Amphibians: Guidelines for the breeding, care, and management of laboratory animals

I Introduction

A. GENERAL COMMENTS

Few animals are as valuable for experimental investigations as amphibians (Kawamura and Nishioka, 1973): They are ubiquitous in nature, transitory between aquatic and terrestrial life, reasonable in size, ectothermic and the only naked vertebrates, accessible to direct examination through all developmental periods, and possess genetic mechanisms amenable to study by biological and mechanical manipulation.

Traditionally, investigators collected these animals in the wild and completed their experiments within days. In spite of their ancient history as experimental animals, care and management of these animals is rudimentary, and few established and tested guidelines for their husbandry are available (Boterenbrood, 1966; Frazer, 1966; Bardach et al., 1972). With few exceptions (e.g., mink, monkeys, hamsters, gerbils), laboratory vertebrates have been chosen from among those animals previously domesticated for agricultural purposes (e.g., chickens, goats, rabbits, swine), for pets (e.g., cats, dogs), or have been symbionts of man (e.g., mice and rats). This is not true of amphibians. While enthusiasts have maintained amphibians in limited numbers, no amphibian, except the axolotl, Ambystoma mexicanum, has a history of mass culture for repeated generations.

B. THE NEED

1. The Demand

Factors that stimulate a demand for amphibians are their utility for current research problems, the increased cost of avian and mammalian research animals, and the increased use of living material in high school and college instructional laboratories.

Best estimates indicate that 20 million preserved and living amphibians

TABLE 1 Common Frog (Rana temporaria) and Common Toad (Bufo bufo): A Summary of the Replies Referring to Changes in Status in Breeding Sites^a

were used for educational purposes alone in the United States in 1971 -1972. Recent studies by the institute of Laboratory Animal Resources (ILAR) report the use of 2 million amphibians per year in research laboratories (ILAR News, 1972). Only rodents exceed this figure. Birds exceed it only if the number of incubated eggs are included in the tally. This heavy use of amphibians escapes the casual observer, because these animals are either used within days of their arrival in a research laboratory or lost prior to the completion of experiments due to inadequate maintenance.

2. The Supply

a. The Natural Resource

Although interest in amphibian resources is long standing (Wright, 1920), accurate figures on the status of amphibians in nature are unavailable for most American populations. This question has recently assumed great interest because of the apparent short supply and diseased state of amphibians collected from nature (Maugh, 1972; anonymous, 1973). American amphibian dealers find it increasingly difficult to identify high-density pop populations that permit economical collection of animals. Informal surveys among herpetologists and others interested in amphibians suggest that in recent times American amphibian populations have decreased significantly.

The best current data dealing with population reduction have been collected for British anurans (Cooke, 1972). This survey, based on questionnaires distributed to educational authorities (schools survey) and professional biologists (breeding sites survey), demonstrated that the changes in these populations were most significantly affected by habitat destruction and were directly proportional to the density of human populations and to increasing industrialization (Tables 1 , 2, and 3).

Amphibian populations fluctuate drastically in response to cataclysmic environmental factors such as food shortage, drought, and early winter. in addition, the problem of short supply is aggravated by large losses that occur during shipment and holding periods (Gibbs et al., 1971). However, no clear evidence exists that the survival of any of the amphibian species commonly used for investigative purposes is endangered by collection practices. The high reproductive potential of these amphibians assures that, given optimal environmental and climatic conditions, the populations rapidly regenerate.

b. Artificial Culture

The demand described above and the increasing difficulty of satisfying these demands from natural sources has led to a renewed interest in the

TABLE 2 Common Frog (Rana temporaria) and Common Toad (Bufo bufo): A Summary of the Reasons for Decreases in Status Given by Correspondents to the Breeding Sites Survey and Schools Survey^a,b

TABLE 3 Comon Frog (Rana temporaria) and Common Toad (Bufo bufo): A Summary of the Reasons for Increases in Status Given by Correspondents to the Breeding Sites Survey and Schools Survey^a,b

artificial culture of amphibians. Serious efforts are currently being made toward this end. However, costs remain high and the quantity of available cultured animals remains inadequate to meet the demand. As the cost of collecting the natural supply increases, artificial culture will certainly become economically feasible, or even necessary, to maintain the supply of healthy amphibians for at least scientific users.

3. Recommendations

We recommend that

a. A survey of the natural amphibian resources, parallel to that conducted in England (Cooke, 1972), be conducted on the North American continent to allow adequate evaluation of the supply base.

b. Increasing efforts be made to develop the culture of amphibians, both for educational and research purposes.

c. Improvements in the care and management of amphibians be instituted as early as possible to minimize the losses that now occur between the supply and the ultimate use of amphibians and to maximize the quality of research animals.

C. USERS OF AMPHIBIANS

Amphibian users are best described by the type of agency that supports their work (Table 4), the location of the research (Table 5), and the topics under investigation (Table 6). These tables were compiled from Science Information Exchange Notices of Research Projects, active as of January 1969, and were originally published in Nace (1970) where they appear with more detail and discussion.

Federal, state, and foundation agencies funded 277 projects relevant to this document (Table 4). In addition, much research on amphibians is conducted on low-budget projects. Though there is no accurate method of estimating the numbers of such projects, reviews of the index journals, including Dissertation Abstracts, would reveal these numbers. At the University of Michigan and at the time in question, however, for each listed project about 15 investigators were conducting research involving the use of amphibians on unlisted projects. This 1:15 ratio of listed to unlisted projects may be conservative when it is recalled that many investigators at smaller colleges not funded by nationwide granting agencies use amphibians. Thus 3,0004,000 amphibian projects may be active, a figure, when divided into the ILAR survey on amphibian use, representing 500-600 amphibians used (received) per project. This figure seems high for

Table 4 Support of Research Using Amphibians as of January 1969^a

TABLE 5 Location of Research Using Amphibians as of January 1969^a

TABLE 6 Topics of Research Using Amphibians as of January 1969^a

many projects, but may be a reasonable average when known projects using 10 times that number are considered. Also, pituitaries from 3 to 10 animals are used for the induced ovulation of a single female frog, and a conservative estimate suggests no more than 50 percent of the amphibians received for research purposes survive the period between receipt and use.

D. LABORATORY-DEFINED AMPHIBIANS

Defined laboratory amphibians (Gay, 1971; Committee on Animal Nutrition, 1972), though essential for high-quality investigations, remain largely a hope for the future. To date, the best definition (nomenclature described in Chapter III) has been attained with the axolotl (Boterenbrood, 1966). Some colonies of laboratory amphibians-such as Xenopus in the laboratory of Fischberg (Blackler and Fischberg, 1968) and Pleurodeles in the laboratories of Gallien (Guillet etal., 1971) and Beetschen (1971)-do exist and meet the criteria defined for laboratory-reared and laboratory-bred animals; however, these have not attained the status of defined laboratory lines. For Rana pipiens efforts are now being made to establish laboratory lines (Nace et al., 1965, 1966; Nace, 1968, 1970; Nace and Richards, 1969, 1972a,b,c), but animals in these lines are not yet available in significant numbers.

The importance of nomenclature cannot be overstressed during this period when defined strains of amphibians are still under initial development. Any nomenclature that is adopted should include a designation of the laboratory where the animals were developed and a key to the criteria satisfied. The geographic designation should be as precise as possible. It should specify a geographic region in sufficient detail to suggest the parent population.

It is hoped that those strains currently under development will soon become sufficiently well defined to justify publication of a standard nomenclature and growth tables as exemplified by those listings that have appeared for mice (States, 1972) and other laboratory animals (Poiley, 1972). Since the laboratory use of amphibians will continue to depend on animals from wild populations, terminology to designate the history of such animals must be introduced. These terms (defined in Chapter III, Section B) include wild, wild-caught nonconditioned, wild-caught conditioned, laboratory-reared, and laboratory-bred. Types of laboratory-reared and laboratory-bred populations and lines are defined in Chapter III, Section C.

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