
Enironmental Enrichment
The primary aim of environmental enrichment is to enhance animal
well-being by providing animals with sensory and motor stimulation,
through structures and resources that facilitate the expression of speciestypical behaviors and promote psychological well-being through physical
1 Gnotobiotic: germ-free animals or formerly germ-free animals in which the compositionof any associated microbial flora, if present, is fully defined (Stedman’s Electronic Medical
Dictionary 2006. Lippincott Williams & Wilkins).

ENVIRONMENT, hOUSING, ANd MANAGEMENT

53

exercise, manipulative activities, and cognitive challenges according to
species-specific characteristics (NRC 1998a; Young 2003). Examples ofenrichment include structural additions such as perches and visual barriers
for nonhuman primates (Novak et al. 2007); elevated shelves for cats
(Overall and Dyer 2005; van den Bos and de Cock Buning 1994) andrabbits (Stauffacher 1992); and shelters for guinea pigs (Baumans 2005), as
well as manipulable resources such as novel objects and foraging devices
for nonhuman primates; manipulable toys for nonhuman primates, dogs,
cats, and swine; wooden chew sticks for some rodent species; and nestingmaterial for mice (Gaskill et al. 2009; Hess et al. 2008; Hubrecht 1993;
Lutz and Novak 2005; Olsson and Dahlborn 2002). Novelty of enrichment
through rotation or replacement of items should be a consideration; however, changing animals’ environment too frequently may be stressful.
Well-conceived enrichment provides animals with choices and a degreeof control over their environment, which allows them to better cope withenvironmental stressors (Newberry 1995). For example, visual barriers allow
nonhuman primates to avoid social conflict; elevated shelves for rabbits andshelters for rodents allow them to retreat in case of disturbances (Baumans
1997; Chmiel and Noonan 1996; Stauffacher 1992); and nesting material anddeep bedding allow mice to control their temperature and avoid cold stressduring resting and sleeping (Gaskill et al. 2009; Gordon 1993, 2004).
Not every item added to the animals’ environment benefits their wellbeing. For example, marbles are used as a stressor in mouse anxiety studies(De Boer and Koolhaas 2003), indicating that some items may be detrimental to well-being. For nonhuman primates, novel objects can increase therisk of disease transmission (Bayne et al. 1993); foraging devices can leadto increased body weight (Brent 1995); shavings can lead to allergies andskin rashes in some individuals; and some objects can result in injury fromforeign material in the intestine (Hahn et al. 2000). In some strains of mice,
cage dividers and shelters have induced overt aggression in groups of males,
resulting in social stress and injury (e.g., Bergmann et al. 1994; Haemisch
et al. 1994). Social stress was most likely to occur when resources weremonopolized by dominant animals (Bergmann et al. 1994).
Enrichment programs should be reviewed by the IACUC, researchers,
and veterinarian on a regular basis to ensure that they are beneficial to animal well-being and consistent with the goals of animal use. They should be
updated as needed to ensure that they reflect current knowledge. Personnel
responsible for animal care and husbandry should receive training in the
behavioral biology of the species they work with to appropriately monitor
the effects of enrichment as well as identify the development of adverse orabnormal behaviors.
Like other environmental factors (such as space, light, noise, temperature, and animal care procedures), enrichment affects animal phenotype

54

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

and may affect the experimental outcome. It should therefore be considered
an independent variable and appropriately controlled.
Some scientists have raised concerns that environmental enrichment
may compromise experimental standardization by introducing variability,
adding not only diversity to the animals’ behavioral repertoire but alsovariation to their responses to experimental treatments (e.g., Bayne 2005;
Eskola et al. 1999; Gärtner 1999; Tsai et al. 2003). A systematic study inmice did not find evidence to support this viewpoint (Wolfer et al. 2004),
indicating that housing conditions can be enriched without compromising
the precision or reproducibility of experimental results. Further research inother species may be needed to confirm this conclusion. However, it has
been shown that conditions resulting in higher-stress reactivity increase
variation in experimental data (e.g., Macrì et al. 2007). Because adequate
environmental enrichment may reduce anxiety and stress reactivity (Chapillon et al. 1999), it may also contribute to higher test sensitivity and reduced
animal use (Baumans 1997).
Sheltered or Outdoor housing
Sheltered or outdoor housing (e.g., barns, corrals, pastures, islands) is a
primary housing method for some species and is acceptable in many situations. Animals maintained in outdoor runs, pens, or other large enclosures
must have protection from extremes in temperature or other harsh weather
conditions and adequate opportunities for retreat (for subordinate animals).
These goals can normally be achieved by providing windbreaks, speciesappropriate shelters, shaded areas, areas with forced ventilation, heat-radiating structures, and/or means of retreat to conditioned spaces, such as anindoor portion of a run. Shelters should be large enough to accommodate
all animals housed in the enclosure, be accessible at all times to all animals,
have sufficient ventilation, and be designed to prevent buildup of wastematerials and excessive moisture. Houses, dens, boxes, shelves, perches,and other furnishings should be constructed in a manner and made of materials that allow cleaning or replacement in accord with generally acceptedhusbandry practices.
Floors or ground-level surfaces of outdoor housing facilities may becovered with dirt, absorbent bedding, sand, gravel, grass, or similar material that can be removed or replaced when needed to ensure appropriate
sanitation. Excessive buildup of animal waste and stagnant water should be
avoided by, for example, using contoured or drained surfaces. Other surfaces should be able to withstand the elements and be easily maintained.
Successful management of outdoor housing relies on stable socialgroups of compatible animals; sufficient and species-appropriate feeding
and resting places; an adequate acclimation period in advance of seasonal

ENVIRONMENT, hOUSING, ANd MANAGEMENT

55

changes when animals are first introduced to outdoor housing; training ofanimals to cooperate with veterinary and investigative personnel (e.g., toenter chutes or cages for restraint or transport); and adequate security via aperimeter fence or other means.
Naturalistic Enironments
Areas such as pastures and islands may provide a suitable environment
for maintaining or producing animals and for some types of research. Theiruse results in the loss of some control over nutrition, health care and surveillance, and pedigree management. These limitations should be balanced
against the benefits of having the animals live in more natural conditions.
Animals should be added to, removed from, and returned to social groups
in this setting with appropriate consideration of the effects on the individual
animals and on the group. Adequate supplies of food, fresh water, and natural or constructed shelter should be ensured.
Space
General Considerations for All Animals An animal’s space needs are complex and consideration of only the animal’s body weight or surface area
may be inadequate. Important considerations for determining space needs
include the age and sex of the animal(s), the number of animals to becohoused and the duration of the accommodation, the use for which theanimals are intended (e.g., production vs. experimentation), and any special
needs they may have (e.g., vertical space for arboreal species or thermal
gradient for poikilotherms). In many cases, for example, adolescent animals,
which usually weigh less than adults but are more active, may require more
space relative to body weight (Ikemoto and Panksepp 1992). Group-housed,social animals can share space such that the amount of space required per
animal may decrease with increasing group size; thus larger groups may behoused at slightly higher stocking densities than smaller groups or individual
animals. Socially housed animals should have sufficient space and structuralcomplexity to allow them to escape aggression or hide from other animals
in the pair or group. Breeding animals will require more space, particularly
if neonatal animals will be raised together with their mother or as a breedinggroup until weaning age. Space quality also affects its usability. Enclosuresthat are complex and environmentally enriched may increase activity andfacilitate the expression of species-specific behaviors, thereby increasing
space needs. Thus there is no ideal formula for calculating an animal’s
space needs based only on body size or weight and readers should takethe performance indices discussed in this section into consideration whenutilizing the species-specific guidelines presented in the following pages.

56

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

Consideration of floor area alone may not be sufficient in determining
adequate cage size; with some species, cage volume and spatial arrangement may be of greater importance. In this regard, the Guide may differ
from the US Animal Welfare Regulations (AWRs) or other guidelines. The
height of an enclosure can be important to allow for expression of speciesspecific behaviors and postural adjustments. Cage height should take into
account the animal’s typical posture and provide adequate clearance forthe animal from cage structures, such as feeders and water devices. Some
species—for example, nonhuman primates, cats, and arboreal animals—use
the vertical dimensions of the cage to a greater extent than the floor. For
these animals, the ability to stand or to perch with adequate vertical space
to keep their body, including their tail, above the cage floor can improvetheir well-being (Clarence et al. 2006; MacLean et al. 2009).
Space allocations should be assessed, reviewed, and modified as necessary by the IACUC considering the performance indices (e.g., health,reproduction, growth, behavior, activity, and use of space) and special
needs determined by the characteristics of the animal strain or species(e.g., obese, hyperactive, or arboreal animals) and experimental use (e.g.,
animals in long-term studies may require greater and more complex space).
At a minimum, animals must have enough space to express their natural
postures and postural adjustments without touching the enclosure walls or
ceiling, be able to turn around, and have ready access to food and water.
In addition, there must be sufficient space to comfortably rest away fromareas soiled by urine and feces. Floor space taken up by food bowls, watercontainers, litter boxes, and enrichment devices (e.g., novel objects, toys,
foraging devices) should not be considered part of the floor space.
The space recommendations presented here are based on professional
judgment and experience. They should be considered the minimum foranimals housed under conditions commonly found in laboratory animal
housing facilities. Adjustments to the amount and arrangement of space
recommended in the following tables should be reviewed and approved by
the IACUC and should be based on performance indices related to animal
well-being and research quality as described in the preceding paragraphs,
with due consideration of the AWRs and PHS Policy and other applicableregulations and standards.
It is not within the scope of the Guide to discuss the housing requirements of all species used in research. For species not specifically indicated,advice should be sought from the scientific literature and from species-relevant experts.
Laboratory Rodents Table 3.2 lists recommended minimum space for commonly used laboratory rodents housed in groups. If they are housed singly
or in small groups or exceed the weights in the table, more space per

5

ENVIRONMENT, hOUSING, ANd MANAGEMENTTABLE 3.2 Recommended Minimum Space for Commonly Used
Laboratory Rodents Housed in Groups*
Weight,g

Floor Area/Animal,a Height,bin. (cm)in.2 (cm2)<10
Up to 15
Up to 25>25

6 (38.7)
8 (51.6)
12 (77.4)≥15 (≥96.7)

5 (12.7)
5 (12.7)
5 (12.7)
5 (12.7)

Larger animals may
require more space tomeet the performancestandards.

51 (330)(recommended
space for the
housing group)

5 (12.7)

Other breeding
configurations may require
more space and will
depend on considerations
such as number of adults
and litters, and size andage of litters.d

17 (109.6)
23 (148.35)
29 (187.05)
40 (258.0)
60 (387.0)≥70 (≥451.5)

7 (17.8)
7 (17.8)
7 (17.8)
7 (17.8)
7 (17.8)
7 (17.8)

Larger animals may
require more space tomeet the performancestandards.

124 (800)(recommended
space for the
housing group)

7 (17.8)

Other breeding
configurations may require
more space and will
depend on considerations
such as number of adults
and litters, and size andage of litters.d<60
Up to 80
Up to 100
>100

10 (64.5)
13 (83.8)
16 (103.2)≥19 (≥122.5)

6 (15.2)
6 (15.2)
6 (15.2)
6 (15.2)

Larger animals may
require more space tomeet the performancestandards.

Guinea pigsc Up to 350>350

60 (387.0)≥101 (≥651.5)

7 (17.8)
7 (17.8)

Larger animals may
require more space tomeet the performancestandards.

Animals
Mice ingroupsc

Female +litterRats ingroupsc<100
Up to 200
Up to 300
Up to 400
Up to 500
>500

Female +litterHamstersc

Comments*The interpretation of this table should take into consideration the performance indicesdescribed in the text beginning on page 55.
aSingly housed animals and small groups may require more than the applicable multiple ofthe indicated floor space per animal.
bFrom cage floor to cage top.
cConsideration should be given to the growth characteristics of the stock or strain as well as thesex of the animal. Weight gain may be sufficiently rapid that it may be preferable to provide
greater space in anticipation of the animal’s future size. In addition, juvenile rodents are highly
active and show increased play behavior.
dOther considerations may include culling of litters or separation of litters from the breedinggroup, as well as other methods of more intensive management of available space to allow
for the safety and well-being of the breeding group. Sufficient space should be allocated formothers with litters to allow the pups to develop to weaning without detrimental effects for themother or the litter.

58

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

animal may be required, while larger groups may be housed at slightly
higher densities.
Studies have recently evaluated space needs and the effects ofsocial housing, group size, and density (Andrade and Guimaraes 2003;
Bartolomucci et al. 2002, 2003; Georgsson et al. 2001; Gonder and Laber
2007; Perez et al. 1997; A.L. Smith et al. 2004), age (Arakawa 2005;
Davidson et al. 2007; Yildiz et al. 2007), and housing conditions (Gordon
et al. 1998; Van Loo et al. 2004) for many different species and strains ofrodents, and have reported varying effects on behavior (such as aggression)and experimental outcomes (Karolewicz and Paul 2001; Laber et al. 2008;
McGlone et al. 2001; Rock et al. 1997; Smith et al. 2005; Van Loo et al.
2001). However, it is difficult to compare these studies due to the study
design and experimental variables that have been measured. For example,
variables that may affect the animals’ response to different cage sizes andhousing densities include, but are not limited to, species, strain (and social
behavior of the strain), phenotype, age, gender, quality of the space (e.g.,
vertical access), and structures placed in the cage. These issues remaincomplex and should be carefully considered when housing rodents.
Other Common Laboratory Animals Tables 3.3 and 3.4 list recommended
minimum space for other common laboratory animals and for avian species. These allocations are based, in general, on the needs of pair- orgroup-housed animals. Space allocations should be reevaluated to provide
for enrichment or to accommodate animals that exceed the weights in thetables, and should be based on species characteristics, behavior, compatibility of the animals, number of animals, and goals of the housing situation(Held et al. 1995; Lupo et al. 2000; Raje 1997; Turner et al. 1997). Singly
housed animals may require more space per animal than that recommended for group-housed animals, while larger groups may be housed atslightly higher densities. For cats, dogs, and some rabbits, housing enclosures that allow greater freedom of movement and less restricted verticalspace are preferred (e.g., kennels, runs, or pens instead of cages). Dogs andcats, especially when housed individually or in smaller enclosures (Bayne2002), should be allowed to exercise and provided with positive humaninteraction. Species-specific plans for housing and management should
be developed. Such plans should also include strategies for environmental
enrichment.
Nonhuman Primates The recommended minimum space for nonhuman primates detailed in Table 3.5 is based on the needs of pair- or group-housed
animals. Like all social animals, nonhuman primates should normally havesocial housing (i.e., in compatible pairs or in larger groups of compatible
animals) (Hotchkiss and Paule 2003; NRC 1998a; Weed and Watson 1998;

5

ENVIRONMENT, hOUSING, ANd MANAGEMENTTABLE 3.3 Recommended Minimum Space for Rabbits, Cats, and Dogs
Housed in Pairs or Groups*
Weight,akg

Floor Area/
Animal,b ft2 (m2)Height,cin. (cm)

Rabbits<2
Up to 4Up to 5.4
>5.4c

1.5 (0.14)
3.0 (0.28)
4.0 (0.37)≥5.0 (≥0.46)

16 (40.5)
16 (40.5)
16 (40.5)
16 (40.5)

Larger rabbits may require
more cage height to allow
animals to sit up.

Cats

<4
>4d

3.0 (0.28)≥4.0 (≥0.37)

24 (60.8)
24 (60.8)

Vertical space with perches
is preferred and may
require additional cageheight.

Dogse<15
Up to 30>30d

8.0 (0.74)
12.0 (1.2)≥24.0 (≥2.4)

—f
—f
—f

Cage height should be
sufficient for the animals
to comfortably stand erectwith their feet on the floor.

Animals

Comments*The interpretation of this table should take into consideration the performance indicesdescribed in the text beginning on page 55.
aTo convert kilograms to pounds, multiply by 2.2.
bSingly housed animals may require more space per animal than recommended for pair- orgroup-housed animals.
cFrom cage floor to cage top.
dLarger animals may require more space to meet performance standards (see text).
eThese recommendations may require modification according to body conformation of individual animals and breeds. Some dogs, especially those toward the upper limit of each weight
range, may require additional space to ensure compliance with the regulations of the Animal
Welfare Act. These regulations (USDA 1985) mandate that the height of each cage be sufficient
to allow the occupant to stand in a “comfortable position” and that the minimal square feetof floor space be equal to the “mathematical square of the sum of the length of the dog in
inches (measured from the tip of its nose to the base of its tail) plus 6 inches; then divide theproduct by 144.”
fEnclosures that allow greater freedom of movement and unrestricted height (i.e., pens, runs,
or kennels) are preferable.

Wolfensohn 2004). Group composition is critical and numerous speciesspecific factors such as age, behavioral repertoire, sex, natural social organization, breeding requirements, and health status should be taken into
consideration when forming a group. In addition, due to conformational differences of animals within groups, more space or height may be required tomeet the animals’ physical and behavioral needs. Therefore, determinationof the appropriate cage size is not based on body weight alone, and professional judgment is paramount in making such determinations (Kaufman etal. 2004; Williams et al. 2000).

60

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALSTABLE 3.4 Recommended Minimum Space for Avian Species Housed inPairs or Groups*
Animals
Pigeons
Quail
Chickens

Weight,akg

Floor area/animal,bft2 (m2)——<0.25
Up to 0.5
Up to 1.5
Up to 3.0
>3.0c

0.8 (0.07)
0.25 (0.023)
0.25 (0.023)
0.50 (0.046)
1.00 (0.093)
2.00 (0.186)≥3.00 (≥0.279)

HeightCage height should be
sufficient for the animals to
comfortably stand erect with
their feet on the floor.

*The interpretation of this table should take into consideration the performance indicesdescribed in the text beginning on page 55.
aTo convert kilograms to pounds, multiply by 2.2.
bSingly housed birds may require more space per animal than recommended for pair- orgroup-housed birds.
cLarger animals may require more space to meet performance standards (see text).

If it is necessary to house animals singly—for example, when justified
for experimental purposes, for provision of veterinary care, or for incompatible animals—this arrangement should be for the shortest duration possible.
If single animals are housed in small enclosures, an opportunity for periodic
release into larger enclosures with additional enrichment items should beconsidered, particularly for animals housed singly for extended periodsof time. Singly housed animals may require more space per animal thanrecommended for pair- or group-housed animals, while larger groups may
be housed at slightly higher densities. Because of the many physical andbehavioral characteristics of nonhuman primate species and the many factors to consider when using these animals in a biomedical research setting,species-specific plans for housing and management should be developed.
Such plans should include strategies for environmental and psychologicalenrichment.
Agricultural Animals Table 3.6 lists recommended minimum space for agricultural animals commonly used in a laboratory setting. As social animals,
they should be housed in compatible pairs or larger groups of compatible
animals. When animals exceed the weights in the table, more space isrequired. For larger animals (particularly swine) it is important that the configuration of the space allow the animals to turn around and move freely(Becker et al. 1989; Bracke et al. 2002). Food troughs and water devices
should be provided in sufficient numbers to allow ready access for all animals. Singly housed animals may require more space than recommended in

61

ENVIRONMENT, hOUSING, ANd MANAGEMENTTABLE 3.5 Recommended Minimum Space for Nonhuman Primates
Housed in Pairs or Groups*

Animals

Weight,akg

Floor
area/animal,bft2 (m2)

Height,cin. (cm)

Monkeysd(including
baboons)
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Group 7
Group 8Chimpanzees(Pan)
Juveniles
Adultsf

Up to 1.5
Up to 3Up to 10
Up to 15
Up to 20
Up to 25
Up to 30>30e

Up to 10>10

2.1 (0.20)
3.0 (0.28)
4.3 (0.4)
6.0 (0.56)
8.0 (0.74)
10 (0.93)
15 (1.40)≥25 (≥2.32)

15 (1.4)≥25 (≥2.32)

30 (76.2)
30 (76.2)
30 (76.2)
32 (81.3)
36 (91.4)
46 (116.8)
46 (116.8)
60 (152.4)

60 (152.4)
84 (213.4)

Comments
Cage height should be
sufficient for the animals
to comfortably stand erectwith their feet on the floor.
Baboons, patas monkeys,
and other longer-legged
species may require moreheight than other monkeys,
as might long-tailed animals
and animals with prehensile
tails. Overall cage volume
and linear perch spaceshould be considerations
for many neotropicaland arboreal species. For
brachiating species cageheight should be such thatan animal can, when fully
extended, swing from thecage ceiling without having
its feet touch the floor. Cage
design should enhancebrachiating movement.
For other apes and largebrachiating species cageheight should be such thatan animal can, when fully
extended, swing from thecage ceiling without having
its feet touch the floor. Cage
design should enhancebrachiating movement.

*The interpretation of this table should take into consideration the performance indicesdescribed in the text beginning on page 55.
aTo convert kilograms to pounds, multiply by 2.2.
bSingly housed primates may require more space than the amount allocated per animal whengroup housed.
cFrom cage floor to cage top.
dCallitrichidae, Cebidae, Cercopithecidae, and Papio.
eLarger animals may require more space to meet performance standards (see text).
fApes weighing over 50 kg are more effectively housed in permanent housing of masonry,
concrete, and wire-panel structure than in conventional caging.

62

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALSTABLE 3.6 Recommended Minimum Space for Agricultural Animals*
Weight,akg

Floor Area/Animal,bft2 (m2)<25
Up to 50>50c

10.0 (0.9)
15.0 (1.35)≥20.0 (≥1.8)

2-5

<25
Up to 50>50c

8.5 (0.76)
12.5 (1.12)≥17.0 (≥1.53)>5<25
Up to 50>50c

7.5 (0.67)
11.3 (1.02)≥15.0 (≥1.35)<15
Up to 25
Up to 50
Up to 100
Up to 200>200c

8.0 (0.72)
12.0 (1.08)
15.0 (1.35)
24.0 (2.16)
48.0 (4.32)≥60.0 (≥5.4)

2-5

<25
Up to 50
Up to 100
Up to 200>200c

6.0 (0.54)
10.0 (0.9)
20.0 (1.8)
40.0 (3.6)≥52.0 (≥4.68)>5<25
Up to 50
Up to 100
Up to 200>200c

6.0 (0.54)
9.0 (0.81)
18.0 (1.62)
36.0 (3.24)≥48.0 (≥4.32)<75
Up to 200
Up to 350
Up to 500
Up to 650
>650c

24.0 (2.16)
48.0 (4.32)
72.0 (6.48)
96.0 (8.64)
124.0 (11.16)≥144.0 (≥12.96)<75
Up to 200
Up to 350
Up to 500
Up to 650
>650c

20.0 (1.8)
40.0 (3.6)
60.0 (5.4)
80.0 (7.2)
105.0 (9.45)≥120.0 (≥10.8)

Animals/Enclosure
Sheep and Goats
1

Swine
1

Cattle12-5

63

ENVIRONMENT, hOUSING, ANd MANAGEMENT>5<75
Up to 200
Up to 350
Up to 500
Up to 650
>650c

18.0 (1.62)
36.0 (3.24)
54.0 (4.86)
72.0 (6.48)
93.0 (8.37)≥108.0 (≥9.72)

Horses—144.0 (12.96)

—
<200
>200c

72.0 (6.48)
60.0 (5.4)≥72.0 (≥6.48)

Ponies1-4>4/Pen*The interpretation of this table should take into consideration the performance indicesdescribed in the text beginning on page 55.
aTo convert kilograms to pounds, multiply by 2.2.
bFloor area configuration should be such that animals can turn around and move freely without
touching food or water troughs, have ready access to food and water, and have sufficient space
to comfortably rest away from areas soiled by urine and feces (see text).
cLarger animals may require more space to meet performance standards including sufficientspace to turn around and move freely (see text).

the table to enable them to turn around and move freely without touchingfood or water troughs, have ready access to food and water, and have sufficient space to comfortably rest away from areas soiled by urine and feces.
Terrestrial Managementbehaioral and Social Management
Actiity Animal Actiity typically implies motor activity but also includes
cognitive activity and social interaction. Animals’ natural behavior andactivity profile should be considered during evaluation of suitable housing
or behavioral assessment.
Animals maintained in a laboratory environment are generally restrictedin their activities compared to free-ranging animals. Forced activity forreasons other than attempts to meet therapeutic or approved protocol
objectives should be avoided. High levels of repetitive, unvarying behavior (stereotypies, compulsive behaviors) may reflect disruptions of normal
behavioral control mechanisms due to housing conditions or management
practices (Garner 2005; NRC 1998a).
Dogs, cats, rabbits, and many other animals benefit from positive humaninteraction (Augustsson et al. 2002; Bayne et al. 1993; McCune 1997; Poole
1998; Rennie and Buchanan-Smith 2006; Rollin 1990). Dogs can be given

64

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

additional opportunities for activity by being walked on a leash, having
access to a run, or being moved into areas for social contact, play, or exploration (Wolff and Rupert 1991). Loafing areas, exercise lots, and pastures are
suitable for large farm animals, such as sheep, horses, and cattle.
Social Enironment Appropriate social interactions among members of thesame species (conspecifics) are essential to normal development and wellbeing (Bayne et al. 1995; Hall 1998; Novak et al. 2006). When selecting
a suitable social environment, attention should be given to whether the
animals are naturally territorial or communal and whether they should behoused singly, in pairs, or in groups. An understanding of species-typicalnatural social behavior (e.g., natural social composition, population density,
ability to disperse, familiarity, and social ranking) is key to successful social
housing.
Not all members of a social species are necessarily socially compatible.
Social housing of incompatible animals can induce chronic stress, injury,and even death. In some species, social incompatibility may be sex biased;for example, male mice are generally more prone to aggression than female
mice, and female hamsters are generally more aggressive than male hamsters. Risks of social incompatibility are greatly reduced if the animals to begrouped are raised together from a young age, if group composition remains
stable, and if the design of the animals’ enclosure and their environmental enrichment facilitate the avoidance of social conflicts. Social stability
should be carefully monitored; in cases of severe or prolonged aggression,
incompatible individuals need to be separated.
For some species, developing a stable social hierarchy will entail
antagonistic interactions between pair or group members, particularly foranimals introduced as adults. Animals may have to be introduced to eachother over a period of time and should be monitored closely during thisintroductory period and thereafter to ensure compatibility.
Single housing of social species should be the exception and justified
based on experimental requirements or veterinary-related concerns aboutanimal well-being. In these cases, it should be limited to the minimum
period necessary, and where possible, visual, auditory, olfactory, and tactile
contact with compatible conspecifics should be provided. In the absenceof other animals, enrichment should be offered such as positive interaction
with the animal care staff and additional enrichment items or addition of acompanion animal in the room or housing area. The need for single housing
should be reviewed on a regular basis by the IACUC and veterinarian.
Procedural habituation and Training of Animals Habituating animals toroutine husbandry or experimental procedures should be encouraged whenever possible as it may assist the animal to better cope with a captive environment by reducing stress associated with novel procedures or people.

ENVIRONMENT, hOUSING, ANd MANAGEMENT

65

The type and duration of habituation needed will be determined by thecomplexity of the procedure. In most cases, principles of operant conditioning may be employed during training sessions, using progressive behavioral
shaping, to induce voluntary cooperation with procedures (Bloomsmith etal. 1998; Laule et al. 2003; NRC 2006a; Reinhardt 1997).
husbandry
Food Animals should be fed palatable, uncontaminated diets that meettheir nutritional and behavioral needs at least daily, or according to their
particular requirements, unless the protocol in which they are being usedrequires otherwise. Subcommittees of the National Research Council Committee on Animal Nutrition have prepared comprehensive reports of thenutrient requirements of laboratory animals (NRC 1977, 1982, 1993, 1994,
1995a, 1998b, 2000, 2001, 2003a, 2006b,c, 2007); these publications
consider issues of quality assurance, freedom from chemical or microbial
contaminants and natural toxicants in feedstuffs, bioavailability of nutrientsin feeds, and palatability.
There are several types of diets classified by the degree of refinement of
their ingredients. Natural-ingredient diets are formulated with agricultural
products and byproducts and are commercially available for all species
commonly used in the laboratory. Although not a significant factor in most
instances, the nutrient composition of ingredients varies, and natural ingredients may contain low levels of naturally occurring or artificial contaminants(Ames et al. 1993; Knapka 1983; Newberne 1975; NRC 1996; Thigpen et al.
1999, 2004). Contaminants such as pesticide residues, heavy metals, toxins,
carcinogens, and phytoestrogens may be at levels that induce few or nohealth sequelae yet may have subtle effects on experimental results (Thigpen
et al. 2004). Certified diets that have been assayed for contaminants are commercially available for use in select studies, such as preclinical toxicology,
conducted in compliance with FDA Good Laboratory Practice standards (CFR2009). Purified diets are refined such that each ingredient contains a single
nutrient or nutrient class; they have less nutrient concentration variability andthe potential for chemical contamination is lower. Chemically defined diets
contain the most elemental ingredients available, such as individual aminoacids and specific sugars (NRC 1996). The latter two types of diet are more
likely to be used for specific types of studies in rodents but are not commonlyused because of cost, lower palatability, and a reduced shelf life.
Animal colony managers should be judicious when purchasing, transporting, storing, and handling food to minimize the introduction of diseases,
parasites, potential disease vectors (e.g., insects and other vermin), andchemical contaminants in animal colonies. Purchasers are encouraged toconsider manufacturers’ and suppliers’ procedures and practices (e.g., storage, vermin control, and handling) for protecting and ensuring diet quality.

66

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALSInstitutions should urge feed vendors to periodically provide data from laboratory-based feed analyses for critical nutrients. The user should know thedate of manufacture and other factors that affect the food’s shelf life. Stalefood or food transported and stored inappropriately can become deficientin nutrients. Upon receipt, bags of feed should be examined to ensure thatthey are intact and unstained to help ensure that their contents have notbeen potentially exposed to vermin, penetrated by liquids, or contaminated.
Careful attention should be paid to quantities received in each shipment,
and stock should be rotated so that the oldest food is used first.
Areas in which diets and diet ingredients are processed or stored should
be kept clean and enclosed to prevent the entry of pests. Food stocks should
be stored off the floor on pallets, racks, or carts in a manner that facilitates
sanitation. Opened bags of food should be stored in vermin-proof containers to minimize contamination and to avoid the potential spread of pathogens. Exposure to elevated storage room temperatures, extremes in relative
humidity, unsanitary conditions, and insects and other vermin hastens fooddeterioration. Storage of natural-ingredient diets at less than 21°C (70°F)
and below 50% relative humidity is recommended. Precautions should betaken if perishable items—such as meats, fruits, and vegetables and somespecialty diets (e.g., select medicated or high-fat diets)—are fed, because
storage conditions may lead to variation in food quality.
Most natural-ingredient, dry laboratory animal diets stored properly
can be used up to 6 months after manufacture. Nonstabilized vitamin C in
manufactured feeds generally has a shelf life of only 3 months, but commonly used stabilized forms can extend the shelf life of feed. Refrigeration
preserves nutritional quality and lengthens shelf life, but food storage timeshould be reduced to the lowest practical period and the manufacturers’
recommendations considered. Purified and chemically defined diets areoften less stable than natural-ingredient diets and their shelf life is usuallyless than 6 months (Fullerton et al. 1982); they should be stored at 4°C(39°F) or lower.
Irradiated and fortified autoclavable diets are commercially available
and are commonly used for axenic and microbiologically defined rodents,
and immunodeficient animals (NRC 1996). The use of commercially fortified
autoclavable diets ensures that labile vitamin content is not compromisedby steam and/or heat (Caulfield et al. 2008; NRC 1996). But consideration
should be given to the impact of autoclaving on pellets as it may affect theirhardness and thus palatability and also lead to chemical alteration of ingredients (Thigpen et al. 2004; Twaddle et al. 2004). The date of sterilization
should be recorded and the diet used quickly.
Feeders should be designed and placed to allow easy access to food andto minimize contamination with urine and feces, and maintained in goodcondition. When animals are housed in groups, there should be enoughspace and enough feeding points to minimize competition for food andENVIRONMENT, hOUSING, ANd MANAGEMENT

6

ensure access to food for all animals, especially if feed is restricted as part ofthe protocol or management routine. Food storage containers should not betransferred between areas that pose different risks of contamination without
appropriate treatment, and they should be cleaned and sanitized regularly.
Management of caloric intake is an accepted practice for long-term
housing of some species, such as some rodents, rabbits, and nonhumanprimates, and as an adjunct to some clinical, experimental, and surgicalprocedures (for more discussion of food and fluid regulation as an experimental tool see Chapter 2 and NRC 2003a). Benefits of moderate caloric
restriction in some species may include increased longevity and reproduction, and decreased obesity, cancer rates, and neurogenerative disorders(Ames et al. 1993; Colman et al. 2009; Keenan et al. 1994, 1996; Lawler et
al. 2008; Weindruch and Walford 1988).
Under standard housing conditions, changes in biologic needs commensurate with aging should be taken into consideration. For example,
there is good evidence that mice and rats with continuous access to foodcan become obese, with attendant metabolic and cardiovascular changes
such as insulin resistance and higher blood pressure (Martin et al. 2010).
These and other changes along with a more sedentary lifestyle and lack of
exercise increase the risk of premature death (ibid.). Caloric management,
which may affect physiologic adaptations and alter metabolic responses ina species-specific manner (Leveille and Hanson 1966), can be achieved by
reducing food intake or by stimulating exercise.
In some species (e.g., nonhuman primates) and on some occasions,varying nutritionally balanced diets and providing “treats,” including fresh
fruit and vegetables, can be appropriate and improve well-being. Scattering
food in the bedding or presenting part of the diet in ways that require theanimals to work for it (e.g., puzzle feeders for nonhuman primates) gives
the animals the opportunity to forage, which, in nature, normally accounts
for a large proportion of their daily activity. A diet should be nutritionallybalanced; it is well documented that many animals offered a choice of
unbalanced or balanced foods do not select a balanced diet and become
malnourished or obese through selection of high-energy, low-protein foods(Moore 1987). Abrupt changes in diet, which can be difficult to avoid at
weaning, should be minimized because they can lead to digestive andmetabolic disturbances; these changes occur in omnivores and carnivores,
but herbivores (Eadie and Mann 1970) are especially sensitive.
Water Animals should have access to potable, uncontaminated drinking
water according to their particular requirements. Water quality and the definition of potable water can vary with locality (Homberger et al. 1993). Periodic monitoring for pH, hardness, and microbial or chemical contamination
may be necessary to ensure that water quality is acceptable, particularly
for use in studies in which normal components of water in a given locality

68

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

can influence the results. Water can be treated or purified to minimize or
eliminate contamination when protocols require highly purified water. The
selection of water treatments should be carefully considered because many
forms of water treatment have the potential to cause physiologic alterations, reduction in water consumption, changes in microflora, or effects on
experimental results (Fidler 1977; Hall et al. 1980; Hermann et al. 1982;
Homberger et al. 1993; NRC 1996).
Watering devices, such as drinking tubes and automated water delivery
systems, should be checked frequently to ensure appropriate maintenance,
cleanliness, and operation. Animals sometimes have to be trained to use
automated watering devices and should be observed regularly until regularusage has been established to prevent dehydration. It is better to replacewater bottles than to refill them, because of the potential for microbiologic
cross contamination; if bottles are refilled, care should be taken to returneach bottle to the cage from which it was removed. Automated watering
distribution systems should be flushed or disinfected regularly. Animals
housed in outdoor facilities may have access to water in addition to thatprovided in watering devices, such as that available in streams or in puddles
after a heavy rainfall. Care should be taken to ensure that such accessory
sources of water do not constitute a hazard, but their availability need not
routinely be prevented. In cold weather, steps should be taken to prevent
freezing of outdoor water sources.
bedding and Nesting Materials Animal bedding and nesting materials arecontrollable environmental factors that can influence experimental data andimprove animal well-being in most terrestrial species. Bedding is used toabsorb moisture, minimize the growth of microorganisms, and dilute andlimit animals’ contact with excreta, and specific bedding materials havebeen shown to reduce the accumulation of intracage ammonia (Perkins
and Lipman 1995; E. Smith et al. 2004). Various materials are used as bothcontact and noncontact bedding; the desirable characteristics and methodsof evaluating bedding have been described (Gibson et al. 1987; Jones 1977;
Kraft 1980; Thigpen et al. 1989; Weichbrod et al. 1986). The veterinarianor facility manager, in consultation with investigators, should select themost appropriate bedding and nesting materials. A number of species, mostnotably rodents, exhibit a clear preference for specific materials (Blom et al.
1996; Manser et al. 1997, 1998; Ras et al. 2002), and mice provided withappropriate nesting material build better nests (Hess et al. 2008). Beddingthat enables burrowing is encouraged for some species, such as mice andhamsters.
No type of bedding is ideal for all species under all management andexperimental conditions. For example, in nude or hairless mice that lackeyelashes, some forms of paper bedding with fines (i.e., very small particles
found in certain types of bedding) can result in periorbital abscesses (WhiteENVIRONMENT, hOUSING, ANd MANAGEMENT

6

et al. 2008), while cotton nestlets may lead to conjunctivitis (Bazille et al.
2001). Bedding can also influence mucosal immunity (Sanford et al. 2002)
and endocytosis (Buddaraju and Van Dyke 2003).
Softwood beddings have been used, but the use of untreated softwoodshavings and chips is contraindicated for some protocols because they
can affect metabolism (Vesell 1967; Vesell et al. 1973, 1976). Cedar shavings are not recommended because they emit aromatic hydrocarbons thatinduce hepatic microsomal enzymes and cytotoxicity (Torronen et al. 1989;
Weichbrod et al. 1986, 1988) and have been reported to increase the incidence of cancer (Jacobs and Dieter 1978; Vlahakis 1977). Prior treatment
with high heat (kiln drying or autoclaving) may, depending on the material
and the concentration of aromatic hydrocarbon constituents, reduce theconcentration of volatile organic compounds, but the amounts remaining
may be sufficient to affect specific protocols (Cunliffe-Beamer et al. 1981;
Nevalainen and Vartiainen 1996).
The purchase of bedding products should take into consideration vendors’ manufacturing, monitoring, and storage methods. Bedding may becontaminated with toxins and other substances, bacteria, fungi, and vermin.
It should be transported and stored off the floor on pallets, racks, or carts
in a fashion consistent with maintenance of quality and avoidance of contamination. Bags should be stored sufficiently away from walls to facilitate
cleaning. During autoclaving, bedding can absorb moisture and as a result
lose absorbency and support the growth of microorganisms. Therefore,
appropriate drying times and storage conditions should be used or, alternatively, gamma-irradiated materials if sterile bedding is indicated.
Bedding should be used in amounts sufficient to keep animals dry
between cage changes, and, in the case of small laboratory animals, it
should be kept from coming into contact with sipper tubes as such contact
could cause leakage of water into the cage.
Sanitation Sanitation—the maintenance of environmental conditions conducive to health and well-being—involves bedding change (as appropriate),cleaning, and disinfection. Cleaning removes excessive amounts of excrement, dirt, and debris, and disinfection reduces or eliminates unacceptableconcentrations of microorganisms. The goal of any sanitation program is tomaintain sufficiently clean and dry bedding, adequate air quality, and clean
cage surfaces and accessories.
The frequency and intensity of cleaning and disinfection should depend
on what is necessary to provide a healthy environment for an animal. Methods and frequencies of sanitation will vary with many factors, including thenormal physiologic and behavioral characteristics of the animals; the type,physical characteristics, and size of the enclosure; the type, number, size,
age, and reproductive status of the animals; the use and type of bedding
materials; temperature and relative humidity; the nature of the materials that0

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

create the need for sanitation; and the rate of soiling of the surfaces of theenclosure. Some housing systems or experimental protocols may require
specific husbandry techniques, such as aseptic handling or modification in
the frequency of bedding change.
Agents designed to mask animal odors should not be used in animal
housing facilities. They cannot substitute for good sanitation practices or forthe provision of adequate ventilation, and they expose animals to volatilecompounds that might alter basic physiologic and metabolic processes.
bedding/Substrate Change Soiled bedding should be removed andreplaced with fresh materials as often as necessary to keep the animals
clean and dry and to keep pollutants, such as ammonia, at a concentration
below levels irritating to mucous membranes. The frequency of bedding
change depends on multiple factors, such as species, number, and sizeof the animals in the primary enclosure; type and size of the enclosure;macro- and microenvironmental temperature, relative humidity, and direct
ventilation of the enclosure; urinary and fecal output and the appearance
and wetness of bedding; and experimental conditions, such as those ofsurgery or debilitation, that might limit an animal’s movement or access toclean bedding. There is no absolute minimal frequency of bedding changes;
the choice is a matter of professional judgment and consultation between
the investigator and animal care personnel. It typically varies from daily toweekly. In some instances frequent bedding changes are contraindicated;
examples include portions of the pre- or postpartum period, research objectives that will be affected, and species in which scent marking is critical andsuccessful reproduction is pheromone dependent.
Cleaning and disinfection of the Microenironment The frequency ofsanitation of cages, cage racks, and associated equipment (e.g., feeders andwatering devices) is governed to some extent by the types of caging andhusbandry practices used, including the use of regularly changed contact
or noncontact bedding, regular flushing of suspended catch pans, and theuse of wire-bottom or perforated-bottom cages. In general, enclosures andaccessories, such as tops, should be sanitized at least once every 2 weeks.
Solid-bottom caging, bottles, and sipper tubes usually require sanitation atleast once a week. Some types of cages and housing systems may requireless frequent cleaning or disinfection; such housing may include large cageswith very low animal density and frequent bedding changes, cages containing animals in gnotobiotic conditions with frequent bedding changes,
individually ventilated cages, and cages used for special situations. Other
circumstances, such as filter-topped cages without forced-air ventilation,
animals that urinate excessively (e.g., diabetic or renal patients), or densely
populated enclosures, may require more frequent sanitation.
The increased use of individually ventilated cages (IVCs) for rodents
has led to investigations of the maintenance of a suitable microenvironment
with extended cage sanitation intervals and/or increased housing densi-

ENVIRONMENT, hOUSING, ANd MANAGEMENT

1

ties (Carissimi et al. 2000; Reeb-Whitaker et al. 2001; Schondelmeyer et
al. 2006). By design, ventilated caging systems provide direct continuous
exchange of air, compared to static caging systems that depend on passive ventilation from the macroenvironment. As noted above, decreased
sanitation frequency may be justified if the microenvironment in the cages,
under the conditions of use (e.g., cage type and manufacturer, bedding,species, strain, age, sex, density, and experimental considerations), is notcompromised (Reeb et al. 1998). Verification of microenvironmental conditions may include measurement of pollutants such as ammonia and CO2,
microbiologic load, observation of the animals’ behavior and appearance,
and the condition of bedding and cage surfaces.
Primary enclosures can be disinfected with chemicals, hot water, or a
combination of both.2 Washing times and conditions and postwashing processing procedures (e.g., sterilization) should be sufficient to reduce levels
or eliminate vegetative forms of opportunistic and pathogenic bacteria,
adventitious viruses, and other organisms that are presumed to be controllable by the sanitation program. Disinfection from the use of hot water
alone is the result of the combined effect of the temperature and the lengthof time that a given temperature (cumulative heat factor) is applied to thesurface of the item. The same cumulative heat factor can be obtained byexposing organisms either to very high temperatures for short periods or tolower temperatures for longer periods (Wardrip et al. 1994, 2000). Effective disinfection can be achieved with wash and rinse water at 143-180°F
or more. The traditional 82.2°C (180°F) temperature requirement for rinse
water refers to the water in the tank or in the sprayer manifold. Detergentsand chemical disinfectants enhance the effectiveness of hot water but
should be thoroughly rinsed from surfaces before reuse of the equipment.
Their use may be contraindicated for some aquatic species, as residue may
be highly deleterious. Mechanical washers (e.g., cage and rack, tunnel,
and bottle washers) are recommended for cleaning quantities of caging andmovable equipment.
Sanitation of cages and equipment by hand with hot water and detergents or disinfectants can also be effective but requires considerable attention to detail. It is particularly important to ensure that surfaces are rinsed
free of residual chemicals and that personnel have appropriate equipment
to protect themselves from exposure to hot water or chemical agents usedin the process.
Water bottles, sipper tubes, stoppers, feeders, and other small pieces ofequipment should be washed with detergents and/or hot water and, where

2 Rabbits and some rodents, such as guinea pigs and hamsters, produce urine with highconcentrations of proteins and minerals. These compounds often adhere to cage surfaces andnecessitate treatment with acid solutions before and/or during washing.

2

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

appropriate, chemical agents to destroy microorganisms. Cleaning withultrasound may be a useful method for small pieces of equipment.
If automated watering systems are used, some mechanism to ensurethat microorganisms and debris do not build up in the watering devices isrecommended (Meier et al. 2008); the mechanism can be periodic flushingwith large volumes of water or appropriate chemical agents followed by athorough rinsing. Constant recirculation loops that use properly maintained
filters, ultraviolet lights, or other devices to disinfect recirculated water are
also effective. Attention should be given to the routine sanitation of automatic water delivery valves (i.e., lixits) during primary enclosure cleaning.
Conventional methods of cleaning and disinfection are adequate formost animal care equipment. However, it may be necessary to also sterilize
caging and associated equipment to ensure that pathogenic or opportunistic
microorganisms are not introduced into specific-pathogen-free or immunocompromised animals, or that experimental biologic hazards are destroyed
before cleaning. Sterilizers should be regularly evaluated and monitored toensure their safety and effectiveness.
For pens or runs, frequent flushing with water and periodic use of detergents or disinfectants are usually appropriate to maintain sufficiently cleansurfaces. If animal waste is to be removed by flushing, this will need to bedone at least once a day. During flushing, animals should be kept dry. The
timing of pen or run cleaning should take into account the normal behavioral
and physiologic processes of the animals; for example, the gastrocolic reflex
in meal-fed animals results in defecation shortly after food consumption.
Cleaning and disinfection of the Macroenironment All components of
the animal facility, including animal rooms and support spaces (e.g., storage
areas, cage-washing facilities, corridors, and procedure rooms) should be
regularly cleaned and disinfected as appropriate to the circumstances andat a frequency based on the use of the area and the nature of likely contamination. Vaporized hydrogen peroxide or chlorine dioxide are effective
compounds for room decontamination, particularly following completion ofstudies with highly infectious agents (Krause et al. 2001) or contamination
with adventitious microbial agents.
Cleaning implements should be made of materials that resist corrosion
and withstand regular sanitation. They should be assigned to specific areasand should not be transported between areas with different risks of contamination without prior disinfection. Worn items should be replaced regularly.
The implements should be stored in a neat, organized fashion that facilitates
drying and minimizes contamination or harborage of vermin.
Assessing the Effectieness of Sanitation Monitoring of sanitation practices should fit the process and materials being cleaned and may include
visual inspection and microbiologic and water temperature monitoring(Compton et al. 2004a,b; Ednie et al. 1998; Parker et al. 2003). The intensityof animal odors, particularly that of ammonia, should not be used as theENVIRONMENT, hOUSING, ANd MANAGEMENT

3sole means of assessing the effectiveness of the sanitation program. A decision to alter the frequency of cage bedding changes or cage washing should
be based on such factors as ammonia concentration, bedding condition,
appearance of the cage and animals, and the number and size of animals
housed in the cage.
Mechanical washer function should be evaluated regularly and include
examination of mechanical components such as spray arms and moving
headers as well as spray nozzles to ensure that they are functioning appropriately. If sanitation is temperature dependent, the use of temperature-sensing devices (e.g., thermometers, probes, or temperature-sensitive indicator
strips) is recommended to ensure that the equipment being sanitized is
exposed to the desired conditions.
Whether the sanitation process is automated or manual, regular evaluation of sanitation effectiveness is recommended. This can be performedby evaluating processed materials by microbiologic culture or the use oforganic material detection systems (e.g., adenosine triphosphate [ATP] bioluminescence) and/or by confirming the removal of artificial soil applied toequipment surfaces before washing.
Waste disposal Conventional, biologic, and hazardous waste should be
removed and disposed of regularly and safely (Hill 1999). There are several
options for effective waste disposal. Contracts with licensed commercial
waste disposal firms usually provide some assurance of regulatory compliance and safety. On-site incineration should comply with all federal, state,
and local regulations (Nadelkov 1996).
Adequate numbers of properly labeled waste receptacles should be
strategically placed throughout the facility. Waste containers should be leakproof and equipped with tight-fitting lids. It is good practice to use disposable liners and to wash containers and implements regularly. There should
be a dedicated waste storage area that can be kept free of insects and othervermin. If cold storage is used to hold material before disposal, a properly
labeled, dedicated refrigerator, freezer, or cold room should be used that isreadily sanitized.
Hazardous wastes must be rendered safe by sterilization, containment,or other appropriate means before their removal from the facility (DHHS
2009 or most recent edition; NRC 1989, 1995b). Radioactive wastes should
be kept in properly labeled containers and their disposal closely coordinated with radiation safety specialists in accord with federal and state
regulations; the federal government and most states and municipalities
have regulations controlling disposal of hazardous wastes. Compliance withregulations concerning hazardous-agent use (see Chapter 2) and disposal isan institutional responsibility.
Infectious animal carcasses can be incinerated on site or collected bya licensed contractor. Use of chemical digesters (alkaline hydrolysis treat-4

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

ment) may be considered in some situations (Kaye et al. 1998; Murphy etal. 2009). Procedures for on-site packaging, labeling, transportation, andstorage of these wastes should be integrated into occupational health andsafety policies (Richmond et al. 2003).
Hazardous wastes that are toxic, carcinogenic, flammable, corrosive,
reactive, or otherwise unstable should be placed in properly labeled containers and disposed of as recommended by occupational health and safety
specialists. In some circumstances, these wastes can be consolidated or
blended. Sharps and glass should be disposed of in a manner that will prevent injury to waste handlers.
Pest Control Programs designed to prevent, control, or eliminate the presence of or infestation by pests are essential in an animal environment. A
regularly scheduled and documented program of control and monitoringshould be implemented. The ideal program prevents the entry of vermin andeliminates their harborage in the facility (Anadon et al. 2009; Easterbrook et
al. 2008). For animals in outdoor facilities, consideration should be given
to eliminating or minimizing the potential risk associated with pests andpredators.
Pesticides can induce toxic effects on research animals and interferewith experimental procedures (Gunasekara et al. 2008). They should be
used in animal areas only when necessary and investigators whose animals
may be exposed to them should be consulted beforehand. Use of pesticides
should be recorded and coordinated with the animal care management staff
and be in compliance with federal, state, or local regulations. Wheneverpossible, nontoxic means of pest control, such as insect growth regulators(Donahue et al. 1989; Garg and Donahue 1989; King and Bennett 1989;
Verma 2002) and nontoxic substances (e.g., amorphous silica gel), should
be used. If traps are used, methods should be humane; traps that catch pests
alive require frequent observation and humane euthanasia after capture(Mason and Littin 2003; Meerburg et al. 2008).
Emergency, Weekend, and holiday Care Animals should be cared for by
qualified personnel every day, including weekends and holidays, both tosafeguard their well-being and to satisfy research requirements. Emergency
veterinary care must be available after work hours, on weekends, and on
holidays.
In the event of an emergency, institutional security personnel and fire orpolice officials should be able to reach people responsible for the animals.
Notification can be enhanced by prominently posting emergency procedures, names, or telephone numbers in animal facilities or by placing themin the security department or telephone center. Emergency procedures forhandling special facilities or operations should be prominently posted andpersonnel trained in emergency procedures for these areas. A disaster plan

ENVIRONMENT, hOUSING, ANd MANAGEMENT

5

that takes into account both personnel and animals should be prepared aspart of the overall safety plan for the animal facility. The colony manager orveterinarian responsible for the animals should be a member of the appropriate safety committee at the institution, an “official responder” in the institution, and a participant in the response to a disaster (Vogelweid 1998).
Population Management
Identification Animal records are useful and variable, ranging from limited information on identification cards to detailed computerized records
for individual animals (Field et al. 2007). Means of animal identification
include room, rack, pen, stall, and cage cards with written, bar-coded,or radio frequency identification (RFID) information. Identification cards
should include the source of the animal, the strain or stock, names andcontact information for the responsible investigator(s), pertinent dates (e.g.,arrival date, birth date, etc.), and protocol number when applicable. Genotype information, when applicable, should also be included, and consistent,
unambiguous abbreviations should be used when the full genotype nomenclature (see below) is too lengthy.
In addition, the animals may wear collars, bands, plates, or tabs or bemarked by colored stains, ear notches/punches and tags, tattoos, subcutaneous transponders, and freeze brands. As a method of identification of smallrodents, toe-clipping should be used only when no other individual identification method is feasible. It may be the preferred method for neonatal mice
up to 7 days of age as it appears to have few adverse effects on behavior
and well-being at this age (Castelhano-Carlos et al. 2010; Schaefer et al.
2010), especially if toe clipping and genotyping can be combined. Under
all circumstances aseptic practices should be followed. Use of anesthesia or
analgesia should be commensurate with the age of the animals (Hankenson
et al. 2008).
Recordkeeping Records containing basic descriptive information are essential
for management of colonies of large long-lived animals and should be maintained for each animal (Dyke 1993; Field et al. 2007; NRC 1979a). These
records often include species, animal identifier, sire and/or dam identifier, sex,
birth or acquisition date, source, exit date, and final disposition. Such animal
records are essential for genetic management and historical assessments ofcolonies. Records of rearing and housing histories, mating histories, andbehavioral profiles are useful for the management of many species, especiallynonhuman primates (NRC 1979a). Relevant recorded information should be
provided when animals are transferred between institutions.
Medical records for individual animals can also be valuable, especially fordogs, cats, nonhuman primates, and agricultural animals (Suckow and Doerning 2007). They should include pertinent clinical and diagnostic information,

6

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

date of inoculations, history of surgical procedures and postoperative care,information on experimental use, and necropsy findings where applicable.
Basic demographic information and clinical histories enhance the valueof individual animals for both breeding and research and should be readily
accessible to investigators, veterinary staff, and animal care staff.
breeding, Genetics, and Nomenclature Genetic characteristics are important with regard to the selection and management of animals for use inbreeding colonies and in biomedical research (see Appendix A). Pedigree
information allows appropriate selection of breeding pairs and of experimental animals that are unrelated or of known relatedness.
Outbred animals are widely used in biomedical research. Founding
populations should be large enough to ensure the long-term genetic heterogeneity of breeding colonies. To facilitate direct comparison of researchdata derived from outbred animals, genetic management techniques should
be used to maintain genetic variability and equalize founder representations(Hartl 2000; Lacy 1989; Poiley 1960; Williams-Blangero 1991). Genetic
variability can be monitored with computer simulations, biochemical markers, DNA markers and sequencing, immunologic markers, or quantitative
genetic analyses of physiologic variables (MacCluer et al. 1986; WilliamsBlangero 1993).
Inbred strains of various species, especially rodents, have been developed to address specific research needs (Festing 1979; Gill 1980). Wheninbred animals or their F1 progeny are used, it is important to periodicallymonitor genetic authenticity (Festing 1982; Hedrich 1990); several methodsof monitoring have been developed that use immunologic, biochemical,
and molecular techniques (Cramer 1983; Festing 2002; Groen 1977; Hoffman et al. 1980; Russell et al. 1993). Appropriate management systems(Green 1981; Kempthorne 1957) should be designed to minimize genetic
contamination resulting from mutation and mismating.
Genetically modified animals (GMAs) represent an increasingly large
proportion of animals used in research and require special considerationin their population management. Integrated or altered genes can interact
with species or strain-specific genes, other genetic manipulations, andenvironmental factors, in part as a function of site of integration, so each
GMA line can be considered a unique resource. Care should be taken topreserve such resources through standard genetic management procedures,
including maintenance of detailed pedigree records and genetic monitoring to verify the presence and zygosity of transgenes and other genetic
modifications (Conner 2005). Cryopreservation of fertilized embryos, ova,
ovaries, or spermatozoa should also be considered as a safeguard against
alterations in transgenes over time or accidental loss of GMA lines (Conner
2002; Liu et al. 2009).

ENVIRONMENT, hOUSING, ANd MANAGEMENTGeneration of animals with multiple genetic alterations often involves
crossing different GMA lines and can lead to the production of offspringwith genotypes that are not of interest to the researcher (either as experimental or control animals) as well as unexpected phenotypes. Carefully
designed breeding strategies and accurate genotype assessment can help
to minimize the generation of animals with unwanted genotypes (Linder
2003). Newly generated genotypes should be carefully monitored andnew phenotypes that negatively affect well-being should be reported to
the IACUC and managed in a manner to ensure the animals’ health andwell-being.
Accurate recording, with standardized nomenclature when available,of both the strain and substrain or of the genetic background of animals
used in a research project is important (NRC 1979b). Several publications provide rules developed by international committees for standardized
nomenclature of outbred rodents and rabbits (Festing et al. 1972), inbred
rats, inbred mice, and transgenic animals (FELASA 2007; Linder 2003). In
addition, the International Committee on Standardized Genetic Nomenclature for Mice and the Rat Genome and Nomenclature Committee maintain
online guidelines for these species (MGI 2009).
AQUATIC ANIMALSThe variety of needs for fish and aquatic or semiaquatic reptiles andamphibians is as diverse as the number of species considered. This section isintended to provide facility managers, veterinarians, and IACUCs with basic
information related to the management of aquatic animal systems (Alworthand Harvey 2007; Alworth and Vazquez 2009; Browne et al. 2007; Browne
and Zippel 2007; Denardo 1995; DeTolla et al. 1995; Koerber and Kalishman 2009; Lawrence 2007; Matthews et al. 2002; Pough 2007). Specificrecommendations are available in texts and journal reviews, and it will benecessary to review other literature and consult with experienced caregivers
for further detail on caring for aquatic species (see Appendix A).
Aquatic Environment
Microenironment and Macroenironment
As with terrestrial systems, the microenironment of an aquatic animal
is the physical environment immediately surrounding it—the primary enclosure such as the tank, raceway, or pond. It contains all the resources withwhich the animals are in direct contact and also provides the limits of theanimals’ immediate environment. The microenvironment is characterizedby many factors, including water quality, illumination, noise, vibration, and8

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

temperature. The physical environment of the secondary enclosure, such asa room, constitutes the macroenironment.
Water Quality
The composition of the water (water quality) is essential to aquatic animal well-being, although other factors that affect terrestrial microenvironments are also relevant. Water quality parameters and life support systems
for aquatic animals will vary with the species, life stage, the total biomass
supported, and the animals’ intended use (Blaustein et al. 1999; Fisher
2000; Gresens 2004; Overstreet et al. 2000; Schultz and Dawson 2003).
The success and adequacy of the system depend on its ability to match thelaboratory habitat to the natural history of the species (Godfrey and Sanders
2004; Green 2002; Lawrence 2007; Spence et al. 2008).
Characteristics of the water that may affect its appropriateness include
temperature, pH, alkalinity, nitrogen waste products (ammonia, nitrite,
and nitrate), phosphorus, chlorine/bromine, oxidation-reduction potential,
conductivity/salinity, hardness (osmolality/dissolved minerals), dissolved
oxygen, total gas pressure, ion and metal content, and the establishedmicrobial ecology of the tank. Water quality parameters can directly affectanimal well-being; different classes, species, and ages in a species may havedifferent water quality needs and sensitivities to changes in water quality
parameters.
Routine measurement of various water characteristics (water quality
testing) is essential for stable husbandry. Standards for acceptable water
quality, appropriate parameters to test, and testing frequency should be
identified at the institutional level and/or in individual animal use protocols
depending on the size of the aquatic program. Staff managing aquatic systems need to be trained in biologically relevant aspects of water chemistry,
how water quality parameters may affect animal health and well-being,
how to monitor water quality results, and how water quality may affect life
support system function (e.g., biologic filtration).
The specific parameters and frequency of testing vary widely (depending on the species, life stage, system, and other factors), from continuous
monitoring to infrequent spot checks. Recently established systems and/or
populations, or changes in husbandry procedures, may require more frequent assessment as the system ecology stabilizes; stable environments may
require less frequent testing. Toxins from system components, particularly
in newly constructed systems, may require special consideration such asleaching of chemicals from construction materials, concrete, joint compounds, and sealants (DeTolla et al. 1995; Nickum et al. 2004). Chlorineand chloramines used to disinfect water for human consumption or todisinfect equipment are toxic to fish and amphibians and must be removed

ENVIRONMENT, hOUSING, ANd MANAGEMENTor neutralized before use in aquatic systems (Tompkins and Tsai 1976;
Wedemeyer 2000).
Life Support System
The phrase life support system refers to the physical structure used tocontain the water and the animals as well as the ancillary equipment used
to move and/or treat the water. Life support systems may be simple (e.g.,a container to hold the animal and water) or extremely complex (e.g., afully automated recirculating system). The type of life support system used
depends on several factors including the natural habitat of the species,
age/size of the species, number of animals maintained, availability andcharacteristics of the water required, and the type of research.
Life support systems typically fall into three general categories: recirculating systems where water (all or part) is moved around a system, flowthrough systems where water is constantly replaced, or static systems wherewater is stationary and periodically replenished or replaced. The water
may be fresh, brackish, or salt and is maintained at specific temperatures
depending on the species’ needs.
The source of water for these systems typically falls into four generalcategories: treated wastewater (e.g., municipal tap water), surface water(e.g., rivers, lakes, or oceans), protected water (e.g., well or aquifer water),or artificial water (e.g., reverse osmosis or distilled water). Artificial saltwater
may be created by adding appropriate salt to freshwater sources. Source
water selection should be based on the provision of a consistent or constant
supply, incoming biosecurity level requirements, water volumes needed,
species selection, and research considerations.
Recirculating systems are common in indoor research settings where
high-density housing systems are often needed. Most recirculating systems
are designed to exchange a specific volume of water per unit time and periodically introduce fresh water into the system. These systems are the mostmechanically advanced, containing biologic filters (biofilters) that promote
conversion of ammonia to nitrite and nitrate via nitrifying bacteria, protein
skimmers (foam fractionators) and particulate filters to remove undissolved
and dissolved proteins and particulate matter, carbon filters to remove
dissolved chemicals, and ultraviolet or ozone units to disinfect the water.
The systems generally contain components to aerate and degas the water(to prevent gas oversaturation) and to heat or cool it, as well as automateddosing systems to maintain appropriate pH and conductivity. Not all elements are present in all systems and some components may accomplish
multiple functions. Recirculating systems may be designed so that multiple
individual tanks are supplied with treated water from a single source, as is
the case with “rack” systems used for zebrafish (danio rerio) and Xenopus

80

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

laeis and X. tropicalis, as examples (Fisher 2000; Koerber and Kalishman
2009; Schultz and Dawson 2003).
The development and maintenance of the biofilter is critical for limiting
ammonia and nitrite accumulation in recirculating systems. The biofilter
must be of sufficient size (i.e., contain a sufficient quantity of bacteria) to
be capable of processing the bioload (level of nitrogenous waste) entering
the system. The microorganisms supported by the biofilter require certain
water quality parameters. Alterations in the aquatic environment (e.g., rapidchanges in salinity, temperature, and pH) as well as the addition of chemicals or antimicrobials may significantly affect the microbial ecology of thesystem and therefore water quality and animal well-being. If damaged,
biofilter recovery may take weeks (Fisher 2000). Changes in water quality
parameters (e.g., pH, ammonia, and nitrite) may negatively affect animal
health and the efficiency of the biofilter, so species sensitive to change inwater quality outside of a narrow range require more frequent monitoring.
Continuous or timed flow-through systems can be used where suitablewater is available to support the species to be housed (e.g., in aquaculture
facilities). These systems may use extremely large volumes of water as it
is not reused. The water may be used “as is” or processed before use, forexample by removing sediments, excessive dissolved gases, chlorine, or
chloramines, and by disinfecting with UV or ozone (Fisher 2000; Overstreet
et al. 2000). Static systems vary in size from small tanks to large ingroundponds, and may use mechanical devices to move and aerate water.
Temperature, humidity, and Ventilation
The general concepts discussed in the Terrestrial Animals section also
apply to the aquatic setting. Most aquatic or semiaquatic species (fish,
amphibians, and reptiles) used in research are poikilotherms, which depend,
for the most part, on the temperature of their environment to sustain physiologic processes, such as metabolism, reproduction, and feeding behavior(Browne and Edwards 2003; Fraile et al. 1989; Maniero and Carey 1997;
Pough 1991). Temperature requirements are based on the natural historyof the species and can vary depending on life stage (Green 2002; Pough
1991; Schultz and Dawson 2003). Water temperature may be controlled atits source, within the life support system, or by controlling the macroenvironment. Some semiopen systems (e.g., raceways by a river) depend on
source water temperature and thus enclosure water temperature will varywith that of the source water.
The volume of water contained in a room can affect room temperature, temperature stability, and relative humidity. Likewise the thermal load
produced by chiller/heater systems can affect the stability of the macroenvironmental temperature. Air handling systems need to be designed to com-

ENVIRONMENT, hOUSING, ANd MANAGEMENT

81

pensate for these thermal and moisture loads. Macroenvironmental relative
humidity levels are generally defined by safety issues and staff comfort,
since room humidity is not critical for aquatic species; however, excessive
moisture may result in condensation on walls, ceilings, and tank lids, which
may support microbial growth and serve as a source of contamination or
create a conducive environment for metal corrosion. In a dry environment(e.g., indoor heating during cold weather or outdoor housing in someclimates/seasons), evaporation rates may be higher, potentially requiring
the addition of large quantities of water to the system and monitoringfor increases in salinity/conductivity, contaminants, or other water quality
aberrations. Some amphibians and reptiles may need elevated microenvironmental humidity (in excess of 50-70% relative humidity), which may
require maintaining elevated macroenvironmental humidity levels (Pough
1991; St. Claire et al. 2005).
Room air exchange rates are typically governed by thermal and moisture loads. For fish and some aquatic amphibians, the microenvironmental
air quality may affect water quality (i.e., gas exchange), but appropriate life
support system design may reduce its importance. Airborne particulates andcompounds (e.g., volatile organic compounds and ammonia) may dissolve
in tank water and affect animal health (Koerber and Kalishman 2009). As theaerosolization of water can lead to the spread of aquatic animal pathogens(e.g., protozoa, bacteria) within or throughout an aquatic animal facility, thisprocess should be minimized as much as possible (Roberts-Thomson et al.
2006; Wooster and Bowser 2007; Yanong 2003).
Illumination
Aquatic and semiaquatic species are often sensitive to changes inphotoperiod, light intensity, and wavelength (Brenner and Brenner 1969).
Lighting characteristics will vary by species, their natural history, and theresearch being conducted. Gradual changes in room light intensity are recommended, as rapid changes in light intensity can elicit a startle response
in fish and may result in trauma. Some aquatic and semiaquatic species
may need full-spectrum lighting and/or heat lamps to provide supplemental
heating to facilitate adequate physiological function (e.g., aquatic turtles
provided with a basking area; Pough 1991).
Noise and Vibration
General concepts discussed in the Terrestrial Animals section apply
to aquatic animals. These animals may be sensitive to noise and vibration, which are readily transmitted through water. Species vary in their
response and many fish species acclimate to noise and vibration, although

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GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

these may cause subclinical effects (Smith et al. 2007). Vibration throughfloors can be reduced by using isolation pads under aquaria racks. Somefacilities elect to place major components of the life support system (e.g.,filters, pumps, and biofilters) outside the animal rooms to reduce vibrationand noise.
Aquatic Housing
Microenironment (Primary Enclosure)
The primary enclosure (a tank, raceway, pond, or pen holding waterand the animal) defines the limits of an animal’s immediate environment.
In research settings, acceptable primary enclosures
•

•
•
•
•
•
•
•
•
•

allow for the normal physiological and behavioral needs of theanimals, including excretory function, control and maintenanceof body temperature, normal movement and postural adjustments,
and, where indicated, reproduction. In some poikilothermic reptiles
and amphibians, microenvironmental temperature gradients may
be needed for certain physiologic functions such as feeding anddigestion.
allow conspecific social interactions (e.g., schooling in fish
species).
provide a balanced, stable environment that supports the animal’s
physiologic needs.
provide the appropriate water quality and characteristics, and permit monitoring, filling, refilling, and changing of water.
allow access to adequate food and removal of food waste.
restrict escape or accidental entrapment of animals or their
appendages.
are free of sharp edges and/or projections that could cause injury.
allow for observation of the animals with minimal disturbance.
are constructed of nontoxic materials that do not leach toxicants or
chemicals into the aquatic environment.
do not present electrical hazards directly or indirectly.

Enironmental Enrichment and Social housing
Environmental enrichment strategies for many aquatic species are notwell established. The implications of a barren versus an enriched environment on well-being, general research, growth, and development are
unknown or poorly defined, as is true of individual versus group (social)

ENVIRONMENT, hOUSING, ANd MANAGEMENT

83

housing for many species. When used, enrichment should elicit speciesappropriate behaviors and be evaluated for safety and utility.
Generally, schooling fish species are housed with conspecifics, andmany amphibians, especially anuran species, may be group housed. Aggression in aquatic animals does occur (van de Nieuwegiessen et al. 2008;
Speedie and Gerlai 2008) and, as for terrestrial animals, appropriate monitoring and intervention may be necessary (Matthews et al. 2002; Torreilles
and Green 2007). Some species need appropriate substrate (e.g., gravel) to
reproduce or need substrate variety to express basic behaviors and maintain health (Overstreet et al. 2000). Improved breeding success in enrichedenvironments has been reported but further research in this area is needed(Carfagnini et al. 2009). For many species (including, e.g., X. laeis), visualbarriers, hides, and shading are appropriate (Alworth and Vasquez 2009;
Torreilles and Green 2007). Most semiaquatic reptiles spend some timeon land (basking, feeding, digesting, and ovipositing) and terrestrial areas
should be provided as appropriate.
Sheltered, Outdoor, and Naturalistic housing
Animals used in aquaculture are often housed in situations that mimic
agricultural rearing and may be in outdoor and/or sheltered raceways,
ponds, or pens with high population densities. In these settings, where
natural predation and mortalities occur, it may be appropriate to measure
animal “numbers” by using standard aquaculture techniques such as final
production biomass (Borski and Hodson 2003).
Space
Space recommendations and housing density vary extensively with thespecies, age/size of the animals, life support system, and type of research(Browne et al. 2003; Green 2009; Gresens 2004; Hilken et al. 1995;
Matthews et al. 2002). In the United States, for example, adult zebrafish(danio rerio) in typical biomedical research settings are generally housed 5
adult fish per liter of water (Matthews et al. 2002), but this housing density
varies when breeding and for housing younger animals (Matthews et al.
2002). This guidance is not necessarily relevant for other species of fish, andmay change as research advances (Lawrence 2007). X. laeis adults may
be housed at 2 liters of water per frog (NRC 1974), but a wide variety of
housing systems are currently used in research settings (Green 2009). Institutions, investigators, and IACUC members should evaluate the appropriate
needs of each species during program evaluations and facility inspections
and continue to review ongoing research in these areas.

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GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

Aquatic Managementbehaior and Social Management
Visual evaluations of aquatic and semiaquatic animals are typicallyused for monitoring. To avoid damage to the protective mucus layers of theskin and negative effects on immune function (De Veer et al. 2007; Subramanian et al. 2007; Tsutsui et al. 2005), handling of these species should
be kept to the minimum required (Bly et al. 1997). Appropriate handlingtechniques vary widely depending on the species, age/size, holding system,and specific research need (Fisher 2000; Matthews et al. 2002; Overstreet
et al. 2000); they should be identified at the facility or individual protocol
level.
Latex gloves have been associated with toxicity in some amphibians(Gutleb et al. 2001). The use of appropriate nets by well-trained personnel can reduce skin damage and thus stress. Nets should be cleaned anddisinfected appropriately when used in different systems and should be
dedicated to animals of similar health status whenever possible.
Exercise and activity levels for aquatic species are minimally describedbut informed decisions may be extrapolated from studies of behavior of
the same or similar species in the wild (Spence et al. 2008). Some aquatic
species do not rest and constantly swim; others may rest all or a significant
portion of the day. Water flow rates and the provision of hides or terrestrial
resting platforms (e.g., for some reptiles and amphibians) need to be appropriate for species and life stage.
husbandry
Food The general principles relating to feeding of terrestrial animals areapplicable to aquatic animals. Food should be stored in a type-appropriatemanner to preserve nutritional content, minimize contamination, and prevent entry of pests. Food delivery methods should ensure that all animals areable to access food for a sufficient period of time while minimizing feeding
aggression and nutrient loss. Feeding methods and frequency vary widely
depending on the species, age/size of species, and type of life support system. Many aquatic or semiaquatic species are not provided with food adlibitum in the tank, and in some cases may not be fed daily.
Commercial diets (e.g., pellets, flakes) are available for certain species
and storage time should be based on manufacturer recommendations or follow commonly accepted practices. In aquatic systems, particularly in fish
rearing or when maintaining some amphibian and reptile species, the useof live foods (e.g., Artemia sp. larva, crickets, or mealworm beetle larvae) is
common. Live food sources need to be maintained and managed to ensure a

ENVIRONMENT, hOUSING, ANd MANAGEMENT

85

steady supply and the health and suitability of the organism as a food. Careshould be taken to feed a complete diet to avoid nutritional deficiencies.
Water (see also section on Water Quality) Aquatic animals need access toappropriately conditioned water. Fully aquatic animals obtain water in theirhabitat or absorb it across their gills or skin. Some semiaquatic amphibians
and reptiles may need “bowls” of water for soaking and drinking, and water
quality should be appropriate (see Terrestrial Animals section). Chlorine orchloramines may be present in tap water at levels that could be toxic tosome species.
Substrate Substrates can provide enrichment for aquatic animals by promoting species-appropriate behavior such as burrowing, foraging, or enhanced
spawning (Fisher 2000; Matthews et al. 2002; Overstreet et al. 2000). They
may be an integral and essential component of the life support system byproviding increased surface area for denitrifying bacteria (e.g., systems with
undergravel filtration), and need routine siphoning (i.e., hydrocleaning) toremove organic debris. System design and species needs should be evaluated to determine the amount, type, and presentation of substrate.
Sanitation Sanitation of the aquatic environment in recirculating systems
is provided through an appropriately designed and maintained life support
system, regular removal of solid waste materials from the enclosure bottom,
and periodic water changes. The basic concept of sanitation (i.e., to provide
conditions conducive to animal health and welfare) is the same for terrestrial and aquatic systems. However, sanitation measures in aquatic systems
differ from those for terrestrial systems because much of the nitrogenous
waste (feces and urine) and respiratory output (carbon dioxide) is dissolvedin the water.
A properly functioning life support system, designed to process the
bioload, will maintain nitrogenous wastes within an acceptable range.
Solids may be removed in a variety of ways, depending on the design ofthe system; generally they are removed by siphoning (hydrocleaning) and/or
filtration. Depending on the type, filters need routine cleaning or replacement or, if self-cleaning, proper maintenance; in saltwater systems dissolved
proteins may be removed by protein skimmers. Reducing organic solids limits the quantities of nitrogen and phosphorus that need to be removed fromthe system, both of which can accumulate to levels that are toxic to fish
and amphibians. The biologic filter (denitrifying bacteria) typically removes
ammonia and nitrite, potential toxins, from aquatic systems. Nitrate, the end
product of this process, is less toxic to aquatic animals but at high levels canbe problematic; it is generally removed through water changes, althoughlarge systems may have a specialized denitrification unit to reduce levels.

86

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALSDisinfection is usually accomplished through water treatment (e.g.,filtration and application of UV light or ozone) and/or water changes. Chlorine and most chemical disinfectants are inappropriate for aquatic systems
containing animals as they are toxic at low concentrations; when used todisinfect an entire system or system components, extreme care must be
taken to ensure that residual chlorine, chemical, and reactive byproducts
are neutralized or removed. The type of monitoring and frequency varies
depending on the disinfection method, the system, and the animals.
Algal growth is common in aquatic systems and increases with thepresence of nitrogen and phosphorus, particularly in the presence of light.
Excessive growth may be an indication of elevated nitrogen or phosphorus
levels. Algal species seen with recirculating systems are generally nontoxic,
although species capable of producing toxins exist. Algae are typicallyremoved using mechanical methods (i.e., scrubbing or scraping). Limiting
algal growth is important to allow viewing of the animals in the enclosure.
Cyanobacteria (commonly called blue-green algae) growth is also possible
and may be common in freshwater aquaculture. The same factors that promote algae growth also promote cyanobacteria growth. As with algae, whilemost species are harmless, some species can produce clinically relevant
toxic compounds (Smith et al. 2008).
Tank (cage) changing and disinfection are conducted at frequencies using
methods that often differ from terrestrial systems. Because waste is dissolvedin the water and/or removed as solids by siphoning or filtration, regularchanging of tanks is not integral to maintaining adequate hygiene in typicalaquatic systems. The frequency of cleaning and disinfection should be determined by water quality, which should permit adequate viewing of the animals, and animal health monitoring. System components such as lids on fishtanks, which may accumulate feed, may require sanitation as often as weekly
depending on the frequency and type of feed and the system’s design.
Cleaning and disinfection of the Macroenironment As with terrestrial
systems, all components of the animal facility, including animal rooms andsupport spaces (e.g., storage areas, cage-washing facilities, corridors, andprocedure rooms), should be regularly cleaned and disinfected as appropriate to the circumstances and at a frequency determined by the use of
the area and the nature of likely contamination. Cleaning agents should bechosen and used with care to ensure there is no secondary contaminationof the aquatic systems.
Cleaning implements should be made of materials that resist corrosion
and withstand regular sanitation. They should be assigned to specific areasand should not be transported between areas with different risks of contamination without prior disinfection. Worn items should be replaced regularly.
The implements should be stored in a neat, organized fashion that facilitates
drying and minimizes contamination or harborage of vermin.

ENVIRONMENT, hOUSING, ANd MANAGEMENT

8

Waste disposal Wastewater treatment and disposal may be necessary insome facilities depending on water volume, quality, and chemical constituents. Local regulations may limit or control the release of wastewater.
Pest Control Terrestrial animal pest control principles apply to aquatic
systems but, due to transcutaneous absorption, aquatic and semiaquatic
species may be more sensitive to commonly used pest control agents thanterrestrial animals. Before use, an appropriate review of chemicals andmethods of application is necessary.
Emergency, Weekend, and holiday Care As with terrestrial species, aquatic
animals should receive daily care from qualified personnel who have a sufficient understanding of the housing system to identify malfunctions and, if
they are unable to address a system failure of such magnitude that it requires
resolution before the next workday, access to staff who can respond to the
problem. Automated monitoring systems are available and may be appropriate depending on system size and complexity. Appropriate emergency
response plans should be developed to address major system failures.
Population Management
Identification Identification principles are similar to those for terrestrial
animals. Identification criteria are based on the species and housing system.
Identification methods available for use in aquatic species include fin clipping, genetic testing (Matthews et al. 2002; Nickum et al. 2004), identification tags, subcutaneous injections of elastomeric or other materials (Nickum
et al. 2004), individual transponder tags (in animals of sufficient size), and,as applicable, external features such as individual color patterns. Because it
can be difficult to individually identify some small aquatic animals throughout their life, group identification may be more appropriate in some situations (Koerber and Kalishman 2009; Matthews et al. 2002).
Aquatic Animal Recordkeeping Adequate recordkeeping is necessary inaquatic system management. In general, the same standards used for terrestrial animals apply to aquatic and semiaquatic species, although modifications may be necessary to account for species or system variations (Koerberand Kalishman 2009).
Although many aquatic animals are maintained using group (vs. individual) identification, detailed animal records are still necessary. Animal
information that may routinely be captured, particularly in biomedical
research with fish, includes species; genetic information (parental stock
identification, genetic composition); stock source; stock numbers in system; tank identification; system life support information; breeding; deaths;

88

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

illnesses; animal transfers within and out of the facility; and fertilization/hatching information (Koerber and Kalishman 2009; Matthews et al.
2002). Records should be kept concerning feeding information (e.g., foodoffered, acceptance), nonexpired food supplies to ensure sustenance ofnutritional profile, and any live cultures (e.g., hatch rates and information to ensure suppliers’ recommendations are being met; Matthews etal. 2002).
Records of water quality testing for system and source water and maintenance activities of the life support system components are important for
tracking and ensuring water quality. The exact water quality parameters
tested and testing frequency should be clearly established and will vary withsuch factors as the type of life support system, animals, and research, as discussed under Water Quality. Detailed tracking of animal numbers in aquatic
systems is often possible with accurate records of transfers, breeding, andmortalities (Matthews et al. 2002). In some cases where animals are housedin large groups (e.g., some Xenopus colonies) periodic censuses may beundertaken to obtain an exact count. In large-scale aquaculture research it
may be more appropriate to measure biomass of the system versus actual
numbers of animals (Borski and Hodson 2003).
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4
Veterinary CareV

eterinary care is an essential part of an animal care and use Program.
The primary focus of the veterinarian is to oversee the well-being
and clinical care of animals used in research, testing, teaching, andproduction. This responsibility extends to monitoring and promoting animal well-being at all times during animal use and during all phases of theanimal’s life. Well-being is determined by considering physical, physiologic,
and behavioral indicators, which vary by species. The number, species, anduse of animals housed in an institution may influence the complexity of theveterinary care program, but a veterinary program that offers a high qualityof care and ethical standards must be provided, regardless of the numberof animals or species maintained.
An adequate veterinary care program consists of assessment of animal
well-being and effective management of
•
•
•
•
•
•
•
•

animal procurement and transportation
preventive medicine (including quarantine, animal biosecurity, andsurveillance)clinical disease, disability, or related health issues
protocol-associated disease, disability, and other sequelae
surgery and perioperative carepain and distressanesthesia and analgesia
euthanasia.

105

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GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALSThe veterinary care program is the responsibility of the attending veterinarian (AV), who is certified or has training or experience in laboratory
animal science and medicine or is otherwise qualified in the care of thespecies being used. Some aspects of the veterinary care program can be
conducted by persons other than a veterinarian, but a mechanism for direct
and frequent communication should be established to ensure that timely
and accurate information is conveyed to the responsible veterinarian about
issues associated with animal health, behavior, and well-being, and thatappropriate treatment or euthanasia is administered. The AV should provide
guidance to investigators and all personnel involved in the care and useof animals to ensure appropriate husbandry, handling, medical treatment,
immobilization, sedation, analgesia, anesthesia, and euthanasia. In addition, the AV should provide guidance and oversight to surgery programs
and perioperative care involving animals.
ANIMAL PROCUREMENT AND TRANSPORTATION
Animal Procurement
All animals must be acquired lawfully, and the receiving institution
should ensure that all procedures involving animal procurement are conducted in a lawful manner. Before procuring animals, the principal investigator should confirm that there are sufficient facilities and expertise to houseand manage the species being acquired. Procurement of animals should belinked to the prior approval of animal use and number by the IACUC (seeChapter 2, Protocol Review). If dogs and cats are obtained from random
sources, such as shelters or pounds, the animals should be inspected fortattoos or identification devices such as subcutaneous transponders (NRC
2009b); such identification might indicate that an animal was a pet, and if
so, ownership should be verified. Attention should also be given to the population status of the species under consideration; the threatened or endangered status of species is updated annually by the Fish and Wildlife Service(DOI 2007). Appropriate records and other forms of documentation should
be maintained for animals acquired by an institution for its investigators.
Potential vendors should be evaluated for the quality of animals they
supply. As a rule, vendors of purpose-bred animals (e.g., USDA Class A
dealers) regularly provide information that describes the genetic and pathogen status of their colonies or individual animals and relevant clinical history (e.g., vaccination status and anthelminthic administration). The use of
purpose-bred and preconditioned animals is therefore preferable when consistent with the research, teaching, and testing objectives. In general, animals used for scientific purposes should not be obtained from pet stores or
pet distributors due to the unknown or uncontrolled background of animals

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from these sources and the potential for introducing health risks to personnel
and other facility animals. Breeding colonies should be established basedon need and managed according to principles of animal reduction such as
cryopreservation for rodent stocks or strains (Robinson et al. 2003).
Transportation of Animals
Transportation of animals is governed by a number of US regulatory
agencies and international bodies. The Animal Welfare Regulations (USDA1985) set standards for interstate and export/import transportation of regulated species; the International Air Transport Association (IATA) updates theLive Animals Regulations annually and IATA member airlines and many
countries agree to comply with these regulations to ensure the safe andhumane transport of animals by air (IATA 2009). The Centers for Disease
Control and Prevention and USDA enforce regulations to prevent the introduction, transmission, or spread of communicable diseases and regulate theimportation of any animal or animal product capable of carrying a zoonotic
disease. The US Fish and Wildlife Service regulates importation/exportationof wild vertebrate and invertebrate animals and their tissues. As the national
authority arm of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), the US Fish and Wildlife Service also
regulates movement of CITES-listed species that are captive bred, including nonhuman primates (DOI 2007). Institutions should contact appropriate authorities to ensure compliance with any relevant statutes and otheranimal transportation requirements that must be met for animals to crossinternational boundaries, including those not of the country of final destination. The NRC publication Guidelines for the humane Transportationof Research Animals provides a comprehensive review of this topic (NRC2006); additional references on transportation of animals are available inAppendix A.
Animal transportation may be intrainstitutional, interinstitutional, or
between a commercial or noncommercial source and a research facility.
For wildlife, transportation may occur between the capture site and fieldholding facilities. Careful planning for all types of transportation should
occur to ensure animal safety and well-being. The process of transportation
should provide an appropriate level of animal biosecurity (see definition on
page 109) while minimizing zoonotic risks, protecting against environmental extremes, avoiding overcrowding, providing for the animals’ physical,
physiologic, or behavioral needs and comfort, and protecting the animals
and personnel from physical trauma (Maher and Schub 2004).
Movement of animals within or between sites or institutions should beplanned and coordinated by responsible and well-trained persons at thesending and receiving sites to minimize animal transit time or delays in

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receipt. Shipping should be coordinated to ensure that animals arrive during normal business hours or, if delivery occurs outside of this time, thatsomeone is available to receive them. Defining and delegating responsibility to the appropriate persons, who are knowledgeable about the needs ofthe species being shipped, will help ensure effective communication andplanning of animal transport (AVMA 2002).
All animals in transit within and between institutions or jurisdictions
should be accompanied by appropriate documentation to minimize delays
in shipping and receipt. Documentation may include health certificates,
sending and receiving institutions’ addresses and contacts, emergency
procedures and veterinary contact information, and agency permits asneeded.
For noncommercial sources of animals, in particular, it is important for
the veterinarian or the veterinarian’s designee to review the health status
and other housing and husbandry requirements before authorizing shipmentof animals. This action will ensure that effective quarantine practices are
implemented for incoming animals and address any special requirements
needed to ensure animal well-being (Otto and Tolwani 2002). Special considerations may be necessary for transporting animals during certain phasesof their life or in certain conditions, such as pregnant, perinatal, and geriatric animals; animals with preexisting medical conditions (e.g., diabetes
mellitus); and animals surgically prepared by the supplier (FASS 2010).
Although ensuring animal biosecurity during transportation is always
important, it is of particular importance for immunocompromised, genetically modified, and specific pathogen-free rodents (Jacoby and Lindsey1998). For these animals, reinforced disposable shipping containers withfilter-protected ventilation openings and internal food and water sources
help ensure that microbial contamination does not occur during transit.
Commercial vendors are experienced in animal transport and typically use
dedicated transport systems and protocols to minimize microbiologic contamination. Noncommercial or interinstitutional transfer of rodents poses ahigher risk of microbial contamination since the individuals involved maylack the required knowledge and animal biosecurity capabilities to maintainthe animals’ health status. Risks due to in-transit microbial contaminationof shipping container surfaces can be reduced by decontaminating the surfaces before placement of the containers in clean sites of animal facilities(NRC 1996, 2006). Transportation of animals in private vehicles is discouraged because of potential animal biosecurity, safety, health, and liability
risks for the animals, personnel, and institution.
For aquatic species and amphibians, special considerations are required
for transportation in an aqueous or sufficiently moist environment, andspecial attention should be given to avoiding temperature extremes forpoikilotherms.

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In all cases, appropriate loading and unloading facilities should be provided for the safe and secure transfer of animals at an institution. Facilities
and procedures should be in place to help ensure that the environment at
the site does not pose risks to animal well-being or personnel safety. During
times of extreme temperatures animal transport may be detrimental to animal well-being and therefore may not be possible unless an appropriately
heated or cooled means of transportation is available (Robertshaw 2004;
Schrama et al. 1996).
PREVENTIVE MEDICINE
Disease prevention is an essential component of comprehensive veterinary medical care and biosecurity programs. Effective preventive medicine
enhances the research value of animals by maintaining healthy animals andminimizing nonprotocol sources of variation associated with disease andinapparent infection, thus minimizing animal waste and potential effects on
well-being. Preventive medicine programs consist of various combinationsof policies, procedures, and equipment related to quarantine and stabilization and the separation of animals by species, source, and health status.
Animal biosecurity
Animal biosecurity refers to all measures taken to identify, contain, prevent, and eradicate known or unknown infections that may cause clinicaldisease or alter physiologic and behavioral responses or otherwise makethe animals unsuitable for research.
Animal biosecurity practices should
be applied to all species, but they
Animal biosecurity includes all
are most important when housing
measures to control known or
unknown infections in laboratory
large numbers of animals in intensive
animals.
housing conditions (e.g., laboratoryrodents). Limiting exposure of animals
to infectious disease agents requiresconsideration of physical plant layout and operational practices. Separationof clean and soiled caging and equipment, and sometimes the associatedstaff, is often fundamental to success.
A successful animal biosecurity program incorporates a number of elements: procedures that ensure that only animals of a desired defined healthstatus enter the facility; personnel and materials, especially consumables,that do not serve as fomites; practices that reduce the likelihood of crosscontamination if an infectious agent is inadvertently introduced; a comprehensive ongoing system for evaluating animals’ health status, including access to all animals; and containment and eradication, if desired, of

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introduced infectious agents. Related components include procedures forevaluating and selecting appropriate animal suppliers (these may include
quarantine and determination of animal health status if unknown); treatment of animals or their products at entry to minimize disease risks (e.g.,surface disinfection of fish eggs); a comprehensive pest control program that
may include evaluation of the health status of feral animals; procedures toensure that all biologics administered to animals are free of contamination;and procedures for intra- and interfacility animal transport (e.g., transportof animals to laboratory and other facilities outside the animal facility canpresent challenges to animal biosecurity) (Balaban and Hampshire 2001).
Additional details pertaining to these topics are available in the sections of
Chapter 2 that deal with occupational health and safety.
Quarantine and Stabilization
Quarantine is the separation of newly received animals from thosealready in the facility, in a way that prevents potential spread of contaminants, until the health and possibly the microbial status of the newly
received animals have been determined. Transportation can be stressful
and may induce recrudescence of subclinical infections harbored by ananimal.
An effective quarantine program minimizes the risk of introduction ofpathogens into an established colony. The veterinary medical staff should
implement procedures for evaluating the health and, if appropriate, thepathogen status of newly received animals, and the procedures shouldreflect acceptable veterinary medical practice and federal and state regulations applicable to zoonoses (Butler et al. 1995). Effective quarantine
procedures are particularly helpful in limiting human exposure to zoonotic infections from nonhuman primates, such as mycobacterial infections,which necessitate specific guidelines for handling of these animals (Lerche
et al. 2008; Roberts and Andrews 2008).
Information from suppliers about animal quality should be sufficient
to enable a veterinarian to establish the length of quarantine, define thepotential risks to personnel and animals in the colony, determine whether
therapy is required before animals are released from quarantine, and, inthe case of rodents, determine whether rederivation (cesarean or embryotransfer) is necessary to free the animals of specific pathogens. Rodents may
not require quarantine if data from the vendor or provider are sufficiently
current, complete, and reliable to define the health status of the incoming
animals and if the potential for exposure to pathogens during transit is considered. When quarantine is indicated, animals from one shipment should
be handled separately or be physically separated from animals from othershipments to preclude transfer of infectious agents between groups.

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Depending on the health status of the colony animals and consistent
with the animal biosecurity program in place, rodents or other animals
being moved outside an animal facility for procedures (e.g., imaging or
behavioral testing) may need to be held separately from their colony oforigin until their health status is evaluated.
Regardless of whether the animals are quarantined, newly received animals should be given a period for physiologic, behavioral, and nutritional
acclimation before their use (Obernier and Baldwin 2006). The length oftime for acclimation will depend on the type and duration of animal transportation, the species, and the intended use of the animals. For animals
not typically housed in research settings, consideration should be given toproviding means to assist with their acclimation (e.g., shearing sheep before
they are brought indoors). The need for an acclimation period has been
demonstrated in mice, rats, guinea pigs, nonhuman primates, and goats, andtime for acclimation is likely important for other species as well (Capitanio
et al. 2006; Conour et al. 2006; Kagira et al. 2007; Landi et al. 1982; Prasad
et al. 1978; Sanhouri et al. 1989; Tuli et al. 1995).
Separation by Health Status and Species
Physical separation of animals by species is recommended to prevent
interspecies disease transmission and to eliminate the potential for anxiety
and physiologic and behavioral changes due to interspecies conflict (Arndt
et al. 2010). Such separation is usually accomplished by housing different
species in separate rooms, but in some instances it may be possible withcubicles, laminar flow units, cages that have filtered air or separate ventilation, or isolators. It may also be acceptable to house different species in thesame room—for example, two species that have a similar pathogen status
and are behaviorally compatible (Pritchett-Corning et al. 2009), or aquatic
species, as long as nets and other animal handling devices remain separate
between systems.
In some species subclinical or latent infections can cause clinical disease if transmitted to another species. A few examples may serve as a guidein determining the need for separate housing by species:••

helicobacter bilis can infect rats and mice and may induce clinical disease in both species (Haines et al. 1998; Jacoby and Lindsey
1998; Maggio-Price et al. 2002).
As a rule, New World (South and Central American), Old World
African, and Old World Asian species of nonhuman primates should
be housed in separate rooms. Simian hemorrhagic fever (Renquist
1990) and simian immunodeficiency virus (Hirsch et al. 1991;
Murphey-Corb et al. 1986), for example, cause only subclinical

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infections in African species but induce clinical disease in Asian
species.
Some species should be housed in separate rooms even though
they are from the same geographic region. For example, squirrel
monkeys (Saimiri sciureus) and tamarins (Saguinus oedipus) may
be latently infected with herpesviruses (herpesirus saimiri and h.
tamarinus, respectively), which could be transmitted to and cause a
fatal epizootic disease in owl monkeys (Aotus triirgatus) (Barahona
et al. 1975; Hunt and Melendez 1966; Murphy et al. 1971).

Intraspecies separation may be essential when animals obtained frommultiple sites or sources, either commercial or institutional, differ in pathogen status—for example, with respect to rat theilovirus in rats, mouse
hepatitis virus in mice, bacterial gill disease in rainbow trout, Pasteurella
multocida in rabbits, Macacine herpesirus 1 (B virus) in macaque species,
and Mycoplasma hyopneumoniae in swine.
Surveillance, Diagnosis, Treatment, and Control of Disease
All animals should be observed for signs of illness, injury, or abnormal
behavior by a person trained to recognize such signs. As a rule, such observation should occur at least daily, but more frequent observations may berequired, such as during postoperative recovery, when animals are ill or
have a physical deficit, or when animals are approaching a study endpoint.
Professional judgment should be used to ensure that the frequency andcharacter of observations minimize risks to individual animals and do notcompromise the research for which the animals are used.
Appropriate procedures should be in place for disease surveillance anddiagnosis. Unexpected deaths and signs of illness, distress, or other deviations from normal in animals should be reported promptly and investigated,
as necessary, to ensure appropriate and timely delivery of veterinary medical care. Animals that show signs of a contagious disease should be isolated
from healthy animals. If an entire room or enclosure of animals is known or
believed to be exposed to an infectious agent (e.g., Mycobacterium tuberculosis in nonhuman primates), the group should be kept intact during theprocess of diagnosis, treatment, and control.
Procedures for disease prevention, diagnosis, and therapy should be
those currently accepted in veterinary and laboratory animal practice.
Health monitoring programs also include veterinary herd/flock health programs for livestock and colony health monitoring programs for aquatic androdent species. Access to diagnostic laboratory services facilitates veterinary
medical care and can include gross and microscopic pathology, hematology, microbiology, parasitology, clinical chemistry, molecular diagnostics,

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and serology. If a disease or infectious agent is identified in a facility orcolony, the choice of therapy should be made by the veterinarian in consultation with the investigator. If the animal is to remain in the study, theselected treatment plan should be therapeutically sound and, when possible, interfere minimally with the research process.
Subclinical microbial infections (see Appendix A, Pathology, Clinical
Pathology, and Parasitology) occur frequently in conventionally maintainedrodents but can also occur in facilities designed and maintained for production and use of pathogen-free rodents if the microbial barrier is breached.
Examples of infectious agents that can be subclinical but that may induce
immunologic changes or alter physiologic, pharmacologic, or toxicologic
responses are noroviruses, parvoviruses, mouse hepatitis virus, lymphocytic
choriomeningitis virus, and helicobacter spp. (Besselsen et al. 2008; Clifford and Watson 2008; NRC 1991a,b,c). Scientific objectives of a particular
protocol, the consequences of infection in a specific strain of rodent, thepotential for zoonotic disease, and the adverse effects that infectious agents
may have on other animals or protocols in a facility should determine thecharacteristics of rodent health surveillance programs and strategies forkeeping rodents free of specific pathogens.
The principal methods for detecting microbial infections in animal
populations are serologic tests (e.g., flow cytometric bead immunoassays,
immunofluorescent assays) but other methods, such as DNA analysis using
polymerase chain reaction (PCR), microbial culture, clinical chemistry (e.g.,lactate dehydrogenase virus), histopathology, and other validated emerging
technologies, can also be used to make or confirm a diagnosis.
Transplantable tumors, hybridomas, cell lines, blood products, andother biologic materials can be sources of both murine and human viruses
that can contaminate rodents or pose risks to laboratory personnel (Nicklas
et al. 1993); rapid and effective assays are available to monitor microbiologic contamination and should be considered before introducing suchmaterial into animals (Peterson 2008).
Because health monitoring programs are dependent on the size and complexity of the Program, the species involved, and the institutional research
focus, it is beyond the scope of the Guide to go into details about healthmonitoring programs for all species; additional references are in Appendix A(under Disease Surveillance, Diagnosis, and Treatment; Pathology, Clinical
Pathology, and Parasitology; and Species-Specific References).
CLINICAL CARE AND MANAGEMENT
Healthy, well-cared-for animals are a prerequisite for good-quality
animal-based science. The structure of the veterinary care program, including the number of qualified veterinarians, should be appropriate to fulfill the

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program‘s requirements, which will vary by institution, species used, andthe nature of the animal use. To be effective in providing clinical care, theveterinarian should be familiar with the species and various uses of animals
in the institutional research, teaching, testing, or production programs andhave access to medical and experimental treatment records.
Medical Management
There should be a timely and accurate method for communication of
any abnormalities in or concerns about animal health, behavior, and wellbeing to the veterinarian or the veterinarian’s designee. The responsibility
for communicating these concerns rests with all those involved with animalcare and use. Reports should be triaged to ensure that animals most in needreceive priority attention, and the veterinarian or veterinarian’s designee
should perform an objective assessment of the animal(s) to determine anappropriate course of action.
Well-planned experiments with clearly delineated scientific and humane
endpoints will help to ensure that a contingency plan is in place for problems that may arise during the study (see Chapter 2, Experimental and
Humane Endpoints). For animals on research protocols, the veterinarian orveterinarian’s designee should make every effort to discuss any problems
with the principal investigator or project director to jointly determine themost appropriate course of treatment or action. Standard operating procedures (SOPs) may be developed for recurrent health conditions to expedite
treatment. Recurrent or significant problems involving experimental animal
health should be communicated to the IACUC, and all treatments and outcomes should be documented (USDA 1997).
Emergency Care
Procedures must be in place to provide for emergency veterinary careboth during and outside of regularly scheduled hours. Such proceduresmust enable animal care and research staff to make timely reports of animal
injury, illness, or death. A veterinarian or the veterinarian’s designee must
be available to expeditiously assess the animal’s condition, treat the animal,
investigate an unexpected death, or advise on euthanasia. In the case of apressing health problem, if the responsible person (e.g., investigator) is notavailable or if the investigator and veterinary staff cannot reach consensus
on treatment, the veterinarian must have the authority, delegated by senioradministration (see Chapter 2, Institutional Official and Attending Veterinarian) and the IACUC, to treat the animal, remove it from the experiment,
institute appropriate measures to relieve severe pain or distress, or performeuthanasia if necessary.

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Recordkeeping
Medical records are a key element of the veterinary care program andare considered critical for documenting animal well-being as well as tracking animal care and use at a facility. A veterinarian should be involved inestablishing, reviewing, and overseeing medical and animal use records(Field et al. 2007; Suckow and Doerning 2007). All those involved in animal care and use must comply with federal laws and regulations regarding
human and veterinary drugs and treatments. Drug records and storage procedures should be reviewed during facility inspections.
SURGERy
Successful surgical outcomes require appropriate attention to presurgical planning, personnel training, anesthesia, aseptic and surgical technique,assessment of animal well-being, appropriate use of analgesics, and animal
physiologic status during all phases of a protocol involving surgery andpostoperative care (see Appendix A, Anesthesia, Pain, and Surgery). The
individual impact of those factors will vary according to the complexity ofprocedures involved and the species of animal used. A team approach to asurgical project often increases the likelihood of a successful outcome byproviding input from persons with different expertise (Brown and Schofield
1994; Brown et al. 1993).
Surgical outcomes should be continually and thoroughly assessed toensure that appropriate procedures are followed and timely corrective
changes are instituted. Modification of standard techniques may be required(for instance, in aquatic or field surgery), but should not compromise thewell-being of the animals. In the event of modification, close assessmentof outcomes may have to incorporate criteria other than clinical morbidity
and mortality. Such assessments rely on continuing communication among
technical staff, investigators, veterinarians, and the IACUC.
Training
Researchers conducting surgical procedures must have appropriate training to ensure that good surgical technique is practiced—that is, asepsis, gentle
tissue handling, minimal dissection of tissue, appropriate use of instruments,
effective hemostasis, and correct use of suture materials and patterns (Brown
et al. 1993; Heon et al. 2006). Training may have to be tailored to accommodate the wide range of educational backgrounds frequently encounteredin research settings. For example, persons trained in human surgery may
need training in interspecies variations in anatomy, physiology, the effects
of anesthetic and analgesic drugs, and/or postoperative care requirements.

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GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALSTechnical staff performing rodent surgery may have had little formal training
in surgical techniques and asepsis and may require general surgical training
as well as training for the specific techniques they are expected to perform(Stevens and Dey 2007).
Training guidelines for research surgery commensurate with an individual’s background are available (ASR 2009) to assist institutions in developing
appropriate training programs. The IACUC, together with the AV, is responsible for determining that personnel performing surgical procedures areappropriately qualified and trained in the procedures (Anderson 2007).
Presurgical Planning
Presurgical planning should include input from all members of the surgical team (e.g., the surgeon, anesthetist, veterinarian, surgical technicians,
animal care staff, and investigator). The surgical plan should identify personnel, their roles and training needs, and equipment and supplies required forthe procedures planned (Cunliffe-Beamer 1993); the location and nature of
the facilities in which the procedures will be conducted; and perioperative
animal health assessment and care (Brown and Schofield 1994). A veterinarian should be involved in discussions of the selection of anesthetic
agents and doses as well as the plan for perioperative analgesic use. If anonsterile part of an animal, such as the gastrointestinal tract, is to be surgically exposed or if a procedure is likely to cause immunosuppression,
preoperative antibiotics may be appropriate (Klement et al. 1987); however,
the routine use of antibiotics should never be considered a replacement for
proper aseptic surgical techniques.
Presurgical planning should specify the requirements for postsurgicalmonitoring, care, and recordkeeping, including the personnel who will
perform these duties. The investigator and veterinarian share responsibility
for ensuring that postsurgical care is appropriate.
Surgical Facilities
Unless an exception is specifically justified as an essential componentof the research protocol and approved by the IACUC, aseptic surgery
should be conducted in dedicated facilities or spaces. When determining
the appropriate location for a surgical procedure (either a dedicated operating room/suite or an area that provides separation from other activities),
the choice may depend on the species, the nature of the procedure (major,
minor, or emergency), and the potential for physical impairment or postoperative complications, such as infection. Most bacteria are carried onairborne particles or fomites, so surgical facilities should be maintained andoperated in a manner that ensures cleanliness and minimizes unnecessary

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traffic (AORN 2006; Bartley 1993). If it is necessary to use an operating
room for other purposes, it is imperative that the room be returned to an
appropriate level of hygiene before its use for major survival surgery.
Generally, agricultural animals maintained for biomedical researchshould undergo surgery with techniques and in facilities compatible withthe guidelines set forth in this section. However, some minor and emergency procedures commonly performed in clinical veterinary practice andin commercial agricultural settings may take place under field conditions.
Even when conducted in an agricultural setting, however, these procedures
require the use of appropriate aseptic technique, sedatives, analgesics, anesthetics, and conditions commensurate with the risk to the animal’s healthand well-being.
Surgical Procedures
Surgical procedures are categorized as major or minor and, in thelaboratory setting, can be further divided into survival and nonsurvival. As
a general guideline, major survival surgery (e.g., laparotomy, thoracotomy,
joint replacement, and limb amputation) penetrates and exposes a body
cavity, produces substantial impairment of physical or physiologic functions, or involves extensive tissue dissection or transection (Brown et al.
1993). Minor survival surgery does not expose a body cavity and causes
little or no physical impairment; this category includes wound suturing,
peripheral vessel cannulation, percutaneous biopsy, routine agricultural
animal procedures such as castration, and most procedures routinely doneon an “outpatient” basis in veterinary clinical practice. Animals recoveringfrom these minor procedures typically do not show significant signs of postoperative pain, have minimal complications, and return to normal function
in a relatively short time. When attempting to categorize a particular surgical procedure, the following should be considered: the potential for pain
and other postoperative complications; the nature of the procedure as wellas the size and location of the incision(s); the duration of the procedure;
and the species, health status, and age of the animal.
Laparoscopic surgeries and some procedures associated with neuroscience research (e.g., craniotomy, neurectomy) may be classified as major orminor surgery depending on their impact on the animal (Devitt et al. 2005;
Hancock et al. 2005; NRC 2003; Perret-Gentil et al. 1999, 2000). For example, laparoscopic techniques with minimal associated trauma and sequelae(e.g., avian sexing and oocyte collection) could be considered minor, whereas
others (e.g., hepatic lobectomy and cholecystectomy) should be consideredmajor. Although minor laparoscopic procedures are often performed on an“outpatient” basis, appropriate aseptic technique, instruments, anesthesia,
and analgesia are necessary. Whether a laparoscopic procedure is deemed

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major or minor should be evaluated on a case-by-case basis by the veterinarian and IACUC.
Emergency situations sometimes require immediate surgical attention
under less than ideal conditions. For example, if an animal maintained
outdoors needs surgical attention, movement to a surgical facility might be
impractical or pose an unacceptable risk to the animal. Such situations often
require more intensive aftercare and may pose a greater risk of postoperativecomplications. The appropriate course of action requires veterinary medical
judgment.
In nonsurvival surgery, an animal is euthanized before recovery fromanesthesia. It may not be necessary to follow all the techniques outlinedin this section if nonsurvival surgery is performed but, at a minimum, thesurgical site should be clipped, the surgeon should wear gloves, and the
instruments and surrounding area should be clean (Slattum et al. 1991). For
nonsurvival procedures of extended duration, attention to aseptic technique
may be more important in order to ensure stability of the model and a successful outcome.
Aseptic Technique
Aseptic technique is used to reduce microbial contamination to thelowest possible practical level (Mangram et al. 1999). No procedure, pieceof equipment, or germicide alone can achieve that objective (Schonholtz1976): aseptic technique requires the input and cooperation of everyone
who enters the surgery area (Belkin 1992; McWilliams 1976). The contribution and importance of each practice varies with the procedure. Regardlessof the species, aseptic technique includes preparation of the patient, suchas hair or feather removal and disinfection of the operative site (Hofmann1979); preparation of the surgeon, such as the provision of appropriate
surgical attire, face masks, and sterile surgical gloves (Chamberlain andHouang 1984; Pereira et al. 1990; Schonholtz 1976); sterilization of instruments, supplies, and implanted materials (Bernal et al. 2009; Kagan 1992b);
and the use of operative techniques to reduce the likelihood of infection(Ayliffe 1991; Kagan 1992a; Lovaglio and Lawson 1995; Ritter and Marmion
1987; Schofield 1994; Whyte 1988).
While the species of animal may influence the manner in which principles of aseptic technique are achieved (Brown 1994; Cunliffe-Beamer
1983; Gentry and French 1994), inadequate or improper technique maylead to subclinical infections that can cause adverse physiologic and behavioral responses (Beamer 1972; Bradfield et al. 1992; Cunliffe-Beamer 1990;
Waynforth 1980, 1987) affecting surgical success, animal well-being, andresearch results (Cooper et al. 2000). General principles of aseptic technique
should be followed for all survival surgical procedures (ACLAM 2001).

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Specific sterilization methods should be selected on the basis of thephysical characteristics of the materials to be sterilized (Callahan et al. 1995;
Schofield 1994) and sterilization indicators should be used to validate thatmaterials have been properly sterilized (Berg 1993). Autoclaving and plasmaand gas sterilization are effective methods most commonly used to sterilize
instruments and materials. Alternative methods, used primarily for rodent
surgery, include liquid chemical sterilants and dry heat sterilization. Liquid
chemical sterilants should be used with appropriate contact times and instruments should be rinsed with sterile water or saline before use. Bead or dryheat sterilizers are an effective and convenient means of rapidly sterilizing
the working surfaces of surgical instruments but care should be taken to
ensure that the instrument surfaces have cooled sufficiently before touching
animal tissues to minimize the risk of burns. Alcohol is neither a sterilant
nor a high-level disinfectant (Rutala 1990) but may be acceptable for someprocedures if prolonged contact times are used (Huerkamp 2002).
Intraoperative Monitoring
Careful monitoring and timely attention to problems increase the likelihood of a successful surgical outcome (Kuhlman 2008). Monitoring includes
routine evaluation of anesthetic depth and physiologic functions and conditions, such as body temperature, cardiac and respiratory rates and pattern (Flegal et al. 2009), and blood pressure (Kuhlman 2008), and should
be appropriately documented. Use of balanced anesthesia, including theaddition of an intraoperative analgesic agent, can help minimize physiologic fluctuations during surgery. Maintenance of normal body temperature
minimizes cardiovascular and respiratory disturbances caused by anesthetic
agents (Dardai and Heavner 1987; Flegal et al. 2009; Fox et al. 2008), andis of particular importance in small animals where the high ratio of surface
area to body weight may easily lead to hypothermia. Fluid replacement may
be a necessary component of intraoperative therapy depending on the duration and nature of the procedure. For aquatic species (including amphibians), care should be taken to keep the skin surfaces moist and minimize
drying during surgical procedures.
Postoperative Care
An important component of postsurgical care is observation of theanimal and intervention as necessary during recovery from anesthesia andsurgery (Haskins and Eisele 1997). The intensity of monitoring will varywith the species and the procedure and may be greater during the immediate anesthetic recovery period. During this period, animals should be in aclean, dry, and comfortable area where they can be observed frequently by

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trained personnel. Particular attention should be given to thermoregulation,
cardiovascular and respiratory function, electrolyte and fluid balance, andmanagement of postoperative pain or discomfort. Additional care may be
warranted, including long-term administration of parenteral fluids, analgesics, and other drugs, as well as care of surgical incisions. Appropriate
medical records should also be maintained.
After recovery from anesthesia, monitoring is often less intense but
should include attention to basic biologic functions of intake and elimination and to behavioral signs of postoperative pain, monitoring for postsurgical infections, monitoring of the surgical incision site for dehiscence,bandaging as appropriate, and timely removal of skin sutures, clips, or
staples (UFAW 1989).
PAIN AND DISTRESS
An integral component of veterinary medical care is prevention or alleviation of pain associated with procedural and surgical protocols. Pain is acomplex experience that typically results from stimuli that damage or havethe potential to damage tissue; such stimuli prompt withdrawal and evasive
action. The ability to experience and respond to pain is widespread in the
animal kingdom and extends beyond vertebrates (Sherwin 2001).
Pain is a stressor and, if not relieved, can lead to unacceptable levelsof stress and distress in animals. Furthermore, unrelieved pain may lead to“wind-up,” a phenomenon in which central pain sensitization results in apain response to otherwise nonpainful stimuli (allodynia; Joshi and Ogunnaike 2005). For these reasons, the proper use of anesthetics and analgesics
in research animals is an ethical and scientific imperative. Recognition
and Alleiation of Pain in Laboratory Animals (NRC 2009a) is an excellent
source of information about the basis and control of distress and pain (seealso Appendix A, Anesthesia, Pain, and Surgery).
Fundamental to the relief of pain in animals is the ability to recognize
its clinical signs in specific species (Bateson 1991; Carstens and Moberg
2000; Hawkins 2002; Holton et al. 1998; Hughes and Lang 1983; Karaset al. 2008; Martini et al. 2000; Roughan and Flecknell 2000, 2003, 2004;
Sneddon 2006). Species vary in their response to pain (Baumans et al. 1994;
Kohn et al. 2007; Morton et al. 2005; Viñuela-Fernández et al. 2007), andcriteria for assessing pain in various species differ. The U.S. Government
Principles for the Utilization and Care of Vertebrate Animals Used in Testing,Research, and Training (see Appendix B) state that in general, unless the contrary is known or established, it should be considered that procedures thatcause pain in humans may also cause pain in other animals (IRAC 1985).
Certain species-specific behavioral manifestations are used as indicators of pain or distress—for example, vocalization (dogs), depression

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(all), anorexia (all), rapid or labored respiration (rodents, birds, fish),lack of grooming (mammals and birds), increased aggression (mammals
and birds), periocular and nasal porphyrin discharge (rodents), abnormal
appearance or posture (all), and immobility (all) (NRC 2008, 2009a).
However, some species may mask signs of pain until they are quite severe(NRC 2009a). It is therefore essential that personnel caring for and using
animals be trained in species-specific and individual clinical, behavioral,
physiologic, and biochemical indicators of well-being (Dubner 1987;
Karas 2002; Murrell and Johnson 2006; Rose 2002; Stoskopf 1994; Valverde and Gunkel 2005).
Distress may be defined as an aversive state in which an animal fails tocope or adjust to various stressors with which it is presented. But distress
may not induce an immediate and observable pathologic or behavioral
alteration, making it difficult to monitor and evaluate the animal’s state
when it is present. Both the duration and intensity of the state are importantconsiderations when trying to prioritize attention to and treatment of animal distress. For example, an injection requiring brief immobilization may
produce acute stress lasting only seconds, while long-term individual housing of a social species in a metabolic cage may produce chronic distress.
Implementation of clear, appropriate, and humane experimental endpoints
for animals, combined with close observation during invasive periods ofexperimentation, will assist in minimizing distress experienced by animals
used in research, teaching, testing, and production. Recognition and Alleiation of distress in Laboratory Animals (NRC 2008) is a resource with
important information about distress in experimental animals.
ANESTHESIA AND ANALGESIA
The selection of appropriate analgesics and anesthetics should reflect
professional veterinary judgment as to which best meets clinical andhumane requirements as well as the needs of the research protocol. The
selection depends on many factors, such as the species, age, and strain or
stock of the animal, the type and degree of pain, the likely effects of particular agents on specific organ systems, the nature and length of the surgicalor pain-inducing procedure, and the safety of the agent, particularly if aphysiologic deficit is induced by a surgical or other experimental procedure(Kona-Boun et al. 2005).
Preemptive analgesia (the administration of preoperative and intraoperative analgesia) enhances intraoperative patient stability and optimizes
postoperative care and well-being by reducing postoperative pain (Coderre
et al. 1993; Hedenqvist et al. 2000). Analgesia may be achieved throughtimely enteral or parenteral administration of analgesic agents as well as byblocking nociceptive signaling via local anesthetics (e.g., bupivacaine).

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Alleviation of chronic pain may be more challenging than postprocedural pain; commercially available opiate slow-release transdermal patches
or implantable analgesic-containing osmotic minipumps may be useful forsuch relief. Because of wide individual variation in response to analgesics,
regardless of the initial plan for pain relief, animals should be closely monitored during and after painful procedures and should receive additional
drugs, as needed, to ensure appropriate analgesic management (Karas et al.
2008; Paul-Murphy et al. 2004). Nonpharmacologic control of pain may
be effective and should not be overlooked as an element of postproceduralor perioperative care for research animals (NRC 2009a; Spinelli 1990).
Appropriate nursing support may include a quiet, darkened recovery or
resting place, timely wound or bandage maintenance, increased ambient
warmth and a soft resting surface, rehydration with oral or parenteral fluids,
and a return to normal feeding through the use of highly palatable foods
or treats.
Most anesthetics cause a dose-dependent depression of physiologic
homeostasis and the changes can vary considerably with different agents.
The level of consciousness, degree of antinociception (lack of response
to noxious stimuli), and status of the cardiovascular, respiratory, musculoskeletal, and thermoregulatory systems should all be used to assess theadequacy of the anesthetic regimen. Interpretation and appropriate responseto the various parameters measured require training and experience with theanesthetic regimen and the species. Loss of consciousness occurs at a lightplane of anesthesia, before antinociception, and is sufficient for purposes ofrestraint or minor, less invasive procedures, but painful stimuli can induce a
return to consciousness. Antinociception occurs at a surgical plane of anesthesia and must be ascertained before surgery. Individual animal responses
vary widely and a single physiologic or nociceptive reflex response may not
be adequate for assessing the surgical plane or level of analgesia (Mason
and Brown 1997).
For anesthesia delivery, precision vaporizers and monitoring equipment(e.g., pulse oximeter for determining arterial blood oxygen saturation levels)
increase the safety and choices of anesthetic agents for use in rodents andother small species. For injectable anesthestic protocols, specific reversal
agents can minimize the incidence of some side effects related to prolonged
recovery and recumbency. Guidelines for the selection and proper useof analgesic and anesthetic drugs should be developed and periodicallyreviewed and updated as standards and techniques are refined. Agents thatprovide anesthesia and analgesia must be used before their expiration dates
and should be acquired, stored, their use recorded, and disposed of legallyand safely.
Some classes of drugs such as sedatives, anxiolytics, and neuromuscular blocking agents may not provide analgesia but may be useful when

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used in combination with appropriate analgesics and anesthetics to provide
balanced anesthesia and to minimize stress associated with perioperative
procedures. Neuromuscular blocking agents (e.g., pancuronium) are sometimes used to paralyze skeletal muscles during surgery in which generalanesthetics have been administered (Klein 1987); because this paralysis
eliminates many signs and reflexes used to assess anesthetic depth, autonomic nervous system changes (e.g., sudden changes in heart rate andblood pressure) can be indicators of pain related to an inadequate depth ofanesthesia. It is imperative that any proposed use of neuromuscular blocking drugs be carefully evaluated by the veterinarian and IACUC to ensure
the well-being of the animal. Acute stress is believed to be a consequenceof paralysis in a conscious state and it is known that humans, if conscious,
can experience distress when paralyzed with these drugs (NRC 2008; Van
Sluyters and Oberdorfer 1991). If paralyzing agents are to be used, theappropriate amount of anesthetic should first be defined on the basis ofresults of a similar procedure using the anesthetic without a blocking agent(NRC 2003, 2008, 2009a).
EUTHANASIA
Euthanasia is the act of humanely killing animals by methods thatinduce rapid unconsciousness and death without pain or distress. Unless a
deviation is justified for scientific or medical reasons, methods should beconsistent with the AVMA Guidelines on Euthanasia (AVMA 2007 or later
editions). In evaluating the appropriateness of methods, some of the criteriathat should be considered are ability to induce loss of consciousness anddeath with no or only momentary pain, distress, or anxiety; reliability; irreversibility; time required to induce unconsciousness; appropriateness for the
species and age of the animal; compatibility with research objectives; andthe safety of and emotional effect on personnel.
Euthanasia may be planned and necessary at the end of a protocol or asa means to relieve pain or distress that cannot be alleviated by analgesics,
sedatives, or other treatments. Criteria for euthanasia include protocol-specific endpoints (such as degree of a physical or behavioral deficit or tumorsize) that will enable a prompt decision by the veterinarian and the investigator to ensure that the endpoint is humane and, whenever possible, thescientific objective of the protocol is achieved (see Chapter 2).
Standardized methods of euthanasia that are predictable and controllable should be developed and approved by the AV and IACUC. Euthanasia
should be carried out in a manner that avoids animal distress. Automatedsystems for controlled and staged delivery of inhalants may offer advantages
for species killed frequently or in large numbers, such as rodents (McIntyre
et al. 2007). Special consideration should be given to euthanasia of fetuses

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and larval life forms depending on species and gestational age (Artwohl etal. 2006).
The selection of specific agents and methods for euthanasia will dependon the species involved, the animal’s age, and the objectives of the protocol. Generally, chemical agents (e.g., barbiturates, nonexplosive inhalantanesthetics) are preferable to physical methods (e.g., cervical dislocation,
decapitation, use of a penetrating captive bolt); however, scientific considerations may preclude the use of chemical agents for some protocols.
Although carbon dioxide (CO2) is a commonly used method for rodent
euthanasia, there is ongoing controversy about its aversive characteristics
as an inhalant euthanasia agent. This is an area of active research (Conlee
et al. 2005; Danneman et al. 1997; Hackbarth et al. 2000; Kirkden et al.
2008; Leach et al. 2002; Niel et al. 2008) and further study is needed to
optimize the methods for CO2 euthanasia in rodents (Hawkins et al. 2006).
The acceptability of CO2 as a euthanasia agent for small rodents should be
evaluated as new data become available. Furthermore, because neonatal
rodents are resistant to the hypoxia-inducing effects of CO2 and require longer exposure times to the agent (Artwohl et al. 2006), alternative methods
should be considered (e.g., injection with chemical agents, cervical dislocation, or decapitation; Klaunberg et al. 2004; Pritchett-Corning 2009).
It is essential that euthanasia be performed by personnel skilled in methods for the species in question and in a professional and compassionate
manner. Special attention is required to ensure proficiency when a physical method of euthanasia is used. Death must be confirmed by personnel
trained to recognize cessation of vital signs in the species being euthanized.
A secondary method of euthanasia (e.g., thoracotomy or exsanguination)
can be also used to ensure death. All methods of euthanasia should be
reviewed and approved by the veterinarian and IACUC.
Euthanizing animals is psychologically difficult for some animal care,veterinary, and research personnel, particularly if they perform euthanasia
repetitively or are emotionally attached to the animals being euthanized(Arluke 1990; NRC 2008; Rollin 1986; Wolfle 1985). When delegating
euthanasia responsibilities, supervisors should be sensitive to this issue.
REFERENCES
Anderson LC. 2007. Institutional and IACUC responsibilities for animal care and use educationand training programs. ILAR J 48:90-95.
ACLAM [American College of Laboratory Animal Medicine]. 2001. Position Statement on Rodent Surgery. Available at www.aclam.org/education/guidelines/position_rodentsurgery.
html; accessed January 7, 2010.
AORN [Association of Operating Room Nurses]. 2006. Recommended practices for traffic
patterns in the perioperative practice setting. AORN J 83:681-686.
Arluke A. 1990. Uneasiness among laboratory technicians. Lab Anim 19:20-39.

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Arndt SS, Lohavech D, van’t Klooster J, Ohl F. 2010. Co-species housing in mice and rats: Effects on physiological and behavioural stress responsivity. Horm Behav 57:342-351.
Artwohl J, Brown P, Corning B, Stein S (ACLAM Task Force). 2006. Report of the ACLAM Task
Force on Rodent Euthanasia. JAALAS 45:98-105.
ASR [Academy of Surgical Research]. 2009. Guidelines for training in surgical research withanimals. J Invest Surg 22:218-225.
AVMA [American Veterinary Medical Association]. 2002. A Report from the American Veterinary
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5
Physical Plant

A

GENERAL CONSIDERATIONS

well-planned, well-designed, well-constructed, properly maintained
and managed facility is an important element of humane animalcare and use as it facilitates efficient, economical, and safe operation (see Appendix A, Design and Construction of Animal Facilities). The
design and size of an animal facility depend on the scope of institutional
research activities, the animals to be housed, the physical relationship to
the rest of the institution, and the geographic location.
Effective planning and design should include input from personnel
experienced with animal facility design, engineering, and operation, as
well as from representative users of the proposed facility. Computational
fluid dynamics (CFD), building information modeling, and literature on
postoccupancy analysis of space use may provide benefits when designingfacilities and caging (Eastman et al. 2008; Reynolds 2008; Ross et al. 2009).
An animal facility should be designed and constructed in accord with allapplicable building codes; in areas with substantial seismic activity thebuilding planning and design should incorporate the recommendations of
the Building Seismic Safety Council (BSSC 2001; Vogelweid et al. 2005).
Because animal model development and use can be expected to change
during the life cycle of an animal facility, facilities should be designed toaccommodate changes in use. Modular units (such as custom-designed trailers or prefabricated structures) should comply with construction guidelines
described in this chapter.

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Building materials for animal facilities should be selected to facilitate
efficient and hygienic operation. Durable, moisture- and vermin-proof, fireresistant, seamless materials are most desirable for interior surfaces, whichshould be highly resistant to the effects of cleaning agents, scrubbing, highpressure sprays, and impact. Paints and glazes should be nontoxic if used on
surfaces with which animals will have direct contact. In the construction of
outdoor facilities, consideration should be given to surfaces that withstandthe elements and can be easily maintained.
Location
Quality animal management and human comfort and health protection
require separation of animal facilities from personnel areas, such as offices
and conference rooms. Separation can be accomplished by having theanimal quarters in a separate building, wing, floor, or room. Careful planning should make it possible to place animal housing areas next to or nearresearch laboratories but separated from them by barriers, such as entry
locks, corridors, or floors. Additional considerations include the impact ofnoise and vibration generated from within the facility and from surrounding
areas of the building, as well as security of the facility.
Animals should be housed in facilities dedicated to or assigned for that
purpose, not in laboratories merely for convenience. If animals must be
maintained in a laboratory to satisfy the scientific aims of a protocol, thatspace should be appropriate to house and care for the animals and its use
limited to the period during which it is required. If needed, measures should
be taken to minimize occupational hazards related to exposure to animals
both in the research area and during transport to and from the area.
Centralization Versus Decentralization
In a physically centralized animal facility, support, care, and use areas
are adjacent to the animal housing space. Decentralized animal housing
and use occur in space that is not solely dedicated to animal care or support
or is physically separated from the support areas and animal care personnel.
Centralization often reduces operating costs, providing a more efficient flowof animal care supplies, equipment, and personnel; more efficient use ofenvironmental controls; and less duplication of support services. Centralization reduces the needs for transporting animals between housing and study
sites, thereby minimizing the risks of transport stress and exposure to diseaseagents; affords greater security by providing the opportunity to control facility access; and increases the ease of monitoring staff and animals.
Decentralized animal facilities generally cost more to constructbecause of the requirement for specialized environmental systems and135

PhySICAL PLANT

controls in multiple sites. Duplicate equipment (e.g., cage washers) may
be needed, or soiled materials may need to be moved distances for processing. But decentralization may be preferred for certain specialized
research services such as imaging, quarantine, and proximity to researchfacilities, or for biosecurity reasons. Decentralization may be necessary toaccommodate large or complex equipment, such as magnetic resonanceimaging, or to permit space sharing by users from multiple facilities or
institutions. The opportunity for exposure to disease agents is much greaterin these situations and special consideration should be given to biosecurity, including transportation to and from the site, quarantine before orafter use of the specialized research area, and environmental and equipment decontamination.
The decisions leading to selection of physically centralized versus
decentralized animal facilities should be made early and carefully andshould involve all stakeholders (NRC 1996; Ruys 1991).
FUNCTIONAL AREAS
Professional judgment should be exercised in the development of apractical, functional, and efficient physical plant for animal care and use.
The size, nature, and intensity of an institutional Program (see Chapter 2)
will determine the specific facility and support functions needed. In facilities that are small, maintain few animals, or maintain animals under special
conditions—such as facilities used exclusively for housing gnotobiotic or
specific pathogen-free (SPF) colonies or animals in runs, pens, or outdoor
housing—some functional areas listed below may be unnecessary or may
be included in a multipurpose area.
Space is required for the following:••••

animal housing, care, and sanitation
receipt, quarantine, separation, and/or rederivation of animals
separation of species or isolation of individual projects whennecessarystorage.

Most multipurpose animal facilities may also include the following:•

specialized laboratories or space contiguous with or near animal housing areas for such activities as surgery, intensive care,necropsy, irradiation, preparation of special diets, experimental
procedures, behavioral testing, imaging, clinical treatment, anddiagnostic laboratory procedures

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•
•
•
•

•
•
•
•
•
•

containment facilities or equipment, if hazardous biologic, physical, or chemical agents are to be usedbarrier facilities for housing of SPF rodents, especially valuablegenetically modified animals, or irreplaceable animal models
receiving and storage areas for food, bedding, pharmaceuticals,
biologics, and supplies
space for washing and sterilizing equipment and supplies and,depending on the volume of work, machines for washing cages,
bottles, glassware, racks, and waste cans; a utility sink; a sterilizer
for equipment, food, and bedding; and separate areas for holding
soiled and clean equipment
space for storing wastes before incineration or removal
space for cold storage or disposal of carcasses
space for administrative and supervisory personnel, including spacefor staff training and education
showers, sinks, lockers, toilets, and break areas for personnel
security features, such as card-key systems, electronic surveillance,
and alarms
areas for maintenance and repair of specialized animal housing
systems and equipment.
CONSTRUCTION GUIDELINES
Corridors

Corridors should be wide enough to facilitate the movement of personnel and equipment; a width of 6 to 8 feet can accommodate the needs ofmost facilities. Floor-wall junctions should be designed to facilitate cleaning. Protective rails or bumpers are recommended and, if provided, should
be sealed or manufactured to prevent vermin access. In corridors leading to
dog or swine housing facilities, cage-washing facilities, and other high-noise
areas, double-door entry vestibules or other noise traps should be considered. Similar entries are advisable for areas leading to nonhuman primate
housing as a means to reduce the potential for escape. Double-door entry
vestibules also permit air locks in these and other areas where directional
airflow is critical for containment or protection. Wherever possible, waterlines, drainpipes, reheat coils and valves, electric service connections, andother utilities should be accessible via interstitial space or through accesspanels or chases in corridors outside the animal rooms. Fire alarms, fire
extinguishers, and telephones should be recessed, installed high enough,or shielded by protective guards to prevent damage from the movement oflarge equipment.

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Animal Room Doors
Doors should be large enough (approximately 42 × 84 in.) to allow
the easy passage of racks and equipment and they should fit tightly in theirframes. Both doors and frames should be appropriately sealed to prevent
vermin entry or harborage. Doors should be constructed of and, whereappropriate, coated with materials that resist corrosion. Self-closing doors
equipped with recessed or shielded handles, sweeps, and kickplates andother protective hardware are usually preferable. Hospital or terminatedstops are useful to aid in cleaning (Harris 2005). For safety, doors should
open into animal rooms; if it is necessary that they open toward a corridor,
there should be a recessed vestibule.
Where room-level security is necessary or it is desirable to limit access(as with the use of hazardous agents), room doors should be equipped withlocks or electronic security devices. For personnel safety, doors should bedesigned to open from the inside without a key.
Doors with viewing windows may be needed for safety and other reasons, but the ability to cover these windows may be considered if exposure
to light or hallway activities would be undesirable (e.g., to avoid disturbing
the animals’ circadian rhythm). Red-tinted windows, which do not transmit
specific wavelengths of visible light between corridors and animal rooms,
have proved useful for mouse and rat holding rooms as both species havea limited ability to detect light in the red portions of the spectrum (Jacobs
et al. 2001; Lyubarsky et al. 1999; Sun et al. 1997).
Exterior Windows
The presence of windows in an animal facility, particularly in animalrooms, creates a potential security risk and should generally be avoided.
Windows also create problems with temperature control of the area andprevent strict control of the photoperiod, which is often required in animalrelated protocols (and is a critical consideration in rodent breeding colonies). However, in specific situations, windows can provide environmental
enrichment for some species, such as nonhuman primates.
FloorsFloors should be moisture resistant, nonabsorbent, impact resistant,and relatively smooth, although textured surfaces may be required in somehigh-moisture areas and for some species (e.g., farm animals). Floors shouldbe easy to repair and resistant to both the action of urine and other biologic
materials and the adverse effects of hot water and cleaning agents. They
should be capable of supporting racks, equipment, and stored items without

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becoming gouged, cracked, or pitted. Depending on their use, floors should
be monolithic or have a minimal number of joints. Some materials that have
proved satisfactory are epoxy resins, hard-surface sealed concrete, methyl
methacrylate, polyurethane, and special hardened rubber-base aggregates.
The latter are useful in areas where noise reduction is important. Correct
installation is essential to ensure the long-term stability of the surface. If sills
are installed at the entrance to a room, they should be designed to allow for
convenient passage of equipment.
Drainage
Where floor drains are used, the floors should be sloped and drain
traps kept filled with liquid. To minimize prolonged increases in humidity, drainage should allow rapid removal of water and drying of surfaces(Gorton and Besch 1974). Drainpipes should be at least 4 in. (10.2 cm)
in diameter, although in some areas, such as dog kennels and agricultural
animal facilities, larger drainpipes (>6 in.) are recommended. A rim- and/or
trap-flushing drain or an in-line comminutor may be useful for the disposalof solid waste. When drains are not in use for long periods, they should becapped and sealed to prevent backflow of sewer gases, vermin, and othercontaminants; lockable drain covers may be advisable for this purpose insome circumstances.
Floor drains are not essential in all animal rooms, particularly thosehousing rodents. Floors in such rooms can be sanitized satisfactorily by wet
vacuuming or mopping with appropriate cleaning compounds or disinfectants. But the installation of floor drains that are capped when not in use
may provide flexibility for future housing of nonrodent species.
Walls and Ceilings
Walls and ceilings should be smooth, moisture resistant, nonabsorbent, and resistant to damage from impact. They should be free of cracks,
unsealed utility penetrations, and imperfect junctions with doors, ceilings,
floors, walls, and corners. Surface materials should be capable of withstanding cleaning with detergents and disinfectants and the impact of water
under high pressure. The use of curbs, guardrails or bumpers, and corner
guards should be considered to protect walls and corners from damage,and such items should be solid or sealed to prevent access and harborageof vermin.
Ceilings formed by the concrete slab above are satisfactory if they aresmooth and sealed or painted. Suspended ceilings are generally undesirable in animal holding rooms unless they are sealed from the space above
with gaskets and clips. When used, they should be fabricated of impervi-

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ous materials, have a washable surface, and be free of imperfect junctions.
Exposed plumbing, ductwork, and light fixtures are undesirable unless thesurfaces can be readily cleaned.
Heating, Ventilation, and Air Conditioning (HVAC)
A properly designed and functioning HVAC system is essential to provide
environmental and space pressurization control. Temperature and humidity
control minimizes variations due either to changing climatic conditions or todifferences in the number and kind of animals and equipment in an animal
holding space (e.g., a room or cubicle). Pressurization assists in controlling
airborne contamination and odors by providing directional airflow between
spaces. Areas for quarantine, housing and use of animals exposed to hazardous materials, and housing of nonhuman primates should be kept under relative negative pressure, whereas areas for surgery or clean equipment storage
should be kept under relative positive pressure with clean air.
HVAC systems should be designed for reliability (including redundancy
where applicable), ease of maintenance, and energy conservation; able tomeet requirements for animals as discussed in Chapter 3; and flexible andadaptable to the changing types and numbers of animals and equipment
maintained during the life of the facility (ASHRAE 2007a). They should becapable of adjustments in and ideally maintain dry-bulb temperatures of±1°C (±2°F). Relative humidity should generally be maintained within arange of 30-70% throughout the year. Although maintenance of humidification within a limited range over extended periods is extremely difficult,daily fluctuations (recognizing the effects of routine husbandry especiallywhen caring for large animal species) in relative humidity should be minimized; if excursions outside the desired range are infrequent, minimal, andof short duration, they are unlikely to negatively affect animal well-being.
Ideally relative humidity should be maintained within ±10% of set point;
however, this may not be achievable under some circumstances.
Constant-volume systems have been mostost commonly used in animal
facilities, but variable-volume (VAV) systems may offer design and operational advantages, such as allowing ventilation rates to be set in accordancewith heat load and other variables. These systems offer considerable advantages with respect to flexibility and energy conservation (see Chapter 3).
Previously specified temperature and humidity ranges can be modified
to meet special animal needs in circumstances in which all or most of theanimal facility is designed exclusively for acclimated species with similar
requirements (e.g., when animals are held in a sheltered or outdoor facility).
In addition, modifications may need to take into account the microenvironment in some primary enclosures, such as rodent isolator cages, where
humidity and temperature may exceed room levels.

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Temperature is best regulated by having thermostatic control for eachholding space. Use of zonal control for multiple spaces can result in temperature variations between spaces in the zone because of differences inanimal densities and heat gain or loss in ventilation ducts and other surfaces
within the zone. Individual space control is generally accomplished by providing each space with a dedicated reheat coil. Valves controlling reheat
coils should fail in the closed position; steam coils should be avoided or
equipped with a high-temperature cut-off system to prevent space overheating and animal loss with valve failure.
Humidification is typically controlled and supplemented on a system orzone basis. Control of humidification in individual holding spaces may bedesirable for selected species with reduced tolerance for low relative (e.g.,
nonhuman primates) or high humidity (e.g., rabbits).
Most HVAC systems are designed for average high and low temperatures and humidities experienced in a geographic area within ±5% variation(ASHRAE 2009). Moderate fluctuations in temperature and relative humidity
outside suggested ranges are generally well tolerated by most species commonly used in research as long as they are brief and infrequent; holding
spaces should be designed to minimize drafts and temperature gradients.
Consideration should be given to measures that minimize fluctuations intemperature and relative humidity outside the recommended ranges due toextremes in the external ambient environment. Such measures can include
partial redundancy, partial air recirculation, altered ventilation rates, or theuse of auxiliary equipment. In the event of an HVAC system or component
failure, systems should at the minimum supply facility needs at a reduced
level, address the adverse effects of loss of temperature control, and, where
necessary, maintain critical pressurization gradients. It is essential that lifethreatening heat accumulation or loss be prevented during mechanicalfailure. Temporary needs for ventilation of sheltered or outdoor facilities canusually be met with auxiliary equipment.
Air handling system intake locations should avoid entrainment of fumes
from vehicles, equipment, and system exhaust. While 100% outside air is
typically provided, when recirculated air is used its quality and quantity
should be in accord with recommendations in Chapter 3. The type andefficiency of supply and exhaust air treatment should be matched to thequantity and types of contaminants and to the risks they pose. Supply air isusually filtered with 85–95% dust spot efficient filters (ASHRAE 2008). In
certain instances, higher efficiency filters (e.g., HEPA) may be beneficial forrecirculated supply air and air supplied to or exhausted from specialized
areas such as surgical and containment facilities (Kowalski et al. 2002).

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Power and Lighting
The electrical system should be safe and provide appropriate lighting,a sufficient number of power outlets, and suitable amperage for specialized
equipment. In the event of power failure, an alternative or emergency power
supply should be available to maintain critical services (e.g., the HVAC
system, ventilated caging systems [Huerkamp et al. 2003], or life support
systems for aquatic species) or support functions (e.g., freezers and isolators)
in animal rooms, operating suites, and other essential areas. Consideration
should be given to outfitting movable equipment for which uninterrupted
power is essential (e.g., ventilated racks), with twist-lock plugs to prevent
accidental removal from the power supply.
Light fixtures, timers, switches, and outlets should be properly sealed
to prevent vermin access. Recessed energy-efficient fluorescent lights
are commonly used in animal facilities. Spectral quality of lights may be
important for some species when maintained in the laboratory; in thesecases full spectrum lamps may be appropriate. A time-controlled lighting
system should be used to ensure a uniform diurnal lighting cycle. Override
systems should be equipped with an automatic timeout or a warning lightto indicate the system is in override mode, and system performance andoverride functions should be regularly evaluated to ensure proper cycling.
Dual-level lighting may be considered when housing species that are sensitive to high light intensity, such as albino rodents; low-intensity lighting
is provided during the light phase of the diurnal cycle, and higher-intensity
lighting is provided as needed (e.g., when personnel require enhanced
visibility). Light bulbs or fixtures should be equipped with protective covers to ensure the safety of the animals and personnel. Moisture-resistantswitches and outlets and ground-fault interrupters should be used in areas
with high water use, such as cage-washing areas and aquarium-maintenance areas.
Storage Areas
Adequate space should be available for storage of equipment, supplies,
food, bedding, and refuse. Corridors are not appropriate storage areas.
Storage space can be decreased when delivery of materials and supplies isreliable and frequent; however, it should be ample enough to accommodate storage of essential commodities to ensure the animals’ uninterrupted
husbandry and care (e.g., if delivery is delayed). Bedding and food should
be stored in a separate area free from vermin and protected from the riskof contamination from toxic or hazardous substances. Areas used for foodstorage should not be subject to elevated temperatures or relative humidity
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storage areas. Refrigerated storage, separated from other cold storage, isessential for storage of dead animals and animal tissue waste; this storage
area should be kept below 7°C (44.6°F) to reduce putrefaction of wastes
and animal carcasses and should be constructed in a manner that facilitatescleaning.
Noise Control
Noise control is an important consideration in an animal facility andshould be addressed during the planning stages of new facility design
or renovation (see Chapter 3). Noise-producing support functions, suchas cage washing, are commonly separated from housing and experimental functions. Masonry walls, due to their density, generally have excellent sound-attenuating properties, but similar sound attenuation can beachieved using many different materials and partition designs. For example,
sanitizable sound-attenuating materials bonded to walls or ceilings may beappropriate for noise control in some situations, whereas acoustic materials applied directly to the ceiling or as part of a suspended ceiling in an
animal room present problems for sanitation and vermin control and are
not recommended. Experience has shown that well-constructed corridor
doors, sound-attenuating doors, or double-door entry vestibules can help tocontrol the transmission of sound along corridors. An excellent resource onpartition design for sound control is available in Noise Control in buildings:
A Practical Guide for Architects and Engineers (Warnock and Quirt 1994).
Attention should be paid to attenuating noise generated by equipment(ASHRAE 2007b). Fire and environmental-monitoring alarm systems andpublic address systems should be selected and positioned to minimize
potential animal disturbance. The location of equipment capable of generating sound at ultrasonic frequencies is important as some species can hearsuch high frequencies. Selecting equipment for rodent facilities that does
not generate noise in the ultrasonic range should be considered.
Vibration Control
Vibration may arise from mechanical equipment, electrical switches,and other building components, or from remote sources (via groundborne
transmission). Regarding the latter, special consideration should be given to
the building structure type especially if the animal facility will be locatedover, under, or adjacent to subways, trains, or automobile and truck traffic.
Like noise, different species can detect and be affected by vibrations of different frequencies and wavelengths, so attempts should be made to identify
all vibration sources and isolate or dampen them with vibration suppression
systems (ASHRAE 2007b).

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Facilities for Sanitizing Materials
A dedicated central area for sanitizing cages and ancillary equipment
should be provided. Mechanical cage-washing equipment is generally
needed and should be selected to match the types of caging and equipmentused. Consideration should be given to such factors as the following:
•
•
•
•
•
•
•
•
•
•
•
•
•

location with respect to animal rooms and waste disposal and storage areas
ease of access, including doors of sufficient width to facilitate
movement of equipment
sufficient space for staging and maneuvering of equipment
soiled waste disposal and prewashing activities
ease of cleaning and disinfection of the area
traffic flow that separates animals and equipment moving between
clean and soiled areas
air pressurization between partitioned spaces to reduce the potential of cross contamination between soiled and clean equipment
insulation of walls and ceilings where necessary
sound attenuation
utilities, such as hot and cold water, steam, floor drains, and electric
power
ventilation, including installation of vents or canopies and provisions for dissipation of steam and fumes from sanitizing processes
vibration, especially if animals are housed directly above, below,
or adjacent to the washing facility
personnel safety, by ensuring that safety showers, eyewash stations,and other equipment are provided as required by code; exposed
hot water and steam lines are properly insulated; procedures witha propensity to generate aerosols are appropriately contained; andequipment, such as cage/rack washers, and bulk sterilizers, which
personnel enter, are equipped with functioning safety devices thatprevent staff from becoming trapped inside.
Environmental Monitoring

Monitoring of environmental conditions in animal holding spaces andother environmentally sensitive areas in the facility should be considered.
Automated monitoring systems, which notify personnel of excursions inenvironmental conditions, including temperature and photoperiod, areadvisable to prevent animal loss or physiologic changes as a result of system malfunction. The function and accuracy of such systems should be
regularly verified.

144

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALSSPECIAL FACILITIESSurgery
The design of a surgical facility should accommodate the species to be
operated on and the complexity of the procedures to be performed (Hessler
1991; see also Appendix A, Design and Construction of Animal Facilities).
The facility, including that used for rodents, by necessity becomes larger
and more complex as the number and size of animals or the complexityof procedures increase. For instance, a larger facility may be required toaccommodate procedures on agricultural species, large surgical teams,
imaging devices, robotic surgical systems, and/or laparoscopic equipment
towers. Surgical facilities for agricultural species may additionally require
floor drains, special restraint devices, and hydraulic operating tables.
For most survival surgery performed on rodents and other small species such as aquatics and birds, an animal procedure laboratory is recommended; the space should be dedicated to surgery and related activities
when used for this purpose, and managed to minimize contamination fromother activities conducted in the room at other times. The association ofsurgical facilities with diagnostic laboratories, imaging facilities, animal
housing, staff offices, and so on should be considered in the overall context
of the complexity of the surgical program. Centralized surgical facilities are
cost-effective in equipment, conservation of space and personnel resources,
and reduced transit of animals. They also enable enhanced personnel safety
and professional oversight of both facilities and procedures.
For most surgical programs, functional components of aseptic surgery
include surgical support, animal preparation, surgeon’s scrub, operating
room, and postoperative recovery. The areas that support those functions
should be designed to minimize traffic flow and separate the related nonsurgical activities from the surgical procedure in the operating room. The
separation is best achieved by physical barriers (AORN 1993) but may also
be achieved by distance between areas or by the timing of appropriate
cleaning and disinfection between activities.
Surgical facilities should be sufficiently separate from other areas tominimize unnecessary traffic and decrease the potential for contamination(Humphreys 1993). The number of personnel and their level of activity havebeen shown to be directly related to the level of bacterial contaminationand the incidence of postoperative wound infection (Fitzgerald 1979). Traffic in the operating room can be reduced by the installation of an observation window, a communication system (such as an intercom system), andjudicious location of doors.
Control of contamination and ease of cleaning should be key considerations in the design of a surgical facility. The interior surfaces should be

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constructed of materials that are monolithic and impervious to moisture.
Ventilation systems supplying filtered air at positive pressure can reduce
the risk of postoperative infection (Ayscue 1986; Bartley 1993; Schonholtz1976). Careful location of air supply and exhaust ducts and appropriate
room ventilation rates are also recommended to minimize contamination(Ayliffe 1991; Bartley 1993; Holton and Ridgway 1993; Humphreys 1993).
To facilitate cleaning, the operating rooms should have as little fixed equipment as possible (Schonholtz 1976; UFAW 1989). Other operating roomfeatures to consider include surgical lights to provide adequate illumination (Ayscue 1986); sufficient electric outlets for support equipment; gases
to support anesthesia, surgical procedures, and gas-powered equipment;
vacuum; and gas-scavenging capability.
The surgical support area should be designed for washing and sterilizing
instruments and for storing instruments and supplies. Autoclaves are commonly placed in this area. It is often desirable to have a large sink in the
animal preparation area to facilitate cleaning of the animal and the operating facilities. A dressing area should be available for personnel to change
into surgical attire; a multipurpose locker room can serve this function.
There should be a scrub area for surgeons, equipped with foot, knee, or
electric-eye surgical sinks (Knecht et al. 1981). To minimize the potential forcontamination of the surgical site by aerosols generated during scrubbing,the scrub area should usually be outside the operating room and animal
preparation area.
A postoperative recovery area should provide the physical environment to support the needs of the animal during the period of anesthetic
and immediate postsurgical recovery and should be sited to allow adequate
observation of the animal during this period. The electric and mechanicalrequirements of monitoring and support equipment should be considered.
The type of caging and support equipment will depend on the species andtypes of procedures but should be designed to be easily cleaned and tosupport physiologic functions, such as thermoregulation and respiration.
Depending on the circumstances, a postoperative recovery area for farmanimals may be modified or nonexistent in some field situations, but precautions should be taken to minimize risk of injury to recovering animals.
barrier Facilities
Barrier facilities are designed and constructed to exclude the introduction of adventitious infectious agents from areas where animals of a defined
health status are housed and used. They may be a portion of a larger facility
or a free-standing unit. While once used primarily for rodent production
facilities and to maintain immunodeficient rodents, many newer facilities
incorporate barrier features for housing specific pathogen-free (SPF) mice

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and rats, especially valuable genetically engineered animals, and SPF animals of other species.
Barrier facilities typically incorporate airlock or special entries (e.g., airor wet showers) for staff and supplies. Staff generally wear dedicated clothing and footwear, or freshly laundered, sterile, or disposable outer garments
such as gowns, head and shoe covers, gloves, and sometimes face masksprior to entry. Consumables, such as feed or bedding, that may harbor
infectious agents are autoclaved or are gamma-irradiated by the supplierand surface decontaminated on entry. Drinking water may be autoclaved or
subject to specialized treatment (e.g., reverse osmosis filtration) to remove
infectious agents. Caging and other materials with which the animals havedirect contact may be sterilized after washing before reuse. Strict operational procedures are frequently established to preclude intermingling ofclean and soiled supplies and personnel groups, depending on work function. Only animals of defined health status are received into the barrier,and once they leave they are prohibited from reentering without retesting.
Personnel entry is restricted and those with access are appropriately trainedin procedures that minimize the introduction of contaminants.
Engineering features may include high-level filtration of supply air (e.g.,
HEPA or 95% efficient filters), pressurization of the barrier with respect tosurrounding areas, and directional airflow from clean to potentially contaminated areas. Specialized equipment augmenting the barrier may include
isolator cages, individually ventilated cages, and animal changing stations.
Detailed information on barrier design, construction, and operations
has been recently published (Hessler 2008; Lipman 2006, 2008).
Imaging
In vivo imaging offers noninvasive methods for evaluating structureand function at the level of the whole animal, tissue, or cell, and allows
for the sequential study of temporal events (Chatham and Blackband 2001;
Cherry and Gambhir 2001). Imaging devices vary in the technology used
to generate an image, body targets imaged, resolution, hazard exposure,and requirements for use. The devices may be self-shielded and require no
modifications of the surrounding structure to operate safely, or they may
require concrete, solid core masonry, lead-, steel-, or copper-lined walls,
or other construction features to operate safely or minimize interferencewith devices and activities in adjacent areas. Because imaging devices areoften expensive to acquire and maintain, and may require specialized support space and highly trained personnel to operate, shared animal imaging
resources may be preferable.
Consideration should be given to the location of the imaging resource.
Whether located in the animal facility or in a separate location, cross

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contamination between groups of animals, different animal species, or
between animals and humans (if the device is used for both animal andhuman subjects) is possible because these devices may be difficult to sanitize (Klaunberg and Davis 2008; Lipman 2006). If the imaging resource islocated outside the animal facility, appropriate transportation methods androutes should be developed to avoid inappropriate exposure of humans toanimals in transit. If possible, animals should not be moved past offices,
lunch rooms, or public areas where people are likely to be present.
As imaging may require the subject to be immobile, often for extended
time periods during image acquisition, provisions should be made for delivery of anesthetics and carrier gas, the scavenging of waste anesthetic gas,
and adequate animal monitoring (Balaban and Hampshire 2001). Remotestorage of gas tanks is generally required in facilities where magnetic resonance (MR) scanners are used as the magnetic field requires ferrous materials to be kept a safe distance away from the magnet. Site selection of MR
scanners requires special attention because of their weight, the fringe field
generated (especially from unshielded magnets), and the impact of ferrous
elements of the building structure or its components, especially those thatare not static (e.g., elevators), as they may affect field homogeneity. Most
MR scanners are superconducting and require the use of cryogens. Because
cryogen boil-off can lead to asphyxiation of both personnel and animals,
rooms with MR scanners or in which cryogen gases are stored must be
equipped with oxygen sensors and a method for increasing room ventilation
to exhaust inert gases during cryogen filling (Klaunberg and Davis 2008).
Many imaging devices, especially those designed for small animals, areself-contained and require no special physical plant considerations. Provisions should be made to locate the operating console away from imaging
devices that emit ionizing or magnetic radiation. Imaging devices with components that are difficult to sanitize should be covered with a disposable orsanitizable material when not in use.
Whole body Irradiation
Total body irradiation of small laboratory animals may be accomplished using devices that emit either gamma- or X-rays. Devices are usually self-shielded and, because of the weight of the shielding material, may
require special site considerations. Devices with gamma-emitting sources
are subject to regulations that require adherence to specific security, monitoring, and personnel clearance requirements (Nuclear Regulatory Commission 2008). The site selected for irradiators should also take into account
whether they are to be used for animals and biologics, as well as the source
and microbial status of the animals to be irradiated. Locating them in the
animal facility may require access for personnel who would normally not

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require it or may necessitate bringing animals into a facility where they are
not normally housed.
Hazardous Agent Containment
The goal of containment is to “reduce or eliminate exposure of laboratory
workers, other persons, and the outside environment to potential hazardous
agents” (DHHS 2010). This is accomplished by employing appropriate practices and equipment, vaccinating personnel if a vaccine is available, andensuring the proper design and operation of the physical plant.
Animal facilities used to study biologic agents that are infectious tohumans are categorized into different biosafety levels of escalating containment requirements as described in biosafety in Microbiological andbiomedical Laboratories (BMBL; DHHS 2009 or most recent version). Eachanimal biosafety level (ABSL) reflects a combination of practices, safety
equipment, and facilities based on risk of human infection. As described in
the 2009 edition of the BMBL, ABSL-1 contains agents not known to cause
human infection; ABSL-2 contains agents of moderate risk that cause humandisease by ingestion or percutaneous or mucosal exposure; ABSL-3 contains
agents that cause serious and potentially lethal infections and have known
potential for aerosol transmission; and ABSL-4 contains nonindigenous(exotic) agents that pose high individual risk of life-threatening disease andfor which there is no available vaccine or treatment. Facility design, engineering criteria, construction methods and materials, commissioning, andvalidation become more important with each increasing level. The BMBL
should be consulted for specific design and engineering requirements.
Considerable care should be taken when selecting the team of professionals
responsible for the design, engineering, construction, and commissioningof a containment facility.
Guidelines have also been developed for containing agricultural pathogens (USDA ARS 2002), recombinant DNA molecules (NIH 2002), arthropod vectors (ACME, ASTMH 2003), and hazardous chemicals (NRC 1995).
Biologic agents and toxins pose a threat to animal and plant health or
public health and safety, and facilities in which they are used must adhere
to APHIS, USDA, and CDC Select Agent Regulations (CFR 2005; CDC andDHHS 1996; PL 107-56; PL 107-188;) and/or other applicable federal, state,
or local regulations. These regulations stipulate, among other requirements,that the institution registered to use select agents establish and adhere to
stringent security measures.
The specific facility features, equipment, and safety practices to be
employed will depend, to a considerable extent, on whether a specifichazard is a particulate, volatile, or both. Facility features applicable to all
hazards include isolation of the animals and their waste, provision of sealed

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monolithic room surfaces that do not promote dust accumulation and areeasy to sanitize, increased air exchange rates to dilute environmental contamination if it occurs, air pressure differentials to ensure that areas containing hazards have negative pressure with respect to surrounding areas,
specialized housing systems, if available, and appropriate safety equipment
such as a biologic safety cabinet or chemical hood (CDC and NIH 2007).
A number of references are available to provide an overview of the issues
related to hazardous material containment (Frazier and Talka 2005; Lehneret al. 2008; Lieberman 1995; NRC 1989, 1995)
behavioral Studies
When planning a behavioral facility, special attention should be given
to all aspects of facility design, construction, equipment, and use that may
generate conditions that inappropriately stimulate the senses of the test
animals. It is frequently necessary to maintain animals in an environment,
especially during periods of testing and observation, with strict control overauditory, visual, tactile, and olfactory stimuli. The facility site, as well as theengineering and construction methods used, should be carefully selected
to minimize airborne transmission of noise and groundborne transmissionof vibration.
Noise and vibration may arise from the building’s structure, its equipment, or from human activities (see section on Noise). The frequencies
and intensity of sound, which stimulate auditory responses in the species
being investigated, should guide the selection of construction materials,
techniques, and equipment to minimize intrusions. For instance, the HVAC
system should be designed and components selected to ensure that noise,
including ultrasonic frequencies, is not generated; fire alarm annunciatorsthat emit sound at a frequency not audible to rodents should be used; hardware should be provided on doors to enable them to close quietly; nonessential noise-generating equipment should be housed outside the study area;and personnel traffic should be minimized both in animal testing areas andin areas contiguous to them (Heffner and Heffner 2007). Attention should begiven to the control of aberrant visual cues, especially in circadian studies.
The selection of the type, intensity, and control of lighting will likely differ
from other animal facility areas. A variety of specialized housing and testing
systems may also need to be accommodated in the facility.
Special construction features may also be desirable. Double-door vestibule entries to the behavioral facility, testing suites, or individual testingrooms may be useful as they can prevent noise, odors, and light from entering
the behavioral testing area. Floor coverings that reduce sound transmission
should be selected. Testing rooms may require floor drains, water sources,
and increased floor loading to support specific behavioral testing apparatus.

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Consideration should be given to the types and amount of electronics andother equipment used to ensure that the HVAC system can accommodate theassociated heat loads. Airlocks and air pressure differentials between spaces
can provide olfactory segregation of species and activities and thus reduce
the risk of altered behavioral responses (ASHRAE 2007c).
When possible, testing equipment should be designed in such a way as
to allow surface disinfection between studies. Components that cannot becleaned or disinfected, such as computers and recording equipment, should
be located in areas where contact with animals is unlikely and should becovered when not in use (the use of computer keyboard covers may also bebeneficial). Provision of sufficient space for storage of behavioral apparatus
and equipment should also be considered. As transportation to and from thetesting area may alter behavioral responses, consideration should be given toproviding housing areas contiguous with those used for testing; if such areas
are provided, they should meet the requirements specified in the Guide.
Aquatic Species Housing
Many of the construction features described above are applicable tothose for aquatic species, but particular consideration should be given to
the housing systems used and the methods for maintaining the aquatic
environment.
The complexity of the life support system depends on the species
housed and the size, type, and number of tanks and animals supported. All
systems require a water source, which may require prior treatment (e.g.,
ultraviolet sterilization and particulate, carbon, and ultrafiltration). Holding areas for aquatic species should be provided with drains of a suitablesize and number to accommodate water released during system operationand maintenance or as a result of life support system or tank failure. Drains
should not permit passage of animals or hazardous materials into the sanitary system without appropriate treatment.
Materials used for floors, walls, and ceilings should be impervious towater while floors should be slip resistant and able to withstand the loads
inherent with large quantities of water. Electrical receptacles or circuits should
be ground-fault interrupted to prevent electrocution of personnel and animals.
Doors and frames, supply diffusers, exhaust registers, lighting fixtures, HVAC
ducts and components (exposed to high levels of moisture or corrosives), andother metallic elements should be made of moisture- and corrosion-resistantmaterials. Housing systems, life support system components, and plumbing
used to distribute water after treatment, including adhesives to connect
components, should be constructed of materials that are nontoxic and biologically inert. If the macroenvironmental/room HVAC system is used as theprimary method for tempering the aquatic environment, sufficient ventila-

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tion should be provided to prevent moisture buildup on room surfaces andmaintain suitable temperatures for the species housed.
SECURITy AND ACCESS CONTROL
Recent episodes of domestic terrorism have heightened awareness of
the importance of animal facility security, but there are other reasons why
security and access control should be provided. Most animals maintained
for research are vulnerable to infection with adventitious agents and therefore access to them should be strictly controlled and made available only
to personnel who have received appropriate training and have a legitimate
need for access. Animals used in studies with hazardous materials require
special precautions for personnel before access, and staff entering the animal facility should have completed the institution’s occupational health andsafety training.
When possible, the animal facility should be located within anotherstructure with its own independent set of security features. Vehicular accessshould be limited and, when provided, controlled and monitored.
Security and access control are generally provided in zones, starting at
the perimeter with areas of highest security located within other zones. Control measures may consist of security personnel, physical barriers, and control
devices. The scope of the security system should depend on the size of the
facility as well as the nature of the activities conducted within. Increasingly,
access control is extended from the facility’s perimeter to each animal holding
room. Microprocessor-controlled security systems are frequently employedbecause of the large number of control points and staff requiring access.
These systems typically use electronic key or proximity cards and associatedreaders, which, in addition to controlling access, enable recording of the time,location, and personal identification of each entry. In more sensitive areas,
biometric reading devices such as thumb or palm readers or retinal scanners
may be more suitable because key cards can be shared. Security may beenhanced with electronic and video surveillance systems. These systems may
be monitored by personnel or motion-activated recording devices.

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Addendum
Guide for the Care and Useof Laboratory Animals
Eighth Edition

ADDENDUM: LIST OF EDITORIAL CHANGESFROM THE PREPUbLICATION VERSION1
1. Page 22. Original sentence: “Personnel training should include
information on laboratory animal allergies, preventive control measures and proper techniques for working with animals (Gordon etat. 1997; Schweitzer et al. 2003; Thulin et al. 2002).” “…earlyrecognition and reporting of allergy symptoms” was added to clarify
the guidance and reflect the cited references.
2. Page 31. Original sentence: “They should therefore be used, whenavailable, for all animal-related procedures (NIH 2008; USDA
1997b).” The NIH reference was removed because it applies only
to the NIH intramural animal research program.
3. Page 32. Original sentence: “Principal investigators conducting fieldresearch should be knowledgeable of relevant zoonotic diseases,
associated safety issues, and, when working in an international
environment, any local laws or regulations that apply.” Compliancewith laws and regulations applies to field investigations irrespectiveof location, so “when working in an international environment”
and “local” were deleted.

1 Page numbers reflect placement of revisions in this report.

155

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4. Page 32. Original sentence: “Appropriate veterinary input is needed
for projects involving capture, individual identification, sedation,
anesthesia, surgery, recovery, holding, transportation, release, or
euthanasia.” Beginning of sentence changed to “Veterinary input
may be needed…” to clarify the intent of the sentence.
5. Page 43. Original sentence: “The ambient temperature range inwhich thermoregulation occurs without the need to increase metabolic heat production is called the thermoneutral zone (TNZ) andis bounded by the upper (UCT) and lower critical temperatures(LCT).” The phrase “or activate evaporative heat loss mechanisms”
and reference to Gordon 2005 were added to provide a more complete definition of thermoneutral zone.
6. Page 45. Original sentence: “In climates where it is difficult to provide a sufficient level of environmental relative humidity, animals
should be closely monitored for negative effects such as excessively
flaky skin in birds and mammals, ecdysis (molting) difficulties in reptiles, and desiccation stress in semiaquatic amphibians.” Because
the significance of excessive flaky skin varies among species, “inbirds and mammals” was deleted.
7. Page 55. Original sentence: “Thus there is no ideal formula for
calculating an animal’s space needs based only on body size or
weight.” The phrase “and readers should take the performanceindices discussed in this section into consideration when utilizing
the species-specific guidelines presented in the following pages”
was added to further clarify the intent of this section.
8. Pages 57-63. The phrase “the interpretation of this table shouldtake into consideration the performance indices described in the
text beginning on page 55” was added as a footnote to tables asadditional guidance to their interpretation. Additionally, the symbol “≥” was restored (due to mistaken deletion) in Table 3.2 (micein groups; rats in groups; hamsters; guinea pigs) and added toTables 3.5 (group 8) and 3.6 (sheep and goats)
9. Page 69. Original sentence: “Cedar shavings are not recommended
because they emit aromatic hydrocarbons that induce hepatic microsomal enzymes and cytotoxicity (Torronen et al. 1989; Weichbrod
et al. 1986, 1988).” Text mistakenly deleted was restored to theend of the sentence: “and have been reported to increase the incidence of cancer (Jacobs and Dieter 1978; Vlahakis 1977).”

AddENdUM

15

10. Page 107. Original sentence: “The Centers for Disease Control
and Prevention enforces regulations to prevent the introduction,transmission, or spread of communicable diseases and regulate
the importation of any animal or animal product capable of carrying a zoonotic disease.” Because USDA also has jurisdiction overimports, it was added to the sentence.
11. Page 107. Original sentence: “The US Fish and Wildlife Service
regulates importation/exportation and interstate trade of wild vertebrate and invertebrate animals and their tissues.” Because US Fish
and Wildlife Services does not regulate interstate trade except for
species listed under the Endangered Species Act, “and interstate
trade” was deleted
12. Page 120. Original sentence: “Additional care might be warranted,
including long-term administration of parenteral fluids (FBR 1987),
analgesics and other drugs; and care of surgical incisions.” Because
this is now standard veterinary medical practice and the cited reference is no longer in print, the reference was removed.
13. Page 120. Original sentence: “In general, unless the contrary is
known or established, it should be considered that procedures thatcause pain in humans may also cause pain in vertebrate species(IRAC 1985).” For consistency with the text of the U.S. Government Principles the phrase “The U.S. Government Principles for
the Utilization and Care of Vertebrate Animals Used in Testing,Research, and Training (see Appendix B) state that…” was added
and the words “in vertebrate species” were replaced with “in otheranimals.”
14. Page 143. Original phrase: “Vibration, especially if animals arehoused directly above the washing facility,” was expanded because
animals housed below or adjacent to the washing facility are also
subject to potential vibration.
15. Added references: pages 163; 169; 179; 182; 192.

Appendicesappendix

A
Additional Selected References

SUbJECT MATTERUSE OF LABORATORY ANIMALS
Alternatives
Ethics and Welfare
Experimental Design and Statistics
Research and Testing Methodology
PROGRAM MANAGEMENT
General References
Laws, Regulations, and Policies
Education
Monitoring the Care and Use of Animals
Occupational Health and Safety
ENVIRONMENT, HOUSING, AND MANAGEMENT
General References
Environmental Enrichment
Genetics and Genetically Modified Animals
Species-Specific References—Environment, Housing, and Management
Agricultural Animals
Amphibians, Reptiles, and Fish
Birds
Cats and Dogs
Exotic, Wild, and Zoo Animals
161

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Nonhuman Primates
Rodents and Rabbits
Other Animals
VETERINARY CARE
Transportation
Anesthesia, Pain, and Surgery
Disease Surveillance, Diagnosis, and Treatment
Pathology, Clinical Pathology, and Parasitology
Species-Specific References—Veterinary Care
Agricultural Animals
Amphibians, Reptiles, and Fish
Birds
Cats and Dogs
Exotic, Wild, and Zoo Animals
Nonhuman Primates
Rodents and Rabbits
DESIGN AND CONSTRUCTION OF ANIMAL FACILITIESUSE OF LAbORATORy ANIMALS
Alternatives
Alternative Methods for Toxicity Testing: Regulatory Policy Issues. EPA 230/12 85 029. NTISPB8 6 113404/AS. Washington: Office of Policy, Planning, and Evaluation, US Environmental Protection Agency.
Alternatives to Animal Use in Research, Testing, and Education. 1986. Office of Technology
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Alternatives to Current Uses of Animals in Research, Safety Testing, and Education. 1986.
Stephens ML. Washington: Humane Society of the United States.
Alternatives to Pain in Experiments on Animals. 1980. Pratt D. New York: Argus Archives.
Alternative Toxicological Methods. 2003. Salem H, Katz S. Boca Raton FL: CRC Press.
Animals and Alternatives in Testing: History, Science, and Ethics. 1994. Zurlo J, Rudacille D,
Goldberg AM. New York: Mary Ann Liebert Publishers.
Future improvements: Replacement in vitro methods. 2002. Balls M. ILAR J 43(Suppl):
S69-S73.
ICCVAM Recommendations on In Vitro Methods for Assessing Acute Systemic Toxicity. 2001.
Available at http://iccvam.niehs.nih.gov/docs/acutetox_docs/finalrpt//finappi2.pdf; accessed January 24, 2010.
Regulatory Testing and Animal Welfare. 2002. ILAR J 43(Supplement).
Implementation of the 3Rs (refinement, reduction, and replacement): Validation and regulatory acceptance considerations for alternative toxicological test methods. 2002.
Schechtman L. ILAR J 43:S85-S94.
Incorporating the 3Rs into regulatory scientific practices. 2002. Sterling S, Rispin A. ILAR
J 43:S18-S20.

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Gluck JP, DiPasquale T, Orlans FB. West Lafayette IN: Purdue University Press.
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Experimental Design and Statistics
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Surgery: Basic principles and procedures. 2003. Waynforth HB, Swindle MM, Elliott H, Smith
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Textbook of Small Animal Surgery, 3rd ed. 2003. Slatter D. Philadelphia: WB Saunders.
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Moberg GP, Mench JA. Wallingford UK: CAB International.
The IACUC Handbook. 2006. Flecknell P, Silverman J, Suckow MA, Murthy S, eds. New York:CRC Press.
The importance of awareness for understanding fetal pain. 2005. Mellor DJ, Diesch TJ, Gunn
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When does stress become distress? 1999. Moberg GP. Lab Anim 28:422-426.

Disease Surveillance, Diagnosis, and Treatment
Clinical Laboratory Animal Medicine. 2008. Hrapkiewicz K, Medina L, eds. San Francisco:Wiley-Blackwell.
Current strategies for controlling/eliminating opportunistic microorganisms. 1998. White WJ,
Anderson LC, Geistfeld J, Martin D. ILAR J 39:391-305.
Drug Dosage in Laboratory Animals: A Handbook. 1989. Borchard RE, Barnes CD, Eltherington
LG. West Caldwell NJ: Telford Press.
FELASA recommendations for the health monitoring of breeding colonies and experimental
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Ferrets, Rabbits and Rodents: Clinical Medicine and Surgery. 1997. Hillyer EV, Quesenberry
KE. Philadelphia: WB Saunders.
Handbook of Veterinary Drugs: A Compendium for Research and Clinical Use. 1975. Rossoff
IS. New York: Springer.
Invertebrate Medicine. 2006. Lewbart GA. Ames: Blackwell Publishing.
Kirk and Bistner’s Handbook of Veterinary Procedures and Emergency Treatment, 8th ed. 2006.
Ford RB, Mazzaferro E. Philadelphia: WB Saunders.
Laboratory Animal Medicine. 2002. Fox JG, Anderson LC, Loew FM, Quimby FW, eds. New
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Mosby’s Fundamentals of Animal Health Technology: Principles of Pharmacology. 1983.
Giovanni R, Warren RG, eds. St. Louis: CV Mosby.
Veterinary Applied Pharmacology and Therapeutics, 5th ed. 1991. Brander GC, Pugh DM,
Bywater RJ. London: Bailliere Tindall.
Veterinary Pharmacology and Therapeutics, 6th rev. ed. 1988. Booth NH, McDonald LE. Ames:
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Pathology, Clinical Pathology, and Parasitology
An Atlas of Laboratory Animal Haematology. 1981. Sanderson JH, Phillips CE. Oxford: Clarendon Press.
An Introduction to Comparative Pathology: A Consideration of Some Reactions of Humanand Animal Tissues to Injurious Agents. 1962. Gresham GA, Jennings AR. New York:
Academic Press.
Animal Clinical Chemistry: A Practical Guide for Toxicologists and Biomedical Researchers,
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Animal Hematotoxicology: A Practical Guide for Toxicologists and Biomedical Researchers.
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Atlas of Experimental Toxicological Pathology. 1987. Gopinath C, Prentice DE, Lewis DJ.
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Blood: Atlas and Sourcebook of Hematology, 2nd ed. 1991. Kapff CT, Jandl JH. 1991. Boston:
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Clinical Biochemistry of Domestic Animals, 4th ed. 1989. Kaneko JJ, ed. New York: Academic
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Clinical Chemistry of Laboratory Animals. 1988. Loeb WF, Quimby FW. New York: Pergamon
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Color Atlas of Comparative Veterinary Hematology. 1989. Hawkey CM, Dennett TB. Ames:
Iowa State University Press.
Color Atlas of Hematological Cytology, 3rd ed. 1992. Hayhoe GFJ, Flemans RJ. St. Louis:
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Comparative Neuropathology. 1962. Innes JRM, Saunders LZ, eds. New York: Academic
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Duncan and Prasse’s Veterinary Laboratory Clinical Pathology. 2003. Latimer KS, Mahaffey EA,
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Essentials of Veterinary Hematology. 1993. Jain NC. Philadelphia: Lea and Febiger.
Flynn’s Parasites of Laboratory Animals, 2nd ed. 2007. Baker DG, ed. Ames: Iowa State University Press.
Handbook of Laboratory Animal Bacteriology. 2000. Hanson AK. Boca Raton FL: CRC Press.
Immunologic Defects in Laboratory Animals. 1981. Gershwin ME, Merchant B, eds. New
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Laboratory Profiles of Small Animal Diseases. 1981. Sodikoff C. Santa Barbara: American
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Natural Pathogens of Laboratory Animals: Their Effects on Research. 2003. Baker DG. Washington: American Society for Microbiology.
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New York: Academic Press.
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Springer-Verlag.
The Pathology of Laboratory Animals. 1965. Ribelin WE, McCoy JR, eds. Springfield IL: Charles
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Veterinary Clinical Parasitology, 6th ed. 1994. Sloss MW, Kemp RL. 1994. Ames: Iowa State
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Veterinary Pathology, 5th ed. 1983. Jones TC, Hunt RD. Philadelphia: Lea and Febiger.

Species-Specific References—Veterinary Care
Agricultural Animals
Basic Surgical Exercises Using Swine. 1983. Swindle MM. 1983. New York: Praeger.
Diseases of Poultry, 9th ed. 1991. Calnek BW, Barnes HJ, Beard CW, Reid WM, Yoder HW,
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Diseases of Sheep. 1974. Jensen R. Philadelphia: Lea and Febiger.
Diseases of Swine, 7th ed. 1992. Leman AD, Straw BE, Mengeline WL, eds. Ames: Iowa State
University Press.
FELASA recommendations for the health monitoring of breeding colonies and experimental
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Swine in the Laboratory: Surgery, Anesthesia, Imaging, and Experimental Techniques, 2nd ed.
Swindle MM, ed. 2007. Boca Raton FL: CRC Press.

12

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALSTechniques in Large Animal Surgery, 3rd ed. 2007. Hendrickson D, ed. 2007. Somerset NJ:
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Textbook of Large Animal Surgery, 2nd ed. 1987. Oehme FW, Prier JE. Baltimore: Williams
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Amphibians, Reptiles, and Fish
An evaluation of current perspectives on consciousness and pain in fishes. 2004. Chandroo
KP, Yue S, Moccia RD. Fish Fisher 5:281-295.
Anesthesia and analgesia in reptiles. Mosley CA. 2005. Semin Avian Exot Pet Medic
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Can fish suffer? Perspectives on sentience, pain, fear and stress. 2004. Chandroo KP, Duncan
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Disease Diagnosis and Control in North American Marine Aquaculture, 2nd rev ed. 1988.
Sindermann CJ, Lichtner DV. New York: Elsevier.
Diseases of Fishes. 1971. Bullock GL. Book 2B: Identification of Fish Pathogenic Bacteria.
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Diseases of Fishes. 1971. Bullock GL, Conroy DA, Snieszko SF. Neptune NJ: TFH Publications.
Diseases of Fishes. 1974. Anderson DP. Book 4: Fish Immunology. Neptune NJ: TFH
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Diseases of Fishes. 1976. Wedemeyer GA, Meyer FP, Smith L. Book 5: Environmental Stressand Fish Diseases. Neptune NJ: TFH Publications.
Do fish have nociceptors? Evidence for the evolution of a vertebrate sensory system. 2003.
Sneddon LU, Braithwaite VA, Gentle MJ. Proc R Soc Lond B 270:1115-1121.
Evaluation of rapid cooling and tricaine methanesulfonate (MS-222) as methods of euthanasia
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Evaluation of the use of anesthesia and analgesia in reptiles. 2004. Read MR. JAVMA
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Fish and welfare: Do fish have the capacity for pain perception and suffering? 2004. Braithwaite VA, Huntingford FA. Anim Welf 13:S87-S92.
Fish, amphibian, and reptile analgesia. Machin KL. 2001. Vet Clin North Am Exot Anim Pract
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Infectious Diseases and Pathology of Reptiles: Color Atlas and Text. 2007. Jacobson E. Boca
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Medicine and Surgery of Tortoises and Turtles. 2004. McArthur S, Wilkinson R, Meyer J. San
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Mycobacteriosis in fishes: A review. 2009. Gauthier DT, Rhodes MW. Vet J 180:33-47.
Pain and Distress in Fish. 2009. ILAR J 50(4).
Fish sedation, analgesia, anesthesia, and euthanasia: Considerations, methods, and typesof drugs. 2009. Neiffer DL, Stamper MA. ILAR J 50:343-360.
Morphologic effects of the stress response in fish. 2009. Harper C, Wolf JC. ILAR J
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Pain perception in fish: Indicators and endpoints. 2009. Sneddon LU. ILAR J
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Parasites, behaviour and welfare in fish. 2007. Barber I. Appl Anim Behav Sci 104:251-264.
Parasites of Freshwater Fishes: A Review of Their Treatment and Control. 1974. Hoffman GL,
Meyer FP. Neptune NJ: TFH Publications.
Recommendations for control of pathogens and infectious diseases in fish research facilities.
2009. Kent ML, Feist SW, Harper C, Hoogstraten-Miller S, Mac Lawe J, Sánchez-Morgado
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The ability of clove oil and MS-222 to minimize handling stressing rainbow trout (Oncorhynchus mykiss Walbaum). 2003. Wagner GN, Singer TD, McKinley RS. Aquacult Res
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The efficacy of clove oil as an anaesthetic for rainbow trout, Oncorhynchus mykiss (Walbaum).
1998. Keene JL, Noakes DLG, Moccia RD, Soto CG. Aquacult Res 29:89-101.
The neurobehavioral nature of fishes and the question of awareness and pain. Rose JD. 2002.
Rev Fish Sci 10:1-38.
The Pathology of Fishes. 1975. Ribelin WE, Migaki G, eds. Madison: University of Wisconsin.

birds
Avian Disease Manual, 6th ed. 2006. Charlton BR, ed. American Association of Avian Pathologists. Jacksonville, FL. Available at www.aaap.info.
Clinical Avian Medicine. 2006. Harrison GJ, Lightfoot TL, eds. 2006. Sphinx Publishing.
Diseases of Wild Waterfowl, 2nd ed. 1997. Wobeser GA. New York: Plenum Press.
Field Manual of Wildlife Disease: General Field Procedures and Diseases of Birds. 1999. Friend
M, Franson JC, eds. US Geological Survey. Available at www.nwhc.usgs.gov/publications/field_manual/field_manual_of_wildlife_diseases.pdf; accessed January 24, 2010.
Handbook of Avian Medicine. 2009. Tully TN, Dorrestein GM, Jones AK, eds. Philadelphia:
WB Saunders.
Pigeon Health and Disease. 1991. Tudor DC. Ames: Iowa State University Press.

Cats and dogs
FELASA recommendations for the health monitoring of breeding colonies and experimental
units of cats, dogs and pigs. 1998. Rehbinder C, Baneux P, Forbes D, van Herck H, Nicklas W, Rugaya Z, Winkler G. Report of the Federation of European Laboratory Animal
Science Associations (FELASA) Working Group on Animal Health. Lab Anim 32:1-17.
Textbook of Veterinary Internal Medicine: Diseases of the Dog and Cat, 6th ed. 2005. Ettinger
SJ, Feldman EC, eds. Philadelphia: WB Saunders.

Exotic, Wild, and Zoo Animals
Biology, Medicine, and Surgery of South American Wild Animals. 2001. Fowler ME, Cubas ZS.
Ames: Iowa State University Press.
CRC Handbook of Marine Mammal Medicine: Health, Disease, and Rehabilitation. 2001.
Dierauf LA, Gulland FMD. Boca Raton FL: CRC Press.
Diseases of Exotic Animals: Medical and Surgical Management. 1983. Philadelphia: WB
Saunders.
Essentials of Disease in Wild Animals. 2005. Wobeser GA. Oxford: Wiley-Blackwell.
Exotic Animal Formulary, 3rd ed. 2004. Carpenter JW. Philadelphia: WB Saunders.
Infectious Diseases of Wild Mammals. 2000. Williams ES, Barker IK, eds. Oxford:
Wiley-Blackwell.
Pathology of Zoo Animals. 1983. Griner LA. San Diego: Zoological Society of San Diego.
Veterinary Clinics of North America: Exotic Animal Practice Series. Elsevier.
Zoo and Wild Animal Medicine: Current Therapy 4. Fowler ME, Miller RE, eds. 1999. Philadelphia: WB Saunders.
Zoo and Wild Animal Medicine, 5th ed. 2003. Fowler E, Miller RE, eds. Philadelphia: WB
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Zoo and Wild Animal Medicine Current Therapy, 6th ed. 2007. Fowler ME, Miller RE, eds.
Philadelphia: WB Saunders.
Zoo Animal and Wildlife Immobilization and Anesthesia. 2007. West G, Heard D, Caulkett
N, eds. Oxford: Wiley-Blackwell.

Nonhuman Primates
Handbook of Primate Husbandry. 2005. Wolfensohn S, Honess P. Oxford: Blackwell
Nonhuman Primates in Biomedical Research: Diseases. 1998. Bennett BT, Abee CR, Henrickson R, eds. New York: Academic Press.
Nursery Rearing of Nonhuman Primates in the 21st Century. 2005. Sackett GP, Ruppenthal
GC, Elias K. Chicago: University of Chicago Press.
Primate Parasite Ecology: The Dynamics and Study of Host-Parasite Relationships. 2009. Huffman MA, Chapman CA, eds. West Nyack NY: Cambridge University Press.
Simian Virology. 2009. Voevodin AF, Marx PA. Ames IA: Wiley-Blackwell.
The Emergence of Zoonotic Diseases. 2002. Burroughs T, Knobler S, Lederberg J, eds. Instituteof Medicine. Washington: National Academies Press.
Zoonoses and Communicable Diseases Common to Man and Animals. 2003. Acha PN,
Szyfres B. Washington: Pan American Health Organization.

Rodents and Rabbits
A Guide to Infectious Diseases of Guinea Pigs, Gerbils, Hamsters, and Rabbits. 1974. National
Research Council. Washington: National Academy of Sciences.
Anesthesia and analgesia for laboratory rodents. 2008. Gaertner D, Hallman T, Hankenson F,
Batchelder M. In: Fish R, Brown M, Danneman P, Karas A, eds. Anesthesia and Analgesia
in Laboratory Animals. San Diego: Academic Press. p 239-298.
Aversion to gaseous euthanasia agents in rats and mice. 2002. Leach MC, Bowell VA, Allan
TF, Morton DB. Comp Med 52:249-257.
Behavioural and cardiovascular responses of rats to euthanasia using carbon dioxide gas. 1997.
Smith W, Harrap SB. Lab Anim 31:337-346.
Biology and Medicine of Rabbits and Rodents. 1989. Harkness JE, Wagner JE. Philadelphia:
Lea and Febiger.
Common Lesions in Aged B6C3F (C57BL/6N x C3H/HeN)F and BALB/cStCrlC3H/Nctr Mice.
1981. Registry of Veterinary Pathology, Armed Forces Institute of Pathology. Washington:
Armed Forces Institute of Pathology.
Common Parasites of Laboratory Rodents and Lagomorphs: Laboratory Animal Handbook.
1972. Owen D. London: Medical Research Council.
Complications of Viral and Mycoplasmal Infections in Rodents to Toxicology Research andTesting. 1986. Hamm TE, ed. Washington: Hemisphere Publishing.
Current Strategies for Controlling/Eliminating Opportunistic Microorganisms. 1998. White WJ,
Anderson LC, Geistfeld J, Martin DG. ILAR J 39:291-305.
Detection and clearance of Syphacia obelata in Swiss Webster and athymic nude mice. 2004.
Clarke CL, Perdue KA. Contemp Top Lab Anim Sci 43:9-13.
Effective eradication of pinworms (Syphacia muris, Syphacia obelata and Aspiculuris tetraptera) from a rodent breeding colony by oral anthelmintic therapy. 1998. Zenner L. Lab
Anim 32:337-342.
Efficacy of various therapeutic regimens in eliminating Pasteurella pneumotropica from themouse. 1996. Goelz MF, Thigpen JE, Mahler J, Rogers WP, Locklear J, Weigler BJ, Forsythe
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Eradication of infection with helicobacter spp. by use of neonatal transfer. 2000. Truett GE,
Walker JA, Baker DG. Comp Med 50:444-451.
Eradication of murine norovirus from a mouse barrier facility. 2008. Kastenmayer RJ, Perdue
KA, Elkins WR. JAALAS 47:26-30.
Euthanasia of neonatal mice with carbon dioxide. 2005. Pritchett K, Corrow D, Stockwell J,
Smith A. Comp Med 55:275-281.
Euthanasia of rats with carbon dioxide: Animal welfare aspects. 2000. Hackbarth H, Kuppers
N, Bohnet W. Lab Anim 34:91-96.
Experimental and Surgical Technique in the Rat. 1992. Waynforth HB, Flecknell PA. New
York: Academic Press.
Experimental Surgical Models in the Laboratory Rat. 2009. Rigalli A, DiLoreto V, eds. Boca
Raton FL: CRC Press.
Gender influences infectivity in C57BL/6 mice exposed to mouse minute virus. 2007. Thomas
ML 3rd, Morse BC, O’Malley J, Davis JA, St Claire MB, Cole MN. Comp Med 57:74-81.
Guidelines for the Euthanasia of Mouse and Rat Feti and Neonates. 2007. Office of Animal
Care and Use, NIH. Avaialable at: http://oacu.od.nih.gov/ARAC/documents/Rodent_Euthanasia_Pup.pdf; accessed January 24, 2010.
helicobacter bilis-induced inflammatory bowel disease in SCID mice with defined flora. 1997.
Shomer NH, Dangler CA, Schrenzel MD, Fox JG. Infect Immun 65:4858-4864.
Humane and practical implications of using carbon dioxide mixed with oxygen for anesthesia or euthanasia of rats. 1997. Danneman PJ, Stein S, Walshaw SO. Lab Anim Sci
47:376-385.
Improving murine health surveillance programs with the help of on-site enzyme-linked immunosorbent assay. 2006. Zamora BM, Schwiebert RS, Lawson GW, Sharp PE. JAALAS
45:24-28.
Infectious Diseases of Mice and Rats. 1991. National Research Council. Washington: National
Academy Press.
Ivermectin eradication of pinworms from rats kept in ventilated cages. 1993. Huerkamp MJ.
Lab Anim Sci 43:86-90.
Large-scale rodent production methods make vendor barrier rooms unlikely to have persistent
low-prevalence parvoviral infections. 2005. Shek WR, Pritchett KR, Clifford CB, WhiteWJ Contemp Top Lab Anim Sci 44:37-42.
Lymphocytic choriomeningitis infection undetected by dirty-bedding sentinel monitoring andrevealed after embryo transfer of an inbred strain derived from wild mice. 2007. Ike F,
Bourgade F, Ohsawa K, Sato H, Morikawa S, Saijo M, Kurane I, Takimoto K, Yamada YK,
Jaubert J, Berard M, Nakata H, Hiraiwa N, Mekada K, Takakura A, Itoh T, Obata Y, Yoshiki
A, Montagutelli X. Comp Med 57:272-281.
Microbiological assessment of laboratory rats and mice. 1998. Weisbroth SH, Peters R, Riley
LK, Shek W. ILAR J 39:272-290.
Microbiological quality control for laboratory rodents and lagomorphs. 2002. Shek WR,
Gaertner DJ. In: Fox, JG, Anderson LC, Loew FM, Quimby FW, eds. Laboratory Animal
Medicine, 2nd ed. London: Academic Press. p 365-393.
Monitoring sentinel mice for helicobacter hepaticus, h. rodentium, and h. bilis by use ofpolymerase chain reaction analysis and serological testing. 2000. Whary MT, Cline JH,
King AE, Hewes KM, Chojnacky D, Salvarrey A, Fox JG. Comp Med 50:436-443.
Mouse parvovirus infection potentiates allogeneic skin graft rejection and induces syngeneic
graft rejection. 1998. McKisic MD, Macy JD, Delano ML, Jacoby RO, Paturzo FX, Smith
AL. Transplantation 65:1436-1446.
Pathology of Aging Rats: A Morphological and Experimental Study of the Age-Associated Lesions in Aging BN/BI, WAG/Rij, and (WAG x BN)F Rats. 1978. Burek JD. Boca Raton FL:
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GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALSPathology of Aging Syrian Hamsters. 1983. Schmidt RE, Eason RL, Hubbard GB, Young JT,
Eisenbrandt DL. Boca Raton FL: CRC Press.
Pathology of Laboratory Rodents and Rabbits, 3rd ed. 2008. Percy DH, Barthold SW. San
Francisco: Wiley-Blackwell.
Pathology of the Syrian Hamster. 1972. Homburger F. Basel NY: Karger.
Preliminary recommendations for health monitoring of mouse, rat, hamster, guinea pig, gerbil
and rabbit experimental units. 1995. Hem A, Hansen AK, Rehbinder C, Voipio HM, Engh
E. Scand J Lab Anim Sci 22:49-51.
Prenatal transmission and pathogenicity of endogenous ecotropic murine leukemia virus AKV.
1999. Hesse I, Luz A, Kohleisen B, Erfle V, Schmidt J. Lab Anim Sci 49:488-495.
Recommendations for the health monitoring of rodent and rabbit colonies in breeding andexperimental units. 2002. Nicklas W, Baneux P, Boot R, Decelle T, Deeny AA, Fumanelli
M, Illgen-Wilcke B. Lab Anim 36:20-42.
Reliability of soiled bedding transfer for detection of mouse parvovirus and mouse hepatitis
virus. 2007. Smith PC, Nucifora M, Reuter JD, Compton SR. Comp Med 57:90-96.
Soiled-bedding sentinel detection of murine norovirus 4. 2008. Manuel CA, Hsu CC, Riley LK,
Livingston RS. JAALAS 47:31-36.
Successful rederivation of contaminated mice using neonatal transfer with iodine immersion.
2005. Watson J, Thompson KN, Feldman SH. Comp Med 55:465-469.
Surgery of the Digestive System in the Rat. 1965. Lambert R. (Translated from the French by B.
Julien). Springfield IL: Charles C Thomas.
The Mouse in Biomedical Research, 2nd ed, vol II: Diseases. 2007. Fox JG, Barthold SW,
Davisson MT, Newcomer CE, Quimby FW, Smith AL, eds. New York: Academic Press.
Transfer of helicobacter hepaticus infection to sentinel mice by contaminated bedding.
1998. Livingston RS, Riley LK, Besch-Williford CL, Hook RR Jr, Franklin CL. Lab Anim
48:291-293.
Viral and Mycoplasmal Infections of Laboratory Rodents: Effects on Biomedical Research.
1986. Blatt PN. Orlando: Academic Press.

DESIGN AND CONSTRUCTION OF ANIMAL FACILITIESAir Handling Systems Ready Reference Manual. 1986. Grumman DL. New York:
McGraw-Hill.
Approaches to the Design and Development of Cost-Effective Laboratory Animal Facilities.
1993. Canadian Council on Animal Care (CCAC) proceedings. Ottawa, CCAC.
Aquatic Facilities. 2008. Diggs HE, Parker JM. In: Hessler J, Lehner N, eds. Planning and Designing Animal Research Facilities. 2008. Orlando: Academic Press. p 323-331.
ARS Facilities Design Standards. 2002. USDA. Available at www.afm.ars.usda.gov/ppweb/
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Biomedical and Animal Research Facilities Design Policies and Guidelines. National Institutesof Health. Available at http://orf.od.nih.gov/PoliciesAndGuidelines/BiomedicalAndAnimalResearchFacilitiesDesignPoliciesAndGuidelines/; accessed January 24, 2010.
Comfortable Quarters for Laboratory Animals, rev 1979. Washington: Animal Welfare
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Control of the Animal House Environment. 1976. McSheely T, ed. 1976. London: Laboratory
Animals Ltd.
Design and Management of Research Facilities for Mice. Lipman NS. 2007. In: Fox JG,
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Design and optimization of airflow patterns. 1994. Reynolds SD, Hughes H. Lab Anim
23:46-49.
Design of Biomedical Research Facilities. 1981. Proceedings of the National Cancer Institute Symposium, National Cancer Institute. Monograph Series, vol 4. NIH Pub. No.
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Design of surgical suites and post surgical care units. 1997. White WJ, Blum JR. In: Kohn DF,
Wixson SK, White WJ, Benson GJ, eds. Anesthesia and Analgesia in Laboratory Animals.
San Diego: Academic Press.
Estimating heat produced by laboratory animals. 1964. Brewer NR. Heat Piping Air Cond
36:139-141.
Guidelines for Construction and Equipment of Hospitals and Medical Facilities, 2nd ed. 1987.
American Institute of Architects Committee on Architecture for Health. Washington:
American Institute of Architects Press.
Guidelines for Laboratory Design: Health and Safety Considerations. 1993. DiBerardinis LJ,
Baum JS, First MW, Gatwood GT, Groden EF, Seth AK. New York: John Wiley and Sons.
Handbook of Facilities Planning, vol 2: Laboratory Animal Facilities. Ruys T, ed. New York:
Van Nostrand.
Laboratory Animal Houses: A Guide to the Design and Planning of Animal Facilities. 1976.
Clough G, Gamble MR. LAC Manual Series No. 4. Carshalton UK: Laboratory Animals
Centre.
Laboratory Animal Housing. 1978. National Research Council. Washington: National Academy of Sciences.
Livestock behavior and the design of livestock handling facilities. 1991. Grandin T. In: Ruys
T, ed. Handbook of Facilities Planning, vol 2: Laboratory Animal Facilities. New York:
Van Nostrand. p 96-125.
Management and Design: Breeding Facilities. 2007. White WJ. In: Fox JG, Barthold SW, Davisson MT, Newcomer CE, Quimby FW, Smith AL, eds. The Mouse in Biomedical Research,
2nd ed, vol III: Normative Biology, Husbandry, and Models. New York: Academic Press.
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Planning and Designing Animal Research Facilities. 2008. Hessler J, Lehner N, eds. Orlando:
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Rodent Facilities and Caging Systems. 2009. Lipman NS. In: Hessler J, Lehner N, eds. Planningand Designing Animal Research Facilities. Orlando: Academic Press. p 265-288.
Structures and Environment Handbook, 11th ed, rev 1987. Ames: Midwest Plan Service, Iowa
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Warning! Nearby construction can profoundly affect your experiments. 1999. Dallman MF,
Akana SF, Bell ME, Bhatnagar S, Choi SJ, Chu A, Gomez F, Laugero K, Sorian L, Viau V.
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Working safely at animal biosafety level 3 and 4: Facility design and management implications.
1997. Richmond JY, Ruble DL, Brown B, Jaax GP. Lab Anim 26:28-35.

appendix

B
U.S. Government Principles
for the Utilization and Care of
Vertebrate Animals Used in
Testing, Research, and TrainingT

he development of knowledge necessary for the improvement of thehealth and well-being of humans as well as other animals requires invivo experimentation with a wide variety of animal species. Whenever U.S. Government agencies develop requirements for testing, research,or training procedures involving the use of vertebrate animals, the following principles shall be considered; and whenever these agencies actually
perform or sponsor such procedures, the responsible Institutional Official
shall ensure that these principles are adhered to:I.

The transportation, care, and use of animals should be in accordancewith the Animal Welfare Act (7 U.S.C. 2131 et. seq.) and otherapplicable Federal laws, guidelines, and policies.1

II.

Procedures involving animals should be designed and performed
with due consideration of their relevance to human or animal health,
the advancement of knowledge, or the good of society.

III.

The animals selected for a procedure should be of an appropriate
species and quality and the minimum number required to obtainvalid results. Methods such as mathematical models, computer simulation, and in vitro biological systems should be considered.

1 For guidance throughout these Principles, the reader is referred to the Guide for the Careand Use of Laboratory Animals prepared by the Institute for Laboratory Animal Research,
National Academy of Sciences.

1

200

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALSIV.

Proper use of animals, including the avoidance or minimization of
discomfort, distress, and pain when consistent with sound scientificpractices, is imperative. Unless the contrary is established, investigators should consider that procedures that cause pain or distress inhuman beings may cause pain or distress in other animals.

V.

Procedures with animals that may cause more than momentary or
slight pain or distress should be performed with appropriate sedation,
analgesia, or anesthesia. Surgical or other painful procedures should
not be performed on unanesthetized animals paralyzed by chemical
agents.

VI.

Animals that would otherwise suffer severe or chronic pain or distressthat cannot be relieved should be painlessly killed at the end of theprocedure or, if appropriate, during the procedure.

VII.

The living conditions of animals should be appropriate for their species and contribute to their health and comfort. Normally, the housing, feeding, and care of all animals used for biomedical purposesmust be directed by a veterinarian or other scientist trained and experienced in the proper care, handling, and use of the species being
maintained or studied. In any case, veterinary care shall be provided
as indicated.

VIII. Investigators and other personnel shall be appropriately qualified andexperienced for conducting procedures on living animals. Adequate
arrangements shall be made for their in-service training, including theproper and humane care and use of laboratory animals.
IX.

Where exceptions are required in relation to the provisions of these
Principles, the decisions should not rest with the investigators directly
concerned but should be made, with due regard to Principle II, by
an appropriate review group such as an institutional animal care anduse committee. Such exceptions should not be made solely for the
purposes of teaching or demonstration.

appendix

C
Statement of Task

T

he use of laboratory animals for biomedical research, testing andeducation is guided by the principles of the Three Rs, replacementof animals where acceptable non-animal models exist, reduction in
the number of animals to the fewest needed to obtain statistically significant data, and refinement of animal care and use to minimize pain anddistress and to enhance animal well-being. The Guide for the Care andUse of Laboratory Animals has been a critical international publication thatprovides information to scientists, veterinarians and animal care personnel
when the decision has been made that animal use is necessary. A committee
will update the 1996 version of the Guide for the Care and Use of Laboratory Animals (the Guide) to reflect new scientific information related to theissues already covered in the Guide, and to add discussion and guidanceon new topics of laboratory animal care and use related to contemporary
animal research programs.
The committee will review the scientific literature published since therelease of the 1996 Guide and determine whether the information in the
Guide concurs with current scientific evidence. The committee will also
review the literature on new technologies related to laboratory animal careand use and determine where new guidance is necessary to ensure thebest scientific outcomes and optimal animal welfare. The committee will
also take into consideration all materials and discussions provided to it,
including those submitted to NIH in response to the Request for Information NOT-OD-O6-011 that requested information related to the need to
update the Guide. Where scientifically warranted, the guidance and recommendations of the 1996 Guide will be changed to reflect new scientific
201

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GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

evidence, while maintaining the performance standards of the 1996 Guide.
The committee will ensure that any recommendations in the Guide will beconsistent with current Public Health Service Policy, the Animal Welfare
Regulations, and the most recent Report of the American Veterinary Medical
Association Panel on Euthanasia.
In addition to the published report, the updated Guide will be postedon the Internet in a pdf or equivalent format such that users will be able tosearch the entire document at one time.

appendix

D
About the Authors

Janet C. Garber (Chair), DVM, PhD, received her Doctor of Veterinary Medicine degree from Iowa State University and her PhD in pathophysiology
from the University of Wisconsin. Her experiences have included infectious
disease research at the U.S. Army Medical Research Institute of Infectious
Diseases (USAMRIID), primate medicine and research, GLP device andmaterials evaluation, and transplantation immunology. Her current interests are in the areas of laboratory animal facility management, infectious
diseases, occupational health and safety, and research program management. She most recently was Vice President, Safety Assessment, at Baxter
Healthcare Corporation and is now a consultant with Garber Consulting,
LLC in North Carolina. Dr. Garber is currently a member of the Council
on Accreditation, AAALAC, International, and previously served as Chair of
the Council. She served on the ILAR Committee to Revise the Guide for theCare and Use of Laboratory Animals and the Committee on Occupational
Health and Safety in the Care and Use of Research Animals.
R. Wayne barbee, PhD, is Professor and Associate Director of Researchat the Department of Emergency Medicine, School of Medicine, Senior
VCURES (Virginia Commonwealth University Reanimation Engineering Science Center) Fellow and Chair of the IACUC at the Virginia Commonwealth
University. Dr. Barbee holds a master’s degree and doctorate in physiology
with three decades of research involving a wide variety of animals (bats,
cats, crabs, dogs, rodents, and swine) in a number of experimental settings.
His research has focused on circulatory shock and resuscitation, acute andchronic rodent surgery, and analysis of rodent hemodynamics. He has
203

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GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

been associated with IACUCs at small, medium, and large institutions forover two decades and is familiar with the oversight of animal care and use
programs. He has served on multiple study sections for both the NIH and
DOD. Dr. Barbee also served as an Oxford, UK 2006 fellow (recipient,
VCU Harris-Manchester Award) where he examined policies, training, andsecurity issues related to animal care and use within the UK.
Joseph T. bielitzki, MS, DVM, is Research Manager, University of Central
Florida. Dr. Bielitzki has worked with non-human primates in the laboratory environment for 20 years. Over this period he has worked withmacaques (pig-tail, long-tail, Japanese, rhesus, stump-tail), baboons (yellow,
green and hybrids) squirrel monkeys, capuchin monkeys, mangabeys, gibbons, chimpanzees, orangutans, bonobos, and gorillas. In the area of
non-human primates his area of expertise is in enteric diseases, nursery
rearing, and colony management. He has also worked with mice and rats
in a variety of international facilities. He was instrumental in the writing
and acceptance of the NASA Bioethical Principles for the Use of Animals
in Research (NPD 8910.1). He speaks frequently on IACUC function andthe importance of ethics in the use of animals. His background includes
experience in academia, industry, and government in the roles of attending
veterinarian, program manager, and researcher.
Leigh Ann Clayton, DVM, is Director of Animal Health at the National
Aquarium in Baltimore where she also chairs the Animal Welfare Committee. Dr. Leigh Clayton has worked in the zoo/aquarium field or the exotic
pet medicine field exclusively since 2000. As she has worked with animals
held in aquatics systems both in recirculating fresh and salt water, she isexperienced in managing disease and accomplishing preventive healthprograms for fishes, amphibians, and reptiles as well as birds and mammals. She is a Diplomate of the American Board of Veterinary Practitioners(Avian). Dr. Clayton has routinely used her knowledge of nitrogen cycling
and the basics of a variety of life support system designs to solve healthissues in these captive settings and help ensure adequate animal health.
She has served on the Executive Board of the Association of Reptilian andAmphibian Veterinarians, a role that allowed her to routinely liaise withleading researchers in the amphibian medicine field.
John C. Donovan, DVM, is President of BioResources Inc. Dr. Donovan
has over 30 years’ experience working as a veterinarian in biomedical
research and is board certified by the American College of Laboratory Animal Medicine (ACLAM). After 7 years in the U.S. Army’s Medical Researchand Development Command, he spent 10 years at the National Institutesof Health, becoming the Director of the National Cancer Institute’s OfficeAPPENdIX d: AbOUT ThE AUThORS

205

of Laboratory Animal Science. He began his career in the pharmaceutical industry in 1994 as Senior Director of Worldwide Laboratory Animal
Resources for Rhone Poulenc-Rorer Pharmaceuticals, leading to his positionas Vice President of Laboratory Animal Science and Welfare for Aventis
Pharmaceuticals in 1999. In 2001 he moved to Wyeth Pharmaceuticals
where he was Vice President of BioResources until his retirement in 2007.
During his career, Dr. Donovan served in numerous professional leadership positions including President of ACLAM and President of the Board of
Directors of the Pennsylvania Society for Biomedical Research (PSBR). He
also served on several boards including those of the National Associationfor Biomedical Research, ACLAM, PSBR, and the New Jersey Association for
Biomedical Research.
Dennis F. Kohn, DVM, PhD, is Professor Emeritus of Clinical Comparative
Pathology at Columbia University. He received his DVM from Ohio State
University and a doctorate in Medical Microbiology from West Virginia
University. He is board certified by the American College of Laboratory
Animal Medicine. He has directed laboratory animal resource/comparative
medicine programs at West Virginia University Medical Center, Universityof Texas Medical School at Houston, and the Health Sciences Division,
Columbia University. His research interests have dealt primarily with thepathogenicity of Mycoplasma pulmonis in the respiratory tract of laboratory
rats, and the experimental pathology induced within the CNS and joints ofrats inoculated with M. pulmonis. He is a past president of the American
College of Laboratory Animal Medicine and the American Society of Laboratory Animal Practitioners, and as chair of a number of American Association of Laboratory Animal Science committees. He has served as a Council
member of AAALAC-International, as a member of the 1986 American Veterinary Medical Association’s Panel on Euthanasia, and the 1996 Instituteof Laboratory Animal Resources’ committee to revise the Guide.
Neil S. Lipman, VMD, is Director of the Center of Comparative Medicine
and Pathology serving the Memorial Sloan-Kettering Cancer Center (MSKCC)
and the Weill Medical College of Cornell University and is Professor of Veterinary Medicine in Pathology and Laboratory Medicine at Weill Cornell
as well as a Laboratory Member at the Sloan-Kettering Institute at MSKCC.
Dr. Lipman is a Diplomate of the American College of Laboratory Animal
Medicine with over 25 years’ experience in laboratory animal medicine
and science. Dr. Lipman has expertise in vivarium design, engineering, andoperations, having designed over 1.5 million gross square feet of vivarium
space and overseen the operation of a number of major academic animal
resource programs. His research interests are principally translational andinclude development and analysis of new technologies especially with

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GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

respect to rodent housing systems and monoclonal antibody production,
the characterization of various animal models, understanding the etiopathogenesis of endocrinologic disorders affecting laboratory animal species, anddevelopment and analysis of novel therapeutic strategies. Throughout his
career, Dr. Lipman has been extensively involved in the postgraduate training of laboratory animal specialists.
Paul Locke, MPH, JD, DrPH, an environmental health scientist and attorney,
is Associate Professor at the Johns Hopkins University Bloomberg Schoolof Public Health in the Department of Environmental Health Sciences,
Division of Toxicology. He holds an MPH from Yale University School of
Medicine, a DrPH from the Johns Hopkins University Bloomberg Schoolof Public Health and a JD degree from Vanderbilt University School of
Law. Prior to joining the Department of Environmental Health, he was the
Deputy Director of the Pew Environmental Health Commission and the
Director of the Center for Public Health and Law at the Environmental Law
Institute. Dr. Locke’s research and practice focus on how decision makers
use environmental health science and toxicology in regulation and policy
making and how environmental health sciences influence the policy-making process. His areas of study include alternatives to animal testing inbiomedical research, with particular emphasis on toxicity testing. He also
maintains an active research program in radiation studies and radiation protection policy. Dr. Locke directs the Doctor of Public Health degree programin the Department of Environmental Health Sciences and is co-director of
the Johns Hopkins certificate program in Humane Science and Toxicology.
From 2004 until 2009 he was a member of the National Academy of Sciences Nuclear and Radiation Studies Board, and has served on five National
Academy of Sciences/National Research Council expert committees. He is
admitted to practice law before the bars of the states of New York and New
Jersey, the District of Columbia, the United States Court of Appeals for theSecond Circuit and the United States Supreme Court.
The Honorable John Melcher, DVM, a graduate of the College of Veterinary Medicine of the Iowa State University, was a practicing veterinarian in
the state of Montana until 1969, in which year he was elected to the U.S.
House of Representatives. He served as a Congressman for 8 years and asa Senator for 12 years. In both the House and the Senate, Senator Melcher
was noted for his interest in agriculture, protection of public lands, notably
Forest Service and Bureau of Land Management lands, and animal welfare
and animal health protections. In 1984 he contributed to the Animal Welfare Act with an amendment requiring consideration of the psychologicalwell being of primates used in medical research. After retiring from Congress, Senator Melcher established a second career as a consultant for the

APPENdIX d: AbOUT ThE AUThORS

20

American Veterinary Medical Association and the American Associationof Veterinary Medical Colleges. Senator Melcher represents the public’s
perspective.
Fred W. Quimby, VMD, PhD, is a board-certified laboratory animal veterinarian, with a doctorate in pathology, specialized in the assessment of
immune function in animals. Prior to his retirement in 2007 he was Associate Vice President at Rockefeller University, while over the past 35 years heoversaw the research animal programs at three Universities (Tufts, Cornell,and Rockefeller) and held the position of Professor at Cornell’s Colleges ofMedicine and Veterinary Medicine. He conducted research and lectured in
the fields of immunology, pathology and environmental toxicology where
he focused his research on toxic shock syndrome, environmental intoxication with polychlorinated biphenyls (PCBs), and immune dysfunction in pet
dogs. As a laboratory animal professional he has designed and overseen theconstruction of five animal facilities for research animals and a zoologicalpark. Dr. Quimby has had broad experience with a wide assortment oflaboratory animals including rodents, dogs, primates, livestock, poultry, andfish, and published on the diseases, care, and /or housing of each of them.
He has served on various NAS/NRC committees including the Guide committee, the Committee on Immunologically Compromised Rodents (Chair),the Transgenic Animal Committee, the Committee to Develop Standards
for Dogs (Chair), the Monoclonal Antibody Production Committee, andthe Committee Evaluating Increasing Veterinary Involvement in Biomedical
Research. He served as a member of the ILAR Council and chaired the
Editorial Committee of the ILAR News. He is currently serving as a memberof the Committee to Assess the Current and Future Workforce Needs in Veterinary Medicine. He was a charter member of the Society for Veterinary
Ethics, a member of the Board of Directors for the National Association for
Biomedical Research and a member of the Strategic Planning Committee
for AAALAC International.
Patricia V. Turner, MS, DVM, DVSc, is Associate Professor and Program
Leader of Laboratory Animal Science in the Department of Pathobiology at the University of Guelph, Canada, where she also is currently
Chair of the Animal Care Committee. She has a doctorate in comparative
pathology and is a Diplomate of both the American College of Laboratory
Animal Medicine and the American Board of Toxicology. Dr. Turner has
experience managing Canadian Council of Animal Care–compliant animal facilities that house a full range of species (fish, rodents and rabbits,
dogs and cats, swine and sheep, nonhuman primates) in both academic
and industry (GLP) settings. Dr. Turner serves as an AAALAC international
ad hoc specialist with excellent knowledge of current US guidelines and208

GUIdE FOR ThE CARE ANd USE OF LAbORATORy ANIMALS

regulations concerning research animal care and use. Her research interests
include innate immunity and infectious disease, toxicologic pathology, andthe interactions between rodents and their environment as they relate todisease susceptibility. In 2007 she was the inaugural recipient of the North
American Animal Welfare Award, co-sponsored by Procter & Gamble andthe Humane Society of the United States.
Geoffrey A. Wood, DVM, PhD, DVSc, is Associate Professor in the Department of Pathobiology at the Ontario Veterinary College, University ofGuelph, Canada. Dr. Wood has a doctorate in cancer biology and a doctorate in veterinary pathology. He has been involved in design or pathologic
characterization of hundreds of genetically engineered rodents, both as theformer Associate Director of Pathology at the Centre for Modeling Human
Disease in Toronto, and in his current position. His lab conducts researchon cancer genetics and the process of metastasis, with a focus on bone andprostate cancer. Dr. Wood’s research collaborations include projects investigating various aspects of a wide range of different cancer types, as well as
studies on stem cell biology, immunity, and inflammation.
Hanno Würbel, Dr.sc.nat, is Professor of Animal Welfare and Ethology at
the Justus Liebig University in Giessen, Germany. He has studied biology (zoology) at the University of Berne, Switzerland and graduated fromthe ETH Zürich, Switzerland with a doctorate in natural sciences. He has
experience in animal behavior and in the scientific assessment of animal
well-being, and has mostly worked with rodents, but also with rabbits, dogs,
poultry, and horses. His research focuses on environment-dependent plasticity of brain and behavior in relation to questions of animal husbandry andanimal welfare. In 2005 Dr. Würbel received the Hessian Animal Welfare
Research Prize and in 2009 the Felix Wankel Animal Welfare ResearchAward. He is a member of the Animal Welfare Council of the German
government, Central Animal Welfare Officer of the University of Giessen,and head of the University’s Central Animal Facility. He is also a council
member of the International Society of Applied Ethology (ISAE), editor of the
Journal of Applied Animal Welfare Science, and editorial board member ofthe journal Applied Animal behaiour Science.

IndexA
Acclimation and adaptation, 111, 188(see also Procedural habitation andtraining)
caloric management and, 67
to noise and vibrations, 81-82
to outdoor housing, 44, 54-55
to physical restraint, 29
Acquisition of animals (see Procurement)
Activity and exercise, xiv, 43, 52-53, 55,
56, 57, 58, 63-64, 67, 84 (see also
Enrichment)
Agricultural animals, 2biomedical vs. agricultural research,
32-33
environment and housing, 29, 33, 43,
44, 137, 176-177
floor drains, 137, 138
information resources, 33, 176-177,
191-192
protocol review, 32-33
space requirements, 60, 62-63
surgery, 117, 144, 145
veterinary care, 145, 191-182
Air pressure, 45, 47, 139, 145-146, 149-150
Air quality and airborne contaminants,
45-47
Airflow (see Ventilation and airflow)
Albino animals, 48-49, 141

Allergens and allergic reactions, 18, 19, 20,
22-23, 45, 155
Alternatives to laboratory animal use, 3, 5,
12, 25, 27, 162-167
American Association for Laboratory Animal
Science, 16
American College of Laboratory Animal
Medicine, 24
Guidelines for Adequate Veterinary Care,xvii, 14Ammonia, 45, 47, 68, 70, 71, 72-73, 78,
79, 80, 81, 85
Amphibians, 41-42, 44-45, 46, 77, 78-79,
80, 81, 82, 83, 84, 85, 108, 119,
156, 178-179, 192 (see also Aquatic
species)Anesthesia and analgesia
considerations in selection of agents, 75,
121
equipment/delivery, 34, 122, 145, 147
euthanasia agent, 124
field conditions, 32, 117monitoring, 119-120, 122, 123
neuromuscular blocking agents, 122-123
oversight, 34, 156
pain management, 12, 121-122
preemptive analgesia, 121
principles governing use, 12, 14, 26
recommended reading, 188-190
recordkeeping, 34

20

210
surgery, 115, 116, 117, 118, 119-120,
122-123, 145, 188-190
training, 17, 106, 115, 122waste gas management, 21, 147
Animal and Plant Health Inspection Service(APHIS), 30, 148
Animal care and use programattending veterinarian, 14
collaborations, 15
defined, 6
disaster planning and emergency
preparedness, 35
IACUC, 14-15, 24-33
institutional official, 13-14
oversight, 24-34
personnel management, 15-24
program management, 13-24
recommended reading, 167-172
regulations, policies, and principles,
12-13
responsibilities, 13-15
Animal care personnel, (see also Personnel)
training and education, 16
Animal training (see Procedural habituationand training)
Animal Welfare Act, 1, 4, 25, 34, 199
Animal Welfare Information Center, xv, 16
Animal Welfare Regulations, 1, 12
housing guidelines, 56, 59
IACUC, 25transportation of animals, 107veterinary care, 30
Antibiotics, 116
Anxiolytics, 122-123
Apes (see Nonhuman primates)
Aquatic species, 2 (see also Amphibians;
Fish; Reptiles)
algal growth, 86
behavior and social management, 84
diseases, 112
emergency, weekend, and holiday care,87
enrichment and social housing, 82-83
environment, 77-82
facilities, 150-151
food and feeding, 84-85
housing, 82-83, 150-151
husbandry, 84-87
identification, 87
illumination, 81
life support system, 79-80
macroenvironment, 77-78, 86

INdEX
management, 84-88
microenvironment, 77-78, 82-83
noise and vibration, 81-82
pest control, 87
population management, 87-88
recommended reading, 178-179,
192-193
recordkeeping, 87-88
sanitation, 85-86
sheltered, outdoor, and naturalistic
housing, 83
space, 83
substrates, 85
temperature, humidity, and ventilation,
80-81
water quality, 78-79, 85
Aseptic practices, 17, 34, 52, 70, 75, 115,
116, 117, 118-119, 144
Attending veterinarian, 3, 4, 13, 14, 15, 17,
21, 24, 28, 106, 114, 116 (see also
Veterinary care)
Autoclaving
bedding material, 69, 146
drinking water, 146
feed, 66, 146
surgical instruments, 119, 145
Avian species (see Birds)bBacterial gill disease, 112
Barriers
access control, 151
facilities, 113, 136, 145-146
separation of housing and research/
surgery areas, 134, 144
visual, 51, 53, 83
Bedding, nesting, and substrates
aquatic species, 85
autoclaving, 69, 136, 146
behavioral aspects, 52changing, 46, 47, 52, 69, 70, 73
disposal, 19, 20
enrichment aspects, 67
environmental aspects, 43, 44, 46, 47,
49, 52, 53, 69, 71
experimental influences, 68, 69
illumination and, 49
materials, 68-69
outdoor facilities, 54
storage, 136, 141
terrestrial species, 52, 68-69

211

INdEX
Behavioral and social management
acclimation, 111
activity and exercise, 63-64, 84
airflow and ventilation and, 150
aquatic species, 82, 83, 84, 85
bedding, nesting, and substrate materials
and, 49, 52, 85
enrichment, 52-53, 54, 60, 83, 85
food and feeding, 65
illumination and, 47, 48, 49
procedural habituation and training, 29,
64-65
recordkeeping, 75
sanitation and, 72
social environment, 51, 58, 59, 64, 111
space considerations, 55, 56, 57, 58, 59
temperature and humidity and, 43, 44,80
terrestrial species, 83-85
training of personnel, 53, 121veterinary care and, 107, 111, 112, 114
Behavioral changes
infections and, 118monitoring animals for, 112
moribund state, 28
pain and distress, 120-121, 123
physical restraint and, 29
sanitation and, 71
stereotypies and compulsive behaviors,
63
transportation of animals and, 150
Behavioral research, 30, 31, 111, 135,
149-150
Bioexclusion, 28 (see also Barriers)
Biologic agents and hazards, 18, 19, 20, 21,111, 112-113, 148, 170-172
Biosecurity, animal, xviii, 105, 107, 108,109-110, 111, 135, 170-172
Birds, 41, 44, 48, 49, 60, 121, 144, 156,
179-180, 193
Bites and scratches, 18, 23
Breeding and reproduction
aquatic animals, 44, 80, 82, 83, 87, 88
caloric restriction and, 67
cryopreservation of fertilized embryos,
ova, ovaries, or spermatozoa, 76,
107
enriched environments and, 83
genetic management, 76, 77
GMAs, 28, 76
lighting and, 48, 137

nesting, bedding and substrates, 52, 70,83nomenclature, 77
nonhuman primates, 59
recordkeeping, 76, 77, 87, 88
rodents and rabbits, xviii, 57, 77, 107
sanitation activities and, 70
space requirements, 55, 56, 57, 33
temperature and humidity and, 44, 80, 82
vibration and, 50
Building Seismic Safety Council, 133

C
Cages and caging (see also Housing;Microenvironment)
cleaning and sanitation, 18, 46, 47, 52,
69, 70-71, 72, 73, 86, 142, 143, 145
dimensions and space allocation, xviii,
56, 57, 58, 59, 60, 61, 133
dividers and shelters in, 53
flooring, 70
isolators, 45, 47, 52, 111, 139, 146
light intensity in, 46, 49
location on cage rack, 46, 49
mechanical washers, 71, 72, 86, 135,
136, 141, 143
metabolic, 121
in secondary enclosures, 47
ventilated, 44, 46, 47, 50, 58, 70-71,
111, 141, 146
vibration of, 50
Caloric management, 31, 57, 67
Canadian Council on Animal Care, 16
Carcass disposal, 20, 73-74, 138, 142
Cardiovascular shock models, 27
Catsbehavioral and social management, 58,
59, 63-64, 180-181
enrichment, 53
environmental requirements, 43, 44, 53,
180-181
housing and space, 49, 56, 58, 59,
180-181procurement, 106
recommended reading, 180-181, 193
recordkeeping, 75-76
veterinary care, 193
Cattle, 62-63, 64, 176-177, 191-192 (seealso Agricultural animals)
Cedar shavings, 69, 156

212
Ceilings, 56, 81, 142
Centers for Disease Control and Prevention,
19, 21, 107, 148, 157
Cercopithecine herpesvirus 1, 23
Chairing, 29
Chemical agentscleaning materials, 71, 72
non-pharmaceutical-grade, 31
hazardous, 18, 19, 20, 21, 136
Chickens, 48, 60
Chimpanzees, xviii, 61
Chlorines and chloramines, 72, 78-79, 80,
85, 86
Circadian rhythm, 43, 48, 49, 137, 149
Cleaning agents, 18, 71, 72 (see also
Sanitation)(see also Vetterinary care)
Clinical care and management
emergency care, 114
medical management, 114
recordkeeping, 115
Clothing and footwear, 20, 21, 146
Cold storage and refrigeration, 66, 73, 136,
142
Cold stress, 43, 44, 53
Collaborations, interinstitutional, 15Computer modelingalternative to animal use, 5, 12, 25, 199
facility design, 46, 133
genetic variability, 76
Conditioned-response protocols, 31
Construction guidelines, 136-143
Containment of hazardous materials, 21,
148-149
Contaminants
airborne, 22, 45, 46-47, 139, 140, 143,
145, 146-147, 149
bedding, 69, 141, 146
cleaning implements, 72, 85
food, 19, 65, 66, 67, 84, 141, 146
genetic, 76
microbial, 72, 81, 108, 109, 110, 113,
118, 144-145
recommended readings
water, 67-68, 81, 86, 138
Convention on International Trade inEndangered Species of Wild Fauna
and Flora, 107
Corridors, 86, 134, 136, 137, 141, 142
Cryogens, 147

INdEX
Cryopreservation, 76, 107
Cyanobacteria, 86

DDefecation, 72
Definitions
animal biosecurity, 109
animal care and use program, 6, 11
animal use, 2attending veterinarian, 14
engineering standard, 6
humane care, 6
institutional animal care and use
committee, 14
institutional official, 13
laboratory animals, 2
life support system, 79
macroenvironment, 42“may,” 8
microenvironment, 42“must,” 8
performance standard, 6-7
policies, 7
practice standard, 7
principles, 7
procedures, 7“should,” 8
thermoneutral zone, 43
three Rs, 4-5
Dehydration, 68
Desiccation stress, 45, 46
Diet and dietary control (see also Food andfeedng)abrupt changes in, 67
aquatic animals, 84, 85
autoclavable, 66
caloric management, 31, 57, 67
certified diets, 65
chemically defined diets, 65, 66
natural-ingredient, 65, 66
nutritionally balanced, 67
purified diets, 65, 66
quality assurance, 65, 66
treats, 67
Disaster planning, 35, 74-75
Disease control, 23, 72, 81, 108, 109, 110,112-113, 118, 144-145, 190
Disease susceptibility, 19, 42

213

INdEX
Disinfection, 20, 51, 52, 68, 69, 70-72, 78,
79, 80, 84, 86, 110, 118, 119, 138,
143, 144, 150
Disposable clothing, 146
Distress (see also Pain and distress)
defined, 121
neuromuscular blocking agents and,122-123
recommended reading, 188-190
Diurnal cycles, 48, 141Dogsbehavioral and social management, 29,
59, 63-64, 180-181
enrichment, 53
environmental requirements, 43, 44,
180-181
housing and space, 49, 58, 59, 136,
138, 180-181
infusion pumps, 29
pain and distress, 121
procurement, 106
recommended reading, 180-181, 193
recordkeeping, 75-76
veterinary care, 193
Doors, 136, 137, 138, 142, 143, 144, 149,
150
Drafts, 46, 140
Drainage, 54, 136, 138, 143, 144, 149,
150

E
Ecdysis (molting), 45, 156
Education (see Training and education)
Electric power, 136, 141, 142, 143, 145,
150
Emergency, weekend, and holiday carehusbandry, 35, 74-75, 87
medical, 114, 118
Enclosures (see Cages and caging; Primary
enclosures; Secondary enclosures)Endangered Species Act, 157
Endpoints (see also Experimental endpoints;
Humane endpoints)
IACUC review, 26, 27-28
Engineering standards, 6, 7
Enrichment, xiv, xviii, 33, 43, 50, 52-54, 55,
56, 58, 60, 64, 82-83, 85, 137, 173175 (see also Social environment)
Entrapment, accidental, 51, 82

Environment (see also Enrichment;
Housing; Macroenvironment;
Microenvironment; Social
environment)
aquatic species, 77-82
recommended reading, 172-175
terrestrial species, 42-50
Eosinopenia, 49
Ergonomic injury, 19
Ethical considerations, xiii, xvii, 4-5, 12
recommended reading, 163-164
training in, 17
Euthanasia (see also Humane endpoints)
AV responsibility, 106, 114, 156
AVMA Guidelines, 123
carbon dioxide for rodents, 124
criteria for, 123
defined, 123
disaster planning, 35
fetuses and larval life forms, 123-124
field studies, 32
methods, 123-124
pest control, 74
preemptive, 28
psychological effects on personnel, 124training, 17, 124
Exercise (see Activity and exercise)
Exotic, wild, and zoo animals, 2, 18, 32, 44,
84, 107, 181-182, 193-194
Experimental design and statistics,
recommended reading, 164-165 (seealso Protocols)
Experimental endpoints, 27-28, 112, 121

F
Facilities (see also Physical plant)
aquatic species housing, 150-151
barrier facilities, 145-146
for behavioral studies, 149-150
hazardous agent containment, 148-149
imaging, 146-147
irradiation, whole-body, 157-158
recommended reading on design andconstruction, 196-197
for sanitizing materials, 143
surgical, 116-117, 144-145
Farm animals (see Agricultural animals)
Field studies, 18, 32, 155 (see also Exotic,
wild, and zoo animals)
Fish (see also Aquatic species)

214

INdEX

air quality considerations, 81
biosecurity, 110
food and feeding, 84-85
GMAs, 28
illumination, 81
noise and vibration, 81-82
pain and distress, 121
recirculating systems, 79-80
recommended reading, 178-179,
192-193
recordkeeping, 87-88
sanitation, 85, 86
schooling, 82, 83
space requirements, 83
temperature, 41-42
toxic substances, 78-79, 85
veterinary care, 192-193
Flaky skin, 45, 156
Floors, 137-138
Food and Drug Administration Good
Laboratory Practice standards, 65
Food and feeding (see also Diet)
aquatic species, 84-85
enrichment activities with, 67
feeders, 66-67
management of caloric intake, 67
restricted or scheduled access, 30-31
sanitation of feeders and waterers, 71-72
storage, vermin control, and handling,
65-66
terrestrial species, 65-67

G
Genetically modified animals (GMAs)
breeding, 76
humane endpoints, 29
monitoring for unexpected outcomes,
28-29
occupational health and safety
considerations, 21
recommended reading, 175-176
Genetics, recommended reading, 175-176
Gerbils, 43, 44, 52
Gnotobiotic animals, 52, 70, 135
Goats, 49, 62, 111, 156, 177, 192
Guinea pigs, 44, 51, 57, 71 n.2, 111, 156(see also Rodents)

H
Hamsters, 44, 57, 64, 68, 71 n.2, 156 (seealso Rodents)
Hazard identification and risk assessment,
18-19
Hazardous agents, 20-21
Health Research Extension Act, 34
Heat stress, 43, 44
Heating and air-conditioning, 139-140
Helicobacter spp, 111, 113
Hepatitis B virus, 22
Herpesvirus, 23, 112
Horses and ponies, 63, 64
Housing (see also Cages andcaging; Macroenvironment;
Microenvironment; Secondary
enclosures; Space requirements; andspecific animals)
aquatic species, 82-83
design considerations, 51
environmental enrichment, 52-54, 8283, 173-175
flooring, 51-52, 54
life support system, 79-80
naturalistic environments, 55, 83
occupational health and safety
considerations, 19-20
recommended reading, 172-173
repair or replacement, 51
sheltered or outdoor, 42, 44, 51, 54-55,
68, 74, 81, 83, 118, 134, 135, 139,
140
social, 51, 55, 58-59, 60, 82-83
terrestrial species, 50-63
Humane care, defined, 6
Humane endpoints, xvii, 5, 12, 27-28, 29,
114, 123, 165-166
Humidity (see Temperature and humidity)
Husbandry (see also Sanitation)aquatic species, 84-87
bedding and nesting materials andsubstrates, 68-69, 85
caloric restriction, 30-31, 67
emergency, weekend, and holiday care,35, 74-75, 87
food, 65-67, 84-85
pest control, 74, 87
terrestrial species, 65-75
waste disposal, 73-74
water, 67-68

215

INdEX
Hydrogen peroxide, 72
Hygiene, personal, 18, 19, 20

I
Identification of animals, 75, 87
Illumination/lighting, 47-49, 81, 141
Imaging facilities, 146-147
Immunization, 22
Immunodeficient animals, 21, 28, 66, 145
Infectious agents (see Biologic agents andhazards
Infectious disease research, 27
Inspection of facilities and equipment, 34
Institute for Laboratory Animal Resources(ILAR), 2, 16
Institutional Animal Care and Use
Committee
agricultural animals, 32-33
animal care and use programs and, 6,
11, 13collaborating institutions, 15
endpoint considerations, 27-28
field investigations, 32
food and fluid regulation, 30-31
investigating and reporting animal
welfare concerns, 23
membership and function of committee,
17, 24-25
multiple survival surgical procedures, 30
non-pharmaceutical-grade chemicals or
substances, 31
occupational health and safety and, 17
physical restraint, 29-30
program management and, 13, 14-15
protocol review, 25-26
recommended reading, 169-170
special considerations during review
process, 27-33
surgery and, 115-118
surgical procedures, 30
training and education, 15
unexpected outcomes, 28-29
Institutional official, 13-14, 17, 23, 24, 25,
30, 35International Air Transport Association, 107
International Committee on Standardized
Genetic Nomenclature for Mice, 77
Invertebrate animals, 2
Irradiation facilities, 157-158

L
Laboratory Animal Welfare and Training
Exchange, 16
Latex toxicity, 84
Laws, regulations, and policies (see also
Animal Welfare Act; Animal Welfare
Regulations; Public Health Service
Policy; other specific acts and policies)
recommended reading, 168
training in, 17Lighting (see Ilumination/lighting)
Lymphocytic choriomeningitis virus, 113

M
Macacine herpesvirus, 23, 112
Macaques, 23, 112
Macroenvironment
aquatic species, 77-78, 86
defined, 42
terrestrial species, 42Management of animals (see Behavioral
and social management; Husbandry;
Population management)
aquatic species, 84-88
medical, 114
recommended reading, 172-173
terrestrial species, 63-77
Mice (see also Rodents)
acclimation, 111
aggression in males, 53, 64
anxiety studies, 53
bedding and nesting materials, 52, 53,
68
breeding animals, 52
caloric restriction, 67
enrichment, 53, 54
GMAs, 28, 68, 145-146
housing, 53, 57, 145-146
identification/nomenclature, 77
illumination, 49, 137
infections, 111, 112
nude or hairless animals, 68
recommended reading, 184-187,
194-196social stress, 53
space requirements, 57, 156
specific pathogen-free mice, 145-146
temperature and humidity, 43, 45
toe-clipping, 75

216

INdEX

Microenvironment (primary enclosure)aquatic species, 77-78, 82-83
defined, 42
terrestrial species, 42, 50-52
Midwest Plan Service, 33
Monitoring and surveillancedisease prevention, 112-113
facilities, equipment, and exposure, 19-20
food and fluid intake, 31
GMAs, 28
intraoperative, 119
postapproval, 33-34
postoperative care, 119
restrained animals, 29
sanitation practices, 72-73
Monkeys (see Nonhuman primates)
Monoclonal antibody production, 27
Moribund state, 28
Mouse hepatitis virus, 112, 113
Mycoplasma hyopneumoniae, 112

N
National Institutes of Health, 19, 155
National Research Council Committee on
Animal Nutrition, 65
Necropsies, 21
Nesting (see Bedding, nesting, andsubstrates)
Neuromuscular blocking agents, 122-123
Nocturnal animals, 48
Noise and vibration
aquatic animals, 81-82
control, 142, 143
hearing protection for personnel, 22, 49
terrestrial animals, 49-50
Non-pharmaceutical-grade chemicals or
substances, 31-32
Nonhuman primatesacclimation, 111
behavioral and social management, xviii,
29, 53enrichment, 53, 67, 137
environment, 43, 44, 45, 50, 139
food and feeding, 67
housing, xviii, 49, 111-112, 136
illumination, 48
infections, 23, 111-112
infusion pumps, 29
noise and vibration effects, 50
personal protective equipment for staff,
21-22

recommended reading, 182-184, 194
recordkeeping, 75-76
restraint, 29
space requirements, xviii, 56, 58-60, 61
temperature and humidity, 43, 44, 45,140transport, 107
ventilation, 47, 139veterinary care, 194
zoonosis surveillance and, 23, 110
Noroviruses, 113

O
Occupational health and safety programadministrative controls, 18
animal housing systems, 19-20
collaborative approach, 17, 21
control and prevention strategies, 18
engineering controls, 18, 19
facility and equipment support, 19-20
field studies or wildlife research, 18
hazard identification and risk
assessment, 18-19
hazardous animal experimentation, 18,
20-21
hazardous material containment, 149
hygiene, 18, 19, 20
medical evaluation and preventive
medicine for personnel, 22-23
monitoring, 19-20
noise exposure, 22, 49
personal protective equipment, 18, 20,
21, 23, 109, 146, 148, 167-172
recommended reading, 17, 19, 21,
170-172
regulations, 17, 18, 20, 21
reporting requirements, 23-24
responsibility, 18
safety committee, 17-18, 21, 32
security, 23
training, 17, 18, 20, 21, 22, 24
Organ or system failure research, 27
Other animals, recommended reading,187-188
Outdoor enclosures/habitats, 42, 44, 51,
54-55, 68, 74, 81, 83, 118, 134, 135,
139, 140
Oversight (see also Institutional Animal Careand Use Committee)
postapproval monitoring, 33-34

21

INdEX
recommended reading, 169-170
responsibility for, 13
Owl monkeys (Aotus trivirgatus), 112

P
Pain and distress (see also Distress)
analgesics and anesthetics, 12, 121-123
chronic, 122
guidelines and, 12, 199-200
humane endpoints, 5, 27, 123
indicators of, 120-121, 123
modeling/studies, 28
monitoring GMAs for, 29
multiple survival surgical procedures, 30
postoperative, 117, 120
prevention or alleviation, 5, 12, 26, 33,
35, 105, 114, 120-121, 200
recommended reading, 120, 188-190
restraint devices, 29
studies of, 34“wind-up” phenomenon, 120
Parvoviruses, 113
Pasteurella multocida, 112
Pedigree management, 55, 76, 87
Performance standard, defined, 6-7
Personnel (see also Occupational healthand safety program; Training andeducation; specific personnel)
animal welfare concerns and, 23-24
hygiene, 18, 19, 20
management, 15-24
medical evaluation and preventive
medicine for, 22-23
occupational health and safety program,
17-23
protection, 21-22
security, 23
Pest control, 74, 87, 110
Pesticides, 65, 74
Photoperiod, 47, 48, 81, 137, 143
Photostressors, 47 (see also
Illumination/lighting)
Phototoxic retinopathy, 48, 49
Physical hazards, 18, 19
Physical plant (see also Facilities)
centralized vs. decentralized, 134-135
construction guidelines, 136-143,
196-197
corridors, 136
doors to animal rooms, 137

drainage, 138
floors, 137-138
functional areas, 135-136
general considerations, 133-135
HVAC, xviii, 46-47, 139-140
location, 134
noise control, 142
power and lighting, 141
recommended reading, 196-197
security and access control, 151
storage areas, 141-142
vibration control, 142
walls and ceilings, 138-139
windows, exterior, 137
Phytoestrogens, 65
Pigeons, 60, 180, 193
Pilot studies, 26, 28
Poikilothermic animals, 41, 44, 55, 80, 82,
108
Policies, defined, 7
Population management
aquatic species, 87-88
breeding, genetics, and nomenclature,
75-76
identification, 75, 87
recordkeeping, 75-76, 87-88
terrestrial species, 75-77
Poultry, 44, 48, 60
Practice standard, 7, 12
Preventive medicine
animal biosecurity, xviii, 79, 105, 107,108, 109-110, 111, 135disease surveillance, diagnosis, andtreatment, 112-113
quarantine and stabilization, 110-111
separation of animals by health status
and species, 111-112
Primary enclosures, 42, 43, 44-45, 46, 5052, 72, 77, 82 (see also Cages andcaging; Microenvironment)
Primates (see Nonhuman primates)
Principal investigator, 5, 16, 18, 27, 28, 32,
114, 155
Principles (see also U.S. Government
Principles)defined, 7
the Guide, 1
Procedural habituation and training, 29,
64-65
Procedures, defined, 7
Procurement of animals, 106-107

218

INdEX

Protocols for animal use
alternatives to animal use, 3, 5, 12, 25,
27, 162-164
conditioned-response, 31
food and fluid regulation, 30-31
IACUC review, 26-33
number of animals used, 5, 12, 15, 25,
26, 28, 106, 199, 201
postapproval monitoring, 33-34
reuse of animals, 5, 30
special considerations, 27-33
Three Rs, xvii, 3, 4-5, 17, 201
topics considered, 25-26
Public Health Service Policy on Humane
Care and Use of Laboratory Animals,
1, 12, 25, 56

Q
Quail, 60, 180, 193
Quarantine and stabilization, 110-111

R
Rabbits
behavioral and social management, 63
enrichment, 54
environment, 43, 44, 49, 54, 140
food and feeding, 67
housing, 51, 53, 58, 59
infections, 112
nomenclature, 77
recommended reading, 184-187
sanitation of cages, 71 n.2
space requirements, xviii, 59
Rabies, 22
Radiation hazards, 18, 20, 73, 147
Rainbow trout, 112
Rat Genome and Nomenclature Committee,
77
Rat theilovirus, 112
Rats (see also Rodents)
acclimation, 111
albino, 48-49
bedding and nesting materials, 52
breeding animals, 52
caloric restriction, 67
diseases, 45, 111, 112
GMAs, 145-146
identification/nomenclature, 77

illumination, 48-49, 137
recommended reading, 184-187,
194-196
space requirements, 57, 156
temperature and humidity, 43, 44, 45
Recordkeeping
clinical care, 115
population management, 75-76, 87-88
Removal of animals from protocol, 26, 29,
31
Reportingaccidents, bites, scratches, and allergic
reactions, 23
animal welfare concerns, 17, 23-24
hazardous conditions or “near miss”
incidents, 19
status of Animal care and use program,
25
Reproduction (see Breeding andreproduction)
Reptiles, 41-42, 44, 45, 77, 80, 81, 82, 83,
84, 85, 156, 178-179, 192-193
Research and testing methodology,
recommended reading, 165-166
Research team, training and education,
16-17
Respirators and respiratory protection, 22,
23
Restraint, 29-30, 55, 122, 144
Reuse of animals, 5
Ringtail, 45
Rodents, laboratory (see also individual
species)
acclimation, 111
aggression in males, 53, 64
albino animals, 48-49, 141anxiety studies, 53
barrier facilities, 145-146
bedding and nesting materials, 52, 53,
68-69
breeding, xviii, 49, 52, 57
caloric restriction, 67
diseases, 111, 112, 113
enrichment, 53, 54
euthanasia, 124
GMAs, 28, 68, 145-146
housing, 53, 57, 145-146
identification/nomenclature, 77
illumination, 48, 49, 137
implanted systems, 29
noise and vibration effects, 49, 50

21

INdEX
pain and distress, 121-122
recommended reading, 184-187,
194-196social stress, 53
space requirements, xviii, 56-58, 156
temperature and humidity, 43, 45
thermoneutral zone, 43

oversight, 34
postoperative care, 44, 119-120
presurgical planning, 116
procedures, 117-118
recommended reading, 188-190
training. 115-116
Swine, 49, 53, 60, 62, 112, 136

S

T

Sanitation
aquatic species, 85-86
assessing effectiveness of, 72-73
bedding/substrate change, 70
defined, 69-70
facilities for sanitizing materials, 143
macroenvironment, 72-73
microenvironment, 70-72
Secondary enclosures, 42, 43, 45-46, 47, 78(see also Macroenvironment)
Security, 23, 55, 151 (see also Biosecurity)
Separation of animals by health status andspecies, 111-112
Sheep, 62, 64, 111, 156, 176-177, 191-192
Simian hemorrhagic fever, 111-112
Simian immunodeficiency virus, 111-112
Social environment, xviii, 51, 53, 64, 82-83,
121 (see also Behavioral and social
management; Enrichment; Housing)Space requirements
agricultural animals, 60-63
aquatic species, 83
general considerations, 55-56
IACUC review, 56
laboratory rodents, xviii, 56-58“minimum,” xviii
nonhuman primates, 58-61
other common species (dogs, cats,rabbits; pigeons, quail, chickens), 58,
59, 60rabbits, xviii
Specific pathogen-free animals, 72, 108,
113, 135, 136, 145-146
Squirrel monkeys (Saimiri sciureus), 112
Statement of task, xix, 201-202
Storage areas, 141-142
Surgery
aseptic technique, 17, 34, 115, 116, 117,
118-119, 144
facilities, 116-117, 144-145
intraoperative monitoring, 119
multiple survival procedures, 30

Tamarins (Saguinus oedipus), 112
Temperature and humidity
aquatic species, 41-42, 80-81
HVAC systems and, 139-140
terrestrial species, 43-45
Terrestrial species
activity, 63-64
bedding and nesting materials, 68-69
behavioral and social management,
83-85
breeding, genetics, and nomenclature,
75-76
emergency, weekend, and holiday care,
74-75
enrichment, 52-54
environment, 42-50
food and feeding, 65-67
housing, 50-63
husbandry, 65-75
identification, 75
illumination, 47-49
macroenvironment, 42
management, 63-77
microenvironment, 42, 50-52
noise and vibration, 49-50
pest control, 74
population management, 75-77
procedural habituation and training,
64-65
recordkeeping, 75-76
social environment, 64
space requirements, 55-63
temperature and humidity, 43-45
ventilation and air quality, 45-47
waste disposal, 73-74
water, 67-68
Tetanus, 22
Thermoneutral zone, 43, 156
Three Rs (replacement, reduction, andrefinement), xvii, 3, 4-5, 17, 201
Toxicology research, 27
Toxins (see Biologic agents and hazards)

220

INdEX

Training and education, 20, 115-116
animal care personnel and technicians,
16, 50, 78
aquatic systems, 78
emergency preparedness, 35
IACUC members, 17
IACUC oversight of, 15
noise reduction, 50
postapproval review as, 34
recommended reading, 168-169
research team, 16-17
U.S. Government Principles, 12,
199-200
veterinary and other professional staff,
15-16
Transportation of animals, 12, 14, 32, 105,
107-109, 110, 111, 134, 135, 147,
150, 156, 188, 199
Trauma research, 27
Tuberculosis, 23, 112
Tumor models/studies, 27, 34

U
Unexpected outcomes, 28-29, 34
US Department of Agriculture, 19, 30, 107,
148, 157
US Fish and Wildlife Service, 106, 107, 157
U.S. Government Principles for theUtilization and Care of Vertebrate
Animals Used in Testing, Research,
and Training, 4, 12, 199-200
Uses and users of the Guide, 4-5

V
Vaccine challenges/studies, 27
Ventilation and airflow (see also Air quality)
aquatic species, 80-81
containment of hazardous agents, 21, 47
filtered, 47
for static isolation caging, 47
HVAC system, 46, 47, 139-140
individually ventilated cages, 44, 46, 50,
58, 70-71, 111, 141, 146preventive maintenance and evaluation,47
recommended reading, 196-197
recycling air, 46-47
terrestrial species, 43, 45-47

Veterinary care (see also Clinical care andmanagement; Preventive medicine;
Surgery)
anesthesia and analgesia, 121-123
euthanasia, 123-124
pain and distress, 120-121
principles, 12
procurement of animals, 106-107
recommended reading, 188-196
responsibilities of attending veterinarian,
14
training and education of professional
staff, 15-16transportation of animals, 12, 32, 33, 55,
107-109, 156, 195
Vibration (see Noise and vibration)

W
Walls and ceilings, 138-139
Waste management (see also Sanitation)
anesthetic gases, 21, 147disposal options, 73-74
drainage, 54, 138
flushing, 72
gases from primary enclosures, 46
hazardous materials, 20, 73-74, 148-149
soiled bedding, 85, 143
storage before incineration or removal,
136, 142
training, 20, 74
wastewater, 80, 87
Water/fluids
devices and delivery systems, 68
outdoor sources, 68
quality for aquatic species, 78-79, 85
regulated, 30-31
terrestrial species, 67-68
Windows, exterior, 137xXenopus spp., 79-80, 83, 88, 178-179,
192-193

Z
Zebrafish, 79, 83
Zoonosis surveillance, 23, 32, 110