Contributors to Volume I NORMAN BLEICHER WILLIAM CARLSON JOSEPH V. JEMSKI ALVIN F. MORELAND WALTER L. NEWTON G. BRIGGS PHILLIPS ALBERT SCHAFFER IRA SINGER DIETRICH C. SMITH GEOFFREY WOODARD Methods of Animal Experimentation EDITED BY WILLIAM I. GAY ANIMAL RESOURCES BRANCH DIVISION OF RESEARCH FACILITIES AND RESOURCES NATIONAL INSTITUTES OF HEALTH BETHESDA, MARYLAND Volume I 1965 ACADEMIC PRESS New York and London COPYRIGHT © 1965, BY ACADEMIC PRESS INC. ALL RIGHTS RESERVED. NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS. ACADEMIC PRESS INC. Ill Fifth Avenue, New York, New York 10003 United Kingdom Edition published by ACADEMIC PRESS INC. (LONDON) LTD. Berkeley Square House, London W.l LIBRARY OF CONGRESS CATALOG CARD NUMBER: 64-24657 PRINTED IN THE UNITED STATES OF AMERICA. List of Contributors Numbers in parentheses indicate the pages on which the authors' contributions begin. Norman Bleicher, Department of Surgery, School of Medicine, Univer- sity of California, Los Angeles, California (103) William Carlson, Department of Radiology and Radiobiology, College of Veterinary Medicine, Colorado State University, Fort Collins, Colo- rado (151) Joseph V. Jemski, Civilian, U.S. Army Biological Laboratories, Fort De- trick, Frederick, Maryland (273) Alvin F. Moreland, University of Florida, College of Medicine, Gaines- ville, Florida ( 1 ) Walter L. Newton,* Laboratory of Germfree Animal Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland (215) G. Briggs Phillips, Civilian,U.S. Army Biological Laboratories, Fort De- trick, Frederick, Maryland (273) Albert Schaffer, State University of New York, Downstate Medical Cen- ter, Brooklyn, New York (43) Ira Singer, Department of Microbiology and Tropical Medicine, George- town University Schools of Medicine and Dentistry, Washington, D.C. (197) Dietrich C. Smith, Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland (167) Geoffrey Woodard, Woodard Research Corporation, Herndon, Virginia (343) * Present Address: National Institute of General Medical Sciences, National Institutes of Health, Bethesda, Maryland. V Preface Although much has been written about methods for the care and production of laboratory animals, there has been a lack of compiled in- formation on the use of animals in various fields of research. In the be- lief that such compilation would benefit investigators conducting re- search dependent upon the use of animals, this book was written. "Methods of Animal Experimentation" provides information on the most common methods for using animals as tools in the search for new bio- logical knowledge. The authors of the individual contributions and the editor believe that the techniques described will facilitate the most efficient use of research animals and provide guidelines for their utmost comfort and welfare. The descriptions of both fundamental and well-developed techniques of animal experimentation in various research fields should be useful to graduate students and experienced scientists who must consider varia- tions in research approaches. The book is a source of information for the scientist administrator who is frequently confronted with different pro- posed approaches to biological research projects utilizing animals. The text is arranged according to specific research methods rather than to organ system or disease category. This approach gives the reader a broad view of the techniques involved in specific fields and describes the range of usefulness of these techniques. This approach also made it possible to select contributors expert in experimental methods and equally well qualified in the care of laboratory animals. The common diseases of laboratory animals are not discussed in the text unless they are related closely to the experimental methods de- scribed. Although not intended to be a general guide on conventional methods of the production, care, and feeding of laboratory animals, the book thoroughly reviews special animal care associated with each ex- perimental technique. The book also discusses hazards involved in using the various experimental techniques and suggests necessary safety pre- cautions. The reader is referred to W. Lane-Petter's "Animals in Re- search" (Academic Press, New York, 1963) for basic information on laboratory animal care and maintenance. The first five chapters of Volume I describe basic information, meth- ods, and principles involved in managing animals for experimental pro- cedures. The remaining chapters in Volume I and those in Volume II deal with special techniques which have been demonstrated to be dis- tinct, useful methods for using laboratory animals as a basic biomédical research tool. vii viii PREFACE The editor is extremely grateful for the splendid cooperation and sup- port given by the contributors of this volume. He is also especially in- debted to Dr. Robert J. Schnitzer for his expert counsel, guidance, and inspiration. December, 1964 WILLIAM I. GAY Contents of Volume II 1. Radiation Hygiene Neal S. Nelson and John H. Rust 2. Uses of Ionizing Radiation for Measuring Biological Phenomena Neal S. Nelson and John H. Rust 3. Tumor Transplantation Kanematsu Sugiura 4. Parabiosis Charles E. Hall 5. Organ Transplantation Roy Y. Calne 6. Care and Use of Hibernating Animals Raymond J. Hock 7. High-Altitude Studies PaoL· S. Timiras 8. Care and Growth of Animals During Chronic Centrifugation Charles C. Wunder 9. Controlled Exercise James E. Corbin 10. Methods of Animal Infections Robert J. Byrne 11. Electronic Monitoring of Physiological Phenomena in Experimental Animals Cesar Λ. Caceres, Juan B. Calatayud, and Patrick A. Gorman Author Index—Subject Index xiii Introduction Volumes on methodology associated with the basic science disciplines have become commonplace. It is surprising that the techniques for using animals in biomédical research have not been previously compiled. This text will provide a cross section of information dealing with the tech- niques of a wide variety of experimental procedures. It is alarming to discover the lack of knowledge of this subject by many graduate students in the biological sciences. The recent trend in many disciplines toward studying isolated phenomena is resulting in a lack of appreciation for the considerable fundamental knowledge which may be gained by making use of experiments utilizing information from the "whole animal." Many students in the biological sciences receive all of their research training using tissue cultures or isolated microorganisms. No doubt these students will reach a point in their investigations where it will be essential to test concepts in experimental animals. Hopefully this text will stimulate incorporation of courses in methods of animal ex- perimentation into the curricula of these students. It has become increasingly obvious that ideas for new avenues of re- search are dependent upon a general knowledge of the experimental methods that have been used in both related and unrelated research areas. Many of the so-called "original" investigations have involved adapting methods used in other types of studies to the individual's problem. This text will doubtless stimulate new approaches by making investigators aware of the methods used in other fields. This text covers a variety of topics by individuals actively using the techniques they describe. The descriptions of the techniques in any spe- cific category is not intended to be exhaustive but each has an ample bibliography and will serve to orient the reader to the pertinent litera- ture. THOMAS B. CLARKSON Bowman Gray School of Medicine, Wake Forest College, Winston-Salem, North Carolina January, 1965 XV CHAPTER 1 Collection and Withdrawal of Body 1 Fluids and Infusion Techniques ALVIN F. MORELAND2 I. Introduction 1 II. Collection and Withdrawal of Body Fluids 2 A. Blood and Lymph Collection 2 B. Urine Collection 18 C. Collection of Body Fluids other than Blood, Lymph, and Urine 21 III. Infusion Techniques 32 A. Administration of Fluids via the Digestive Tract 32 B. Techniques for Parenteral Administration of Fluids 37 References 40 I. Introduction The purpose of this chapter is to present a résumé of the more com- monly used techniques for the collection and withdrawal of body fluids and infusion techniques with experimental animals. Through the years many techniques have been used and described in periodical literature. It is hoped that this chapter will assimilate much of this data in a readily accessible form. The reader must be aware, however, that all available techniques are not discussed. Much of the material presented will be quite elementary. This is necessary since this book will be used as a reference guide by some in- dividuals who have not had previous experience with animal experi- mentation. Such basic information for these people is invaluable. ΧΑ11 illustrations for this chapter have been drawn by George Lynch, medical il- lustrator at Wake Forest College, The Bowman Gray School of Medicine, Winston- Salem, North Carolina. 2Fellow in Laboratory Animal Medicine, The Bowman Gray School of Medicine, Wake Forest College, Winston-Salem, North Carolina. The author is presently Assist- ant Professor of Experimental Medicine and Director of the Animal Department, J. Hillis Miller Health Center, University of Florida, Gainesville, Florida. 1 2 ALVIN F. MORELAND II. Collection and Withdrawal of Body Fluids A. Blood and Lymph Collection 1. The Mouse a. Blood Collection. The blood volume of a 30-gm mouse is approxi- mately 2.4 ml. However, one is seldom able to obtain more than approxi- mately one-half this volume. Heart puncture. This technique requires that the animal be anes- thetized. It usually is accomplished by placing the mouse in a jar which contains cotton saturated with ether and leaving him until his activity ceases and he breathes with moderately deep and regular respiratory movements. The animal is then restrained in dorsal recumbency. Figure 1 illustrates a board with clips fastened at the four corners which can be attached to the animal's legs and tension can be applied. A 1- or 2-ml syringe with a 25- to 27-gauge, %- to 1-inch needle is used. The thorax is swabbed with disinfectant and the apex beat of the heart is palpated with the index finger. This is located immediately anterior to the xiphoid cartilage. The needle is introduced into the area of the heart and aspira- tion applied. This is not an easy technique, and the old adage "practice makes perfect" applies. Tail venipuncture. This technique can be applied without the use of anesthesia providing the mouse is restrained. However, the technique is more easily accomplished if anesthesia is used. Place the mouse on a flat surface under a bright light, preferably of the white "spotlight vari- ety, and grasp the tail between the thumb and the index finger. Swab the tail first with xylol, follow with disinfectant, and dry with a sponge. A 1- or 2-ml syringe with a 25- to 27-gauge, %- to 1-inch needle is used. The veins are visualized laterally and dor sally on the tail. The needle is introduced near the distal portion of the tail with the bevel up. This al- lows one to observe directly as the needle enters the vein, and gentle aspiration can be applied. If subcutaneous hemorrhage occurs, it is better to move the site anteriorly along the vein. This is a very difficult technique and requires much practice to gain proficiency. Jugular venipuncture. Kassel and Levitan (1953) state that samples may be drawn repeatedly from this site for several weeks if necessary. Briefly, the mouse is held by the skin at the back of the neck and the head is extended by fastening gauze over its upper incisors and hold- ing this between the fingers of the left hand. The hair of the neck and upper thorax is removed and both jugulars are easily seen. A 26-gauge needle is used to enter the distended vessel. It is stated that weanling mice and larger may be bled in this manner.