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Amphibi n Guidelines for the breeding, care, and management of laboratory animals A Report of the Subcommittee on Amphibian Standards Committee on Standards Institute of Laboratory Animal Resources National Research Council NATIONAL ACADEMY OF SCIENCES Washington,D.C. | 1974

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NOTICE: The project that is the subject of this report was approved by the Govern- ing Board of the National Research Council, acting in behalf of the National Acad- emy of Sciences. Such approval reflects the Board's judgment that the project is of national importance and appropriate with respect to both the purposes and re- sources of the National Research Council. The members of the committee selected to undertake this project and prepare this report were chosen for recognized scholarly competence and with due consider- ation for the balance of disciplines appropriate to the project. Responsibility for the detailed aspects of this report rests with that committee. Each report issuing from a study committee of the National Research Council is reviewed by an independent group of qualified individuals according to proce- dures established and monitored by the Report Review Committee of the National Academy of Sciences. Distribution of the report is approved, by the President of the Academy, upon satisfactory completion of the review process. Lee primary support for this publication was provided by Contract VlOOlP-243 within the Veterans Administration. Additional support was provided by Contract PH43~4-44 with the Drug Research and Development, Division of Cancer Treat- ment, National Cancer Institute and the Animal Resources Branch, National Insti- tutes of Health, U.S. Public Health Service; Contract AT (11-1)-3369 with the Atomic Energy Commission; Contract N00014~7-A-0244-0016 with the Office of Naval Research, U.S. Army Medical Research and Development Command, and U.S. Air Force; Contract 12-16-140-155-91 with the Animal and Plant Health Inspection Service, U.S. Department of Agriculture; Contract NSF-C310, Task Order 173, with the National Science Foundation; Grant RC-10 from the American Cancer Society, Inc.; and contributions from pharmaceutical companies and other industry. Library of Congress Cataloging in Publication Data National Research Council. Institute of Laboratory Animal Resources. Subcommittee on Amphibian Standards. Amphibians: guidelines for the breeding, care, and management of laboratory animals. 1. Amphibians as laboratory animals. I. Title. [DNLM: 1. Amphibia. 2. Animals, Laboratory. QY50 N272a 19741 SF407.A45N37 1974 636'.37 74-925 ISBN 0-309-0221X Available from Printing and Publishing Office, National Academy of Sciences 2101 Constitution Avenue, N.W., Washington, D.C. 20418 Printed in the United States of America

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Preface Amphibians have a long history of use in biomedical research, but have re- ceived little formal attention as laboratory animals. The need for amphib- ians and the increasing difficulty encountered in obtaining suitable animals collected from the wild have produced a demand for recommendations for the breeding, care, and management of these laboratory animals. With the exception of useful but limited chapters in books on over topics and of privately circulated handout sheets, this document is a first attempt to as- semble the available, practical information on the breeding, care, and man- agement of amphibians for the laboratory. Major emphasis is on the an" urans (frogs and toads) used by investigators in North America, with some attention given to the urodeles (salamanders and newts). In many respects, this effort is premature. Though the situation is somewhat better for urodeles, so recent is the attempt to maintain anurans in long-term laboratory culture that relatively little is known about the critical factors involved. Adequate objective criteria on which to base the evaluation of quality or uniformity are simply unavailable. Although there has been an increase in the knowledge of amphibian genetics recently, the control or reproductive cycles, the evaluation of nutritional regimens, the regulation of growth, and the management of disease in amphibians are only now under serious concerted investigation. As a consequence of these deficiencies in our knowledge, this guide is heavily dependent on the lore that has developed among amphibian biolo- gists. Therefore, the recommendations and guidelines suggested here should be considered as tentative. Specific procedures are subject to mod- ification in accordance with individual situations, colony size, and personal preference. A serious handicap to the development of adequate care of amphibians is the relative inaccessibility to investigators of jargon-free, yet adequate, ...

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iv accounts of the "whole animal" biology of amphibians. While this docu- ment cannot contain more than brief reference to some of these matters, we hope it will provide essential information to those who are not experi- enced with amphibians. We also hope that it will lead those with greater experience to devise new procedures or discover new principles, whose adoption would greatly improve breeding, care, and management of am- phibians for laboratory purposes. This document should serve as a useful guide to all users of amphibians, should lead to success in the normal maintenance of amphibian colonies, and should stimulate efforts toward improving the quality of utilization of these animals. In addition to guidelines for animal care and quality, cer- tain terminology is suggested. If accepted in standard practice, this termi- nology should lead to more reliable products from biomedical investigators using amphibians.

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SUBCOMMITTEE ON AMPHIBIAN STANDARDS COMMITTEE ON STANDARDS G EO R G E W. N AC E, Chairman, University of Michigan DU D ~ EY D. C U ~ ~ KY, Louisiana State University MARVIN B. EMMONS, E. G. Stei~ilber and Company ERICH L. GIBBS, Ultrascience Inc. VICTOR H. HUTCHISON, University of Oklahoma ROBERT G. McKINNE~L, University of Minnesota v

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Contents I Introduction A. General Comments B. The Need 1. The Demand, 1 2. The Supply, 3 a. The Natural Resource, 3 b. Artificial Culture, 3 3. Recommendations, 7 C. Users of Amphibians D. Laboratory-Defined Amphibians 11 Classification and Description of Amphibians Commonly Used for Laboratory Research A. Phylogeny and Classification B. Description of Species Commonly Used in Laboratory Research 1. Urodeles, 13 a. Ambystoma, 13 b. Notophthalmus viridescent, 15 c. Necturusmaculosus, 16 2. Anurans, 16 a. Xenopus Levis, 16 b. Rana catesbeiana, 17 c. Rana grylio, 18 d. Rana clamitans, 18 e. Rana pipiens, 21 f. Rana palustris, 23 g. Rana sylvatica, 24 h. Bulo marinas, 24 i. Bombina orientalis, 25 l 7 11 12 12 13 vi

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e vll 111 Definition and Description of Experimental Amphibians A. Introduction B. Definition 1. Wild, 28 2. Wild Caught, 28 a. Nonconditioned, 28 b. Conditioned, 30 3. Laboratory Reared, 31 a. Standard, 31 b. Miscellaneous, 31 4. Laboratory Bred, 32 a. Standard, 32 b. Miscellaneous, 32 C. Description of Laboratory-Reared and Laboratory Bred Amphibians 1. Types of Populations and Lines, 32 a. Random Mating Lines, 33 b. Heterozygous Isogenic Clones, 33 c. Heterozygous Marked Lines, 33 d. Mutant Lines, 34 e. Inbred Lines, 34 f. Gynogenetic Diploid Lines, 34 g. Homozygous Lines, 34 h. Haploid Animals, 35 i. Polyploid Animals, 35 2. Sex Determination and Its Manipulation, 36 3. Species of Laboratory-Reared or Laboratory-Bred Amphibians Available in the United States, 37 a. Anurans, 37 b. Urodeles, 38 IV Sources A. Selection B. Dealer Care C. Ordering, Shipping, and Receiving 1. Ordering, 40 2. Shipping, 41 3. Receiving, 42 a. Aquatic Amphibians, 42 b. TerrestrialAmphibians,43 D. Legal Aspects V Physical Facilities A. Relation to Natural Habitat B. The Amphibian Quarters 1. General Description, 47 a. Isolation Quarters, 47 b. Heating, Ventilation, and Size Specifications for Rooms, 48 27 27 28 32 39 39 40 40 45 46 46 47

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viii c. Description of Ancillary Rooms, 49 d. GeneralSpecif~cations,49 2. Environmental Control, 50 a. Water, 50 b. Temperature, 57 c. Lighting, 57 C. Enclosures 1. Embryos: Fertilization through Initiation of Feeding, 58 2. Larvae, 59 3. Juveniles, 60 a. At Metamorphic Climax, 60 b. Postmetamorphic,61 4. Adult Enclosures (Evaluation Criteria), 62 5. Hibernation Quarters, 63 6. Enclosure Designs, 64 a. The Amphibian Facility of The University of Michigan, 64 b. The R. catesbeiana Facility at Louisiana State University, 68 c. Southern Frog Company, 71 d. The Aquatic Animal Facility of Arizona State University, 72 Vl Amphibian Management and Laboratory Care A. General Comments B. Anurans 1. Ranidae, 76 a. R. pipiens, 76 b. R. catesbeiana, 84 c. Other Ranid Species, 87 2. Other Anurans, 88 a. Xenopus, 88 b. Bufo, 90 c. Bombina orientalis, 90 C. Urodela 1. Axalotls, 91 a. Early Larvae, 91 b. Mature Larvae, 92 2. Other Urodeles, 93 Vl I Breeding A. Anurans 1. General Comments, 95 2. Artificial induction of Ovulation, 95 3. Amplexus, 96 4. Artificial Insemination, 97 5. Parthenogenetic Lines and Haploid Animals, 99 6. Homozygous Lines and Androgenesis, 100 7. Polyploid Animals, 100 8. Mosaic Animals, 101 9. Nuclear Transplantation, 101 10. Xenopus, 101 58 75 75 76 91 95 95

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ix B. Urodeles 1. Mating, 103 2. Spawning, 104 3. Artificial Insemination, 106 4. Initial Care of Embryos, 107 Vl 11 Records and Information Control A. Identification of Individuals 1. Numbering, 108 2. Tattooing, 109 3. Branding, 109 4. Toe Clipping, 110 5. Over Marking Systems, 110 6. Drawings and Photographs, 111 B. Information Control Systems IX Amphibian Medicine A. General Comments B. Bacterial Diseases 1. Pathogens, 116 2. Drug Selection and Administration, 117 3. Identifying Diseased Frogs, 120 4. The Need for Treatment, 121 C. Viral Diseases 1. General Comments, 121 2. Lucke Tumor Herpesvws (LTHV), 122 3. Amphibian Polyhedral Cytoplasmic Deoxyribovirus (PCDV), 122 4. Lymphosarcoma Virus of Xenopus, 123 D. Parasitic Diseases E. Mycotic Diseases F. Euthanasia and Anesthesia X Personnel A. General Comments B. Safety Hazards 1. Physical, 128 2. Biological, 128 Appendix A Status of "Endangered" Amphibians 103 108 108 111 115 115 116 121 123 124 124 127 127 128 131 Appendix B Control Laws by States and Canadian Provinces 133 References 143

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