Amphibians as Laboratory Animals

Dale DeNardo, D.V.M., Ph.D., is an Associate Veterinarian at the University of California, Berkeley. Amphibians include over 4,500 species within 3 major lineages---caecilians, salamanders, and anurans (Stebbins and Cohen 1995). These lineages are linked by several unique physiological traits. The most prominent is the indirect lifestyle of many amphibians--the aquatic gill-breathing larval stage and the aquatic or terrestrial lung or skin-breathing adult stage. A second feature of amphibians is the presence of 2 types of skin glands: mucous and granular. Mucous glands keep the skin surface moist to allow transcutaneous respiration, but they also put the animals at risk of desiccation. The granular glands secrete toxins and function primarily for defense (Zug 1993). The more than 160 species of caecilians are not diverse, all being legless and inhabiting tropical regions. This poorly understood group of amphibians is used in research relatively infrequently, and, therefore, will not be discussed further (see Wake 1994, for review of caecilian husbandry). The salamanders are represented by nearly 400 species, some of which have a four-legged aquatic larval stage, while others (primarily the Plethodontidae) pass the larval stage within eggs that are laid in moist places on land (Stebbins and Cohen 1995). Some salamanders retain the larval gills through their adult stage and depend on oxygenated water throughout their lives. While most salamanders have legs, they have become vestigial in some. The anurans, or frog and toads, are the most successful lineage of amphibians with approximately 4,000 species distributed around the world in a multitude of habitats. The larval stage of most, but not all, anurans includes a legless tadpole or polliwog. The degree of dependency on water varies--species range from purely aquatic to completely terrestrial. Frogs are not only the most diverse group of amphibians; they also are by far the most commonly used amphibians in research. While space constraints limit detailed discussion of amphibian biology and taxonomy here, the topic is thoroughly covered elsewhere (Moore 1964-1976; Frost 1985; Duellman and Trueb 1986; Duellman 1993; Zug 1993). Only a small number of the approximately 4,500 species of amphibians are available commercially. Fortunately, it is with these few commercially available species that the vast majority of laboratory research with amphibians is conducted. Many amphibians have a seasonal availability that must be planned for or around. “Professionalism,” in terms of reliability and quality, can vary greatly among amphibian vendors. One must realize and accept the fact that vendors of amphibians do not conduct business or house animals using the high standards of most rodent vendors. However, this does not mean that one should accept unhealthy or inhumanely treated animals. Potential vendors should be assessed by obtaining information about how animals are acquired and housed. Once obtained and deemed acceptable, the accuracy of this information can usually be validated when an initial shipment of animals is received. Animals that are of poor quality upon arrival can be a result of either unhealthy animals being shipped or improper shipping of originally healthy animals. It is important to differentiate these two causes, in order to prevent obtaining animals in similar condition in the future. Problems in shipping can be corrected by using alternative packing methods or transport services, while problems originating from the vendor are usually best corrected by switching to another source. Of the limited number of amphibians readily available commercially, only a handful of these are actually bred in captivity. Species commonly used in research and routinely cap-tive-bred include the clawed frog (Xenopus laevis), the African dwarf frog (Hymenochirus boettgeri), the fire-bel-lied toad (Bombina orientalis), the axolotl (Ambystoma mexicanum), and the tiger salamander (A. tigrinum). Some additional species are selectively bred at institutions. The remaining amphibians used in research are collected from wild populations by investigators or by commercial vendors, as is the case with commonly used amphibians such as the bullfrog (Rana catesbeiana), grass frog (R. pipiens), cane toad (Bufo marinus), and mud puppy (Nectutus maculosus). Many states require permits to collect amphibians. Fishing licenses may suffice in some states, while scientific collecting permits may be necessary in others. Amphibians can be collected in numerous way. Procedures concerning the collection of amphibians have been thoroughly reviewed elsewhere (Heyer and others 1994), so only a short discussion will be included here. The most common method is to collect by hand amphibians that are secluded under rocks, logs, or other debris. Active amphibians can often be collected during rains or at night with the use of flashlights and lanterns. Slowly driving roads at night, especially during rains, can be a very productive way to capture many species of amphibians. Numerous devices have been used to aid in the collection of amphibians. Larvae and aquatic species can be captured with nets and seines. More recently, aquatic amphibians have been collected using electroshock. When using electroshock, one must consider the potential health risk to not only the target species, but also to other species present. Predominantly terrestrial amphibians are commonly captured using fence lines with pitfall traps. The fence lines are placed around breeding ponds, trapping individuals as they move to and from the ponds. Pitfall traps must be shaded from the sun and contain a moist substrate to prevent mortality. There are limited restrictions that affect the importation, exportation, and possession of amphibians in the United States. Quarantine is not required when importing amphibians into the United States. Some states, however, have restrictions for certain species. The most commonly restricted species are the clawed frogs (Xenopus spp.) and large toads (such as Bufo marinus, B. horribilis, and B. paracnemis). State permits may be required to own these anurans, and they may also require special housing conditions. Investigators should consult their state wildlife department for information regarding specific species restrictions. Regardless of the source, one's ability to successfully maintain amphibians starts with acquiring healthy animals. It is far easier to prevent than to treat disease in amphibians. Clinical disease in amphibians can be frustrating to researchers and veterinarians alike, since individuals do not often respond to treatment and infectious diseases can spread rapidly through a collection. When choosing a vendor, one must consider reliability, location, transportation method, and, of course, condition of the animals upon arrival. Reliability refers to receiving individuals of the proper size and correct species at the appropriate time. Many species of amphibians are prone to “shipping stress,” so time in transport should be minimized. Choosing a nearby vendor and using air freight or, at the very least, overnight mail, can help shorten the shipping time. Packaging should be of solid construction for physical protection and should maintain adequate moisture levels for the entire time of transit. It is often best to withhold food, but not water, for 24 hours prior to shipping, since this will reduce waste production while in transit. Shipping during extreme weather conditions, especially heat, should be avoided. Housing for newly acquired amphibians should be functional prior to their arrival, so that the animals can be quickly unpacked. Newly arrived individuals should be given a thorough visual evaluation and housed separately from individuals already present in the colony. A minimum 30-day quarantine period is suggested. During quarantine, animals should be carefully monitored for morbidity and mortality and evaluated for endoparasites. Any clinical disease should be properly diagnosed and aggressively treated. It is best to locate recently arrived amphibians, particularly ranid frogs, in a room with low traffic since they are easily agitated. When frightened, these frogs will jump excessively and may traumatize themselves on the cage lid. Using a deep opaque cage will minimize the trauma to the frogs. Placing opaque plastic over cages housing newly acquired frogs will also minimize the amount of jumping. Being “lower vertebrates,” amphibians are exempt from U.S. Department of Agriculture (USDA) regulations. The Guide for the Care and Use of Laboratory Animals (Guide) (NRC 1996) does not provide specific recommendations for the maintenance of amphibians. However the Guide does state, “An appropriate environment should be provided for nontra-ditional species...Expert advice on the natural history and behavior of nontraditional species should be sought when such animals are to be introduced into a research environment“ (NRC 1996, p 14 of prepublication copy). To assure that humane issues are being properly addressed, one must use available guidelines (NRC 1974; ASIH 1987; Pough 1991, 1992); possess a good understanding of amphibian biology; and apply, with adaptations, procedures used for traditional species. Institutional animal care and use committees (IACUCs) need to establish institutional guidelines for dealing with the use of amphibians, both in the field and the laboratory. These guidelines should be developed by a cooperative process, incorporating input from written guidelines, committee members, faculty using amphibians, campus veterinarians, and consultants. Guidelines contrived in such a positive manner tend to be stronger and hold up better in this complex field where much is still not well understood. A wide variety of caging is used for housing amphibians. Standard rodent cages with modified lids work well for smaller species, while large plastic or fiberglass tubs are used for larger species. Certain criteria should be addressed when choosing proper caging. First, the cage should be easily sanitized. Cages made of plastic or fiberglass with built-in drains seem to work best for most amphibians. Most amphibian cages need lids because some species can jump quite high and many others, including some salamanders, can climb smooth vertical surfaces. Metal screen lids can be abrasive to frogs that readily jump, especially new arrivals. Alternate smoother surfaces (such as plexiglass with holes or plastic mesh) can be used along with taller cages to decrease the likelihood of the frog abrading itself. Amphibians can be housed singly or in groups, provided sanitary conditions can be maintained. Some amphibian species are aggressive or territorial, and should be housed individually. Additionally, care must be taken not to transfer among cages pheromones that are present in the skin secretions (Jaeger 1992). Most amphibians prefer relatively cool temperatures (10-20°C). However, it is important to know the preferred temperature for the species in question, since some montane species require much cooler temperatures (5-10°C) and tropical species may require warmer temperatures (25-30°C) (Brattstrom 1970). Overall, amphibians are more sensitive to heat stress than cold stress. A few minutes at an excessively high temperature can be sufficient to cause immediate death or stress related opportunistic infectious disease. Control systems such as temperature sensitive alarms and flow solenoids are very useful in preventing catastrophes related to physical plant failures. Many animal rooms maintain air flow rates that are appropriate for mammals (10-15 air changes per hour), but can rapidly desiccate amphibians. This is of for species that are since they require moist conditions but not air or using solid cage lids can all prevent desiccation. Most amphibians are either under or are is not and animal room is usually adequate for most amphibians. in are and require Most amphibians are or which are available commercially. It is important to use that have been on a that have been during should themselves be for prior to being to an and can be commercially available is one such or (such as species that are used as for amphibians include and (see and for the of many common While most amphibians require to some species will on commercially available from such as or which in numerous and either or used amphibian species that the successfully include and aquatic often a in amphibians that can to of Problems can be by housing only similar individuals more and throughout the are a from that of food, and in water are commonly used for quality is the most of amphibian This is especially for larval amphibians, including terrestrial species, are also by poor water When water quality, it is to both water and cage The most common with water is and can also be to amphibians. Amphibians are housed in water, but this may be for species or in certain many water the of their water and the amount of (such as and in the water their changes can have on amphibian water can be made by using an The to be on to assure proper levels in the water it been through the can help the of a can also be from water by it in an for 24 but other may not be using this species, especially many water is not such either commercially available water or made from water can be used and water is it should be used the of conditions can cause and death in amphibians. water used for housing amphibians in from water used for housing amphibians in from the water is one can still problems with water quality in the The of cage water are and animal and often to poor cage water quality and, therefore, clinical disease. The most important to in cage water are in as a waste is by the group of to which is also is rapidly by the group of to 1993). of or may either or both of these are not present. This is often with new cage and that have been treated with new or treated with substrate from healthy will help a and levels should be monitored routinely to on species and to provide of in the for as well as other (such as levels and are available from and and A to cage water quality is the evaluation of Animals should in that are sufficient to maintain good but are not in when is properly cage water quality is at to the presence of food, to in many amphibians. many aquatic species, it is to most amphibians, including aquatic species, are air water is usually However, water may be necessary for larval or species that retain gills as It is important to that can to and death in many amphibians It is to of all available written and and new species. 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