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Vl Amphiblan Management anct Laboratory Care A. GENERAL COMMENTS Care of amphibians is necessarily complex because of the different envi- ronmental requirements at different stages in their life cycles. In their pre- metamorphic or larval stages, they live entirely in an aquatic environment; upon metamorphosis they become partially or wholly terrestrial, with the exception of the newt which undergoes a second change and returns to water. Thus, any institution developing or maintaining laboratory colonies of amphibians must design housing facilities to provide for this dual re- quirement of aquatic and terrestrial environments. Aquatic facilities require more care than terrestrial environments (see Chapter V). Sanitation and waste removal are crucial as the materials often are soluble and may directly affect exposed amphibians. Thus, large vol- umes of water for flushing are required or, if the water is to be recycled, treatment facilities must be installed and maintained. Water quality must be rigidly controlled as sudden physical or chemical changes may severely stress the animals. It is often necessary to develop special feed formula- tions since conventional feed may rapidly deteriorate and contaminate the environment when added to the water. The period of transformation from an aquatic to a terrestrial mode of living requires both aquatic and terrestrial environments. Some species con- tinue to feed in the aquatic environment; others become terrestrial in feed" ing habits; and still others may feed in either environment. Nutritional habits and requirements change as the animal moves from an aquatic to a terrestrial environment. Different methods of disease control must be de- veloped depending on the type of environment. Housing, surgical methods, humane disposal, shipping and receiving, quarantining, and protection from injury must all be handled differently for animals in the two environments. 75

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76 Finally, extreme care must be exercised in providing optimal environmental conditions during two delicate stages in amphibian life cycles, i.e., during embryonic development and metamorphosis. Rapid physiological changes occur at both periods and sudden environmental changes may prove detri- mental or lethal. Even though many problems in amphibian culture still exist, sufficient progress has been made to warrant description of culture techniques for several species (see also Boterenbrood, 1966; Frazer, 1966~. Procedures for R. pipiens and R. catesbeiana will be described as prototypes. Where known variations exist between these and other species, these will be noted. These techniques do work as attested to by current success in maintaining colo- nies of amphibians. Nevertheless, there is much room for improvement and existing techniques will be improved and new techniques will be developed as our knowledge of the optimum conditions for rearing amphibians in- creases. Most of the methods described here are designed for facilities maintain- ing less than 10,000 animals. Although more animals could be maintained with these methods, commercial dealers cannot economically afford to do so at present. However, incorporation of some of the husbandry principles could improve commercial stocks at little extra cost, and attention to the objective of stock improvement would certainly result in the development of improved techniques. A better quality animal commands a higher price. Once these animals and prices become established, commercial dealers should improve their facilities to include larger numbers of high-quality amphibians. B. ANURANS 1. Ranidae a. R. pipiens ( 1 ) Premetamorphosis (a) Enclosures See Chapter V, Sections C.1,2,3, and 6. (b) Environmental Controls See Chapter V, Section B. (c) General Care Until feeding stages are reached, embryos are held at low densities in shallow pans. To provide a maximum surface- volume ratio, the medium should not exceed a depth of 15-20 mm (0.6- 0.75 ink. Dead embryos should be removed regularly to avoid contamina- tion, and the medium changed at least every third day or more frequently

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77 if it becomes turbid. Before hatching, 200-300 eggs/liter can develop successfully. After hatching and when feeding begins, larvae should be thinned to 50 or less/liter; overcrowding will stunt growth (Richards, 1958, 1962; Rose and Rose, 1965; Gromko et al., 1973~. As the tadpoles grow, they should be continually thinned or given more medium so that near meta- morphosis there are only four to six tadpoles/liter. The embryos and tad- poles should be housed in a container that provides a large surface area for gas exchange. If the volume-to-surface-area ratio is not adequate, the use of an air line to oxygenate the water is advisable. During this period the use of artificial medium is recommended [for example, a 10 percent concentration of Steinberg's or Barth's modi- fications of Holtfreter's (Holtfreter, 1931; Rugh, 1965) medium or De Boers solution (see Chapter VII, Section A.104] . The formulations of Steinberg's and Barth's media may be found in Johnson and Volpe (1973~. At this and all other stages, great care must be exercised for the clean- liness of instruments used in manipulating the embryos and larvae. Con- tamination with formalin, etc., must be rigorously avoided. Indeed, it is good practice to reserve sets of instruments for each clutch of animals. For larger numbers of later larval stages, the tadpoles may be housed in any of several types of enclosures (see Chapter V, Sections C.2 and 6~. Preference should be given to those types requiring minimum daily atten- tion and minimal handling of the stock in order to reduce stress to the animals and the chances for error in labeling or in mixing tadpoles from different clutches. (d) Food Supply Ranid larvae should be fed when strands of fecal material appear in the water some days after they hatch from their jelly. Young larvae tend to be vegetarians and the larger tadpoles are om- nivorous. Because larvae may be raised on a variety of foodstuffs (Ham- burger, 1960; Rugh, 1965; Di Berardino, 1967), the choice should be determined by the type of tadpole enclosure. Without special care, the food will float to the surface and produce a scum that inhibits gas ex- change (see Chapter V, Section C.6.c). If the animals are housed in a water bottle system (see Chapter V, Section C.6.a), food must not be of such fine grain as to settle in the dividing screen or be lost as a result of the contin" ual water flow. The food must not be allowed to disintegrate and decay in the medium-a problem that is particularly applicable to static water enclosures. For colonies with laboratory-reared or laboratory-bred animals, food of uniform quality must be available throughout the year. Spinach must be avoided because it causes kidney stones in a number of amphibian species (Berns, 1965~. Romaine or escarole lettuces have been

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78 found most suitable though their nutritional adequacy remains uncertain. Raw lettuce will not be eaten, but it may be readily softened in a pressure cooker and may be stored in a frozen state and dispensed to the animal containers with ease. Some caution in feeding is required since tadpoles are killed if their containers are overloaded with wilted lettuce. Since large tadpoles eat enormous quantities, it may be necessary to supply the con- tainers with lettuce twice a day. Even in flowing-water bottle systems un- eaten lettuce must be removed to prevent fouling of the water. Protein supplement is provided by feeding cubes of raw or boiled liver two or three times a week. Tadpoles will also eat pulverized rabbit chow, "dry" dogfood, or a variety of other commercially available animal feeds. They will also sur- vive on a variety of other diets, including powdered or hard-boiled egg yolk, raw liver, or liverwurst. These diets, however, are generally inap- propriate because the foodstuffs rapidly disintegrate and decay in water. Thus not only is the water quality reduced but the material may be caught in the dividing screen if the flowing-water bottle system is used. Tadpole feeds that incorporate commercial feeds and binders to pre- vent disintegration have been developed. These feeds avoid the diffi- culties encountered with the feeds described above. Such a feed was described by Hirschfeld, Richards, and Nace (1970~. It is prepared by adding 250 g of pulverized chow, 20 g granular agar, and 14 g unflavored Knox gelatin/liter of water; the mixture is brought to about 100 C (212 F), after which it is allowed to solidify in flat pans. It can then be sliced for use or stored at -20 C (-4 F). It may be stored indefi- nitely at this temperature or for up to 14 days at refrigerator tem- peratures but should not be held at room temperature. Neither agar nor gelatin alone is satisfactory as a matrix. Rabbit chow incorporated in such binders has now been used for some years. It has good holding qualities in the flowing-water bottle system at the temperatures used for raising R. pipiens. Experience has indicated that recently hatched tadpoles should first be fed wilted lettuce in the "embryo enclosures" for several days; then they should receive both lettuce and the agar-gelatin diets for several additional days, after which the agar-gelatin diet alone is sufficient. This diet does not cloud the water and requires minimal attention. A full 1- to 2-day ra- tion can be administered at one time in that it is not excessive for a single feeding, and no debris remains for manual removal. [See Section B.1 .b for a description of a similar preparation used for R. catesbeiana (Culley and Meyers, 1972~.] Various diets must be tested on the larvae grown in each facility. Vari- ations may exist between animals from different original sources. Further

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79 more, postmetamorphic survival and maturation efficiency are greatly improved by adequate larval nutrition. (2) Postmetamorphosis (a) Enclosures See Chapter V, Sections C.3,4,5, and 6. (b) Environmental Controls See Chapter V, Section B. (c) Feeding Juveniles The most successful diet for juvenile R. pipiens hats been sowbugs or crickets, selected in sizes appropriate to the animals to be fed. (See below for information on handling these arthropods.) Another suitable diet is mosquitos. Kawamura of the Hiroshima Lab- oratory for Amphibian Biology has routinely introduced mosquito tum- blers into the containers with metamorphosed rapids. Upon eclosion, the, are readily eaten by even the smallest froglets. The mosquito is an excel- lent nutrient source and can be readily retained within the containers. At the University of Michigan Amphibian Facility Culex pipiens, a bird mos- quito that is easily raised according to standard procedures (Keppler et al., 1965) with Japanese quail as blood donor, has been used successfully. Other dietary regimens at this stage must be viewed with caution. Force- feeding of small, recently metamorphosed frogs has been found to be nu- tritionally inadequate and time-consuming. Live insects should be provided. Mealworms are frequently recommended, but separation of large numbers of small mealworms from their culture medium is onerous and they quickly die when placed in a moist environment. Furthermore, mealworms have been reported as nutritionally inadequate for R. pipiens (Cairns et al., 1967~. If mealworms are used, the frogs must be transferred to dry con- tainers during the feeding period. This handling is deleterious and time- consuming. The low biomass of Drosophila, their inability to survive near water, and the difficulty in containing them does not support the recom- mendation they frequently receive as frog food. (d) Management of Active Adults The laboratory manage- ment and care of active adult R. pipiens require both aquatic and terres- trial environments as implied by the description of enclosures (see Chapter V, Sections C.4 and 6~. All animals should initially be treated as described in Chapter IV, Section C.2, pertinent information should be recorded (Chapter VIII), and special attention should be given to the possibility of disease (Chapter IX). Those animals to be used within a few days for any objective other than egg production may be held for this short period in a box or covered aquarium at room temperature. The enclosure should

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80 contain enough water to permit the animals to submerge completely. It also should be provided with a table or shelf about 10 mm (0.5 in.) above water level or the enclosure should be sufficiently slanted to provide both aquatic and terrestrial areas. Northern frogs received between October and late March and to be used as active frogs may be stored in hibernation as described below (see Chap- ter V, Sections C.5 and 6~. Northern frogs to be used for egg production and received during these same months must be stored in hibernation. Northern frogs received in other months cannot be used for egg produc- tion without provision for feeding and management designed to support oogenesis. Attempts should not be made to force northern "summer" frogs into hibernation unless the chilling is part of a specific, knowledgeable ex- perimental design. They should be held as described above if they are to be used within a few days and as described below if they are to be held for longer periods. Southern frogs must never be refrigerated or they will die. Attempts should not be made to obtain eggs from them until more is learned con- cerning their reproductive cycle. Southern frogs at any season of the year may be held temporarily as described above; if they are to be held for longer periods, they must be managed as described below for active frogs. Active adults that are to be retained for more than a few days are best contained where the aquatic and terrestrial areas can be adjusted in ac- cordance with the frog's known behavior. Such containers are described more fully in Chapter V, Sections C.4 and 6. Water in the aquatic portion of the adult enclosures should be suffi- ciently deep to permit the animals to float in their typical position with eyes and nostrils above water, legs dangling and relaxed. Such a water depth should be provided even for primarily terrestrial animals; in the confinement of an enclosure, they obtain needed exercise by swimming rather than by jumping. This aquatic area also provides a suitable place for them to rest. Water should flow through the enclosure gently (two to three changes per day) but in sufficient volume to maintain bacterial counts below tolerance levels [see Chapter V, Section B.2.a.~7~] . Contrary to the circumstances for larvae, chlorinated water (4-6 ppm) for the reduc- tion of bacterial counts may be used. Although not as desirable as other types of enclosure (Chapter V, Section C.6), plastic vegetable crispers may be used. For these it is diff~- cult to arrange flowing water and, if static water is used, caution is re- quired because of the toxicity of some commonly used materials [see Chapter V, Section B.2.a.~10~] . A smaller opaque crisper with one end removed should be inverted inside a larger one to provide a terrestrial area; this constitutes a feeding platform on top and a hiding place below.

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81 Matting made from a solid neoprene mesh should be placed on exposed surfaces. Not only does this keep the frog from direct contact with the plastic surface but also permits desirable air circulation about the animal. Adult R. pipiens, especially from specific collection sites in the northern states, are highly susceptible to the Lucke renal adenocarcinoma. Whether as a result of stress or other factors, the development of this tumor is fa- cilitated when the animals are held for long periods at room temperature under crowded conditions (Rafferty, 1962~. Thus, experience suggests that 12 frogs are an appropriate number for a vegetable crisper measuring about 0.36 X 0.25 X 0.12 m (14 X 10 X 4.S ink. Aquaria or terraria can also be used. If the frogs are not In running wa- ter, the water should be changed frequently to prevent it from becoming fouled. Since frogs are normally secretive, several shards of unglazed clay flower pot placed in the container provide places to hide. Additional pieces of pottery in the water, but extending above it, provide a cool respite. Northern R. pipiens eat well and are active at 22-24 C (72- 75 F); those from Mexico do best if held at higher temperatures, e.g., 25-27 C (77-80 F) Optimal management principles to support and synchronize oogenesis in laboratory-reared and laboratory-bred R. pipiens are currently under investigation and as yet cannot be specified. However, a significant pro- portion of mature females will produce eggs if maintained as described above and fed as described below. No criteria have yet been established to recognize when eggs in living females are mature; however, mature eggs may be found 3 - months following a previous ovulation. Such eggs will be resorbed within a few days of their maturity if the females are not artificially ovulated. This is also true of wild-caught gravid females if not returned to hibernation immediately. Pigment from such resorbed eggs is stored in the liver, which becomes intensely black as a result. Preparations of active sperm have been made from male R. pipiens at all seasons of the year when they are managed following the procedures described here. (e) Food for Adults At metamorphosis R. pipiens shift from an omnivorous to a carnivorous diet comprised of food that must be mov- ing. They differ from R. catesbeiana and R. clamitans in that they do not or cannot take food while submerged. Reports in the literature that `'frogs and toads" will take food from mechanical devices such as "lazy susans" (Kaess and Kaess, 1960) do not apply to R. pipiens. A few individuals will strike at food presented in this manner, and an even smaller number will swallow it. Attempts are in progress to learn the criteria for motion, taste, and texture that must be met to present prepared foods to R. pipiens. At

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82 present, results do not warrant inclusion of a protocol for prepared foods in this document. Adult frogs can survive for extended periods (3 - weeks) without feed- ing if their quarters are clean, but long-term survival requires feeding the equivalent of 10-12 full-grown crickets two to three times a week. Frogs can be force-fed raw liver or hamburger, but not only is this time-consum- ing but quantity and timing are hard to judge and growth rates are poor. Therefore, when possible, use of live food is recommended. Definitive studies of R. pipiens dietary requirements have not been done because of the dependence on live food or force-feeding. Should devices for the pres- entation of prepared diets be successfully developed, such studies will be possible. At the University of Michigan Amphibian Facility, bone mal- formations that resulted from dietary insufficiencies have been observed. These have been alleviated, at least partially, by dusting the arthropod dietary items with commercial vitamin and protein supplements. Some adult amphibians do not readily accept live food. These animals must be force-fed with special food mixtures from a syringe through a stomach catheter. Careful insertion of the catheter and a slow adminis- tration of the food will help prevent regurgitation. One ration that has been used successfully is prepared by homogenizing beef liver, eggs, and lettuce (all boiled) in a blender in a ratio of 4: 1: 1. A small amount of bone meal, one drop of cod liver oil/ml mixture, and commercial vitamin supplements may be added. Antibiotics may also be added in proper amounts (see Chapter IX, Section B), and the mixture frozen for later use. Canned dog food mixed with warm water and homogenized in a blender was another adequate force-feeding diet; when used for Necturus, cod liver oil was added (Kaplan and Glaczenski, 1965~. Food items from the wild, such as many arthropods and snails, can be used. One effective collection method is "light trapping" for beetles, moths, etc. Frogs fed on such diets, however, cannot be classified as laboratory reared or laboratory bred. To meet the criteria for these classifications, the animals must be fed living materials that have been reared in confinement (see Chapter III, Sections B.3 and 4), such as crickets, sowbugs, earth- worms, beetles, flies, caterpillars, and moths. Even newborn or young mice will be readily taken. A word of caution must be added concerning the use of fly maggots: Because maggots of many fly species are not killed in the digestive tract of the frog, such maggots may destroy the frog. The problem of supplying a living diet, perhaps, has been the greatest single deterrent to developing laboratory-maintained cultures of R. p~piens; by proper selection of food types and standardization of food culture, how- ever, this is not a prohibitively difficult problem and becomes increas- ingly feasible as the colony enlarges. Many potential food items may be obtained commercially. Thus, al

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83 though crickets and earthworms may be cultured, they may also be purl chased from bait dealers and arrangements can be made for delivery on a regular basis. Sowbugs, which can be found in nature under old boards and dry leaves, are readily raised. They are recommended because they frequently appear on checklists of wild R. pipiens stomach contents, are "clean,' and do not drown in the aquatic portion of the frog container. Indeed, they crawl out of the water onto the flower pots or up the sides of the container. Sowbugs may be raised at temperatures between 25 and 30 C (77-86 F) in roaster pans with a half inch of damp peat moss. The peat moss should be covered with a moist rectangle of corrugated card- board and with a plastic lid from a vegetable crisper on the cardboard to reduce loss of moisture by evaporation. Sowbugs feed on crumbled rabbit chow and crushed blackboard chalk, which should be placed in a dry cor- ner to retard molding. Both the rabbit chow and the peat moss bedding should be heat treated to kill organisms such as weevils. (The weevils not only compete with the sowbug colony but may actually destroy it.) Under these conditions, a colony of Sowbugs will double its number about every 40 days. Some frogs will kill themselves by overeating (e.g., the Japanese frog R. nigromaculata), but this does not occur with R. pipiens. Nevertheless, highly fed laboratory R. pipiens do develop muscle glycogen levels ap- preciably higher than those found in nature (Smith-Farrar, 1972~. (f) Hibernation Hibernation is essential for holding Northern gravid females in the gravid state and is helpful in the winter months for low-cost holding of northern adults to be used for other purposes. Gravid females, if held under correct conditions, may be ovulated as late as July, 3 months after the time of normal ovulation. If correct conditions, how- ever, are not met, the energy reserves in the eggs are mobilized and the eggs regress. Even a few days of improper conditions will lead to reduced fertility. Summer frogs forcibly submerged without proper preparation for hi- bernation will drown. After entering hibernation, however, they remain submerged below the ice. During hibernation a respiratory shift from aerobic to anaerobic may occur to some extent, although cutaneous respiration does occur. With these factors in mind refer to Chapter V, Sections C.5 and 6 for descriptions of hibernation quarters. Note that for wild-caught northern R. pipiens water temperature should be be- tween 3 and 4 C (37-39 F) in October and November, 1.5-2 C (34- 36 F) from December, and 3 - C (37-39 F) for 2 - weeks prior to removal from hibernation, unless the frogs are being retained at room temperature only long enough to have ovulation induced. Southern frogs should never be placed under hibernation conditions.

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84 Criteria to recognize when an animal is in hibernation as distinct from cold torpor have not been devised. Such information is critically needed. Also, criteria for recognizing true emergence from hibernation are miss- ing. b. R. catesbeiana (1) Premetamorphosis B.1.a above. (a) Enclosures See Chapter V, Sections C. 1-6 and Section (b) Environmental Controls See Chapter V, Section B. The management of embryonic and larval stages of R. catesbeiana is simi- lar to that given forR. pipiens isee Section B.1.a(1~], except in rate of development and animal size. Because of their large size R. catesbeiana tadpoles are placed in the frog enclosures as soon as the forelimbs emerge. During this stage mortality is reduced if the animal can move out of the water. The cause is not known but may be related to stress (see Chapter V, Sections C.2 and 3 and Sec- tion B.1.a of this chapter). (c) Food Supply The discussion of food supply for R. pipiens larvae is equally applicable here [see Section B.1.a(1~] . In addition to the prepared diet described, unflavored Knox gelatin has proven to be fairly effective as a binder at water temperatures below 24 C (75.2 F) (Culley and Meyers, 1972~. The binding quality is best retained when the food re- tains its moisture. As more is learned about tadpole nutrition, undoubtedly new foods will become available. The rabbit chow-gelatin mixture serves as an adequate food for several species of amphibian larvae. However, for R. catesbeiana this diet should be supplemented with a boiled leafy lettuce (not head lettuce). The reason is unclear, but growth appears to be better when lettuce is added. R. cates- beiana larvae from stock collected in the Gulf states have been successfully reared from egg through metamorphosis on the rabbit chow-gelatin-lettuce diet in 3-4 months at 30 C (86 F). At metamorphosis, the larvae weigh about 10 g (0.35 oz). (2) Postmetamorphosis (a) Rearing Facilities The most successful rearing containers for the frog stage are those that have a wet and dry area and a continuous supply of fresh water. Crowding of adults creates no serious problems as long as sanitation is maintained. Juvenile bullfrogs placed in containers

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85 with as little as 120-150 mm2 (1.86-2.33 in.2) of floor area per frog have been successfully reared for over 2 years with the same degree of crowd- ing. Specifications for enclosures are given in Chapter V, Sections C.3,4, and 6. (b) Environmental Controls The same water quality criteria holds for frogs as for tadpoles (Chapter V, Section B.), with the exception of chlorine. As long as chlorine is less than 3 - mg/liter, the bullfrogs are not affected. Long-term exposure to concentrations above 4 mg/liter may be detrimental. Since chlorine in water supplies is occasionally as high as 4 mg/liter at the tap, the supply should be checked periodically. Although wide spectrum light is recommended for laboratory-reared frogs, bullfrogs maintained for over 3 years under fluorescent lights have shown no signs of vitamin D deficiency (Culley, 1973~. (c) Food Supply The diet of wild southern bullfrogs consists mainly of crayfish, supplemented with fish and, at times, large quantities of tadpoles. Given a choice, however, bullfrogs prefer tadpoles and fish. Cray- fish are nutritionally adequate for bullfrogs, but have much less food value than fish of the same weight. Tadpoles of either R. catesbeiana or R. pipiens may be used as food for bullfrogs; however, these tadpoles must be of a size that the frog can swal- low, a factor of particular concern when feeding juvenile bullfrogs. Though wild-caught tadpoles may be used, they may affect the classification cate- gory of the frog (see Chapter III, Section Bob. Although tadpoles appear to provide an adequate diet for short~term feeding, a long-term diet of bullfrog tadpoles for young bullfrogs may not be nutritionally adequate. Tests have shown that after 2 months young bullfrogs on this diet feed erratically, become sluggish, and lose their son brilliance. The use of crickets, mealworms, or earthworms as single diets or in combination have not been nutritionally adequate. In recent studies at the Louisiana State University Ensue facility, bullfrogs on this diet developed bone deformations within 3 months. Thus, only fish have proven to be nutritious and accepted on a long- term basis. Several bait minnows have been utilized in feeding bullfrogs, although it must be cautioned that using minnows raised in outdoor ponds may affect the standard classification of the frogs (see Chapter III, Sections B.2 and 3~. Using growth as the criterion, minnows, so far, have proven to be nutritionally adequate. Two species of small minnows-mosquito fish (Gambusia aff nis) and sailfin molly (Mollienisia latipinna)-have been used at Lou. However, both become infested with a nematode (Eustrongylides wenrichi) that attains a length of 70-100 mm (3= ink. When infested

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86 fish are consumed, the nematodes burrow through the stomach wall of the frog and migrate randomly. Many locate just under the skin and in the mus- cle tissue, apparently causing little harm to the frogs. Others move into the liver, kidneys, or heart and death results. The nematode has been found only during the summer and early fall in the fish in the Mu area. It is advisable, therefore, not to use these fishes as a source of frog food, par- ticularly during the seasons indicated. So far this nematode has not been found in goldfish (Carassius auratus), fathead minnows (Pimephales promelas), or the golden shiner (Notemigonus crysoleucas)-common bait minnows that have been used for feeding frogs. One of the most successful regimes for bullfrogs has been a combination of crickets, earthworms, and golden shiners fed daily. Although inadequate when used alone, worms and crickets have been successfully used to sup- plement the nutritionally adequate shiners and results in some cost reduc- tion. Where shiners have not been available, excess live juvenile and adult mice and hatchling chicks and quail from the animal facility have proved to be an adequate substitute. Because bullfrogs feeding under water have difficulty catching their food if the water is deeper than 10-20 mm (0.39-0.78 in.), a depth at which fish can escape, water depth in their enclosures must be controlled. If water depths are not optimal and if different-sized frogs are in the same enclosure, cannibalism may result. As long as food is abundant and readily obtained, cannibalism seldom occurs. For frogs under 2 months of age, the water depth should not exceed 10-20 mm (0.39-0.78 in.) and not cover over one fourth of the floor. Ex- cess food should be added to facilitate capture, and dead food should be removed daily. These young frogs will not be very successful in taking live food over 20 mm (0.78 in.) in length. After 2 months growth, food up to 40 mm (1.56 in.) can be captured, and after 4 months food size is not critical, except for the slower growing frogs. (d) Hibernation Little information is available on hiberna- tion requirements for R. catesbeiana. However, techniques now employed with R. pipiens may be applicable to R. catesbeiana collected in the North. iSee Chapter V, Sections C.S and 6 and Section B.1.a(2) of this chapter.] To keep southern R. catesbeiana under hibernating temperatures for periods up to 6 weeks, 10 juveniles or 5 adults can be placed in nylon netting sandwiched between moist layers of sphagnum moss in a 3-gal plastic container. The frogs are placed in these containers at ambient air temperatures and then stored under refrigeration. The container will cool to temperatures of 5-7 C (41.0 44.6 F) in about 24 h. In this tempera- ture range mortality will not occur if 5-7 C (41.0-44.6 F) water is added

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87 to the container for a few minutes every 2 weeks and then drained off. Wood excelsior or peat moss is inadequate as a substitute for sphagnum moss. The wood excelsior develops heavy fungal growth and the peat moss packs too tightly. - The animals should be removed slowly from storage at low temperatures by placing the container at room temperature for approximately 24 h. The only obvious detrimental effect noted has been the development of tempo- rary skin lesions on the webbing within 48 h after removal from refriger- ation. c. Other Ranid Species The care of other ranid species encountered in American laboratories fol- lows either the procedures for R. pipiens or R. catesbeiana, depending on their affinities or behavior patterns. R. grylio and R. clamitans resemble R. catesbeiana in their habits and may be handled following the proce- dures for R. catesbeiana R. palustrzs, which may be hybridized in the lab- oratory with R. pipiens, may be treated as R. pipiens. R. sylvatica is, as the name implies, a woods frog. It is very secretive and spends little time in the water except to breed. All indications are that they hibernate, not in water, but burrowed under the forest litter. Consequently, they should be kept in containers with a minimum of water, a maximum of terrestrial space, and appropriate places to hide (unglazed flower pot shards). R. sylvatica are difficult to maintain in the laboratory, especially during the period from metamorphosis to sexual maturity. Further studies are needed to identify the reasons for these difficulties since a similar spe- cies, R. japonica, has proved to be highly adaptable to laboratory condi- tions in the Laboratory for Amphibian Biology of the University of Hiroshima, Japan (Kawamura and Nishioka, 1972~. In addition to the divisions that have been noted above in describing these several species, they can be further divided into two categories: those that breed immediately upon emerging from hibernation and those that mate following a period of nutritional intake between hiber- nation and breeding. The former include R. pipiens, R. palustris, R. sylvatica; the latter, R. catesbeiana and R. clamitans. Artificial breeding of northern representatives of the first group is readily accomplished through much of the year because they are gravid at the time of entering hibernation and may be bred at any time when removed from hibernation (see Chapter VII). For the latter group, how- ever, this is not yet possible. Additional study of endocrinology and nu" trition is needed to bring the artificial fertilization of these species under control (Sarkar and Appasvvamy Rao, 1971~.

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88 The conditions for hibernation of the several species show some dif- ferences, especially for those species with extended latitudinal distribu- tion. Representatives collected north of the line separating ice-free from ice-covered ponds may be maintained as described for R. pipiens. Animals collected soup of this line have not been critically tested. R. nigromacu- lata, which has been studied in Japan (Kawamura and Nishioka, 1972), is native to a territory where ponds seldom freeze. They burrow in the dikes between rice paddies and under forest litter. Hibernation or dor- mancy temperatures for these animals are between 7 and 10 C (45-50 F). Presumably, American animals collected from similar climatic areas could best be maintained under similar temperature conditions (see Chapter V, Section C.5~. 2. Other Anurans a. Xenopus The care of Xenopus has been extensively described (Weiz,1945a,b; Nieukopp and Faber, 1956; Frazer, 1966; Gurdon, 1967; Brown, 1970; Deuchar, 1972~. However, a brief review may be useful. (1) Larvae Development of Xenopus is rapid. At 20-22 C (68- 72 F) cleavage and gastrulation take place within 1 day. Hatching oc- curs on the second and third day. The larvae should be fed when they begin to swim along the bottom at an angle of about 45 with their heads down and tails up. Gasche (1943, 1944) introduced powdered nettle as the food for larval Xenopus. A1- though good, it is not necessary. Dried green pea soup or other finely ground food is satisfactory. The food is mixed with water and allowed to settle, and the supernatant containing fine food particles is decanted. The decanted suspension is delivered to the tadpoles in measured amounts. Care must be exercised as coarse food particles congest the filter-feeding mechanism of these larvae and can produce death (Weiz, 1945a,b). The quantity of food administered should permit clearing of the water by the tadpoles about =5 h after feeding. A fresh food slurry should be made each day; since growth is facilitated by a second daily feeding, the same slurry can be utilized a second time on the same day. The larvae may be transferred from the amplexus chamber (see Chap- ter VII, Section A.10) to larvae enclosures after they have fed for a few days. They should be siphoned or dipped but not netted until they have

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89 grown for about a week. Plastic vegetable crispers are satisfactory as larval enclosures if the density of the larvae does not exceed 6-8 per liter. The water should be changed and the enclosures cleaned at 3-day intervals. Xenopus larvae can also be raised in the flowing-water bottle system used for ranid larvae if the screen in the neck of the bottle (see Chapter V, Section C.6.a) is covered to retain the suspended food. Under the conditions of temperature, feeding, and water changing de- scribed above, metamorphosis should begin S-6 weeks following fertiliza- tion. Metamorphosis, itself, requires 15-20 days. Shortly after forelimb eruption, the larvae stop feeding. This is frequently first detected by the failure of the animals to clear the water after feeding. After their tail is half resorbed, the animals will eat finely diced meat, mosquito larvae, Tubifex (redworms), etc. A suitable diet has been suggested made of ground fat-free beef heart and powdered milk blended together to yield a dry, crumbly material. This may be used as steady diet, whereas Tubifex or mosquito larvae used alone will result in poor growth or death of the young adults, presumably as a result of nutritional deficiency. Care must be taken to remove uneaten food and to keep the water clear. After a month or so the juvenile animals may be treated as adults. (2) Adults Adult Xenopus are maintained in aquaria or other suit- able enclosures with a volume adequate to the numbers of animals (one animal/2 liters) (see Chapter V, Section C.6J. The walls of the enclosures should be sufficiently high or the enclosures should be covered to prevent escape. In their natural habitat, the frogs are subject to considerable tem- perature variation. Thus, although they can tolerate tepid water, water at normal laboratory temperature is suitable. The water must be dechlori- nated and should be at least 0.15 m (6 in.) deep. It is convenient to equip the enclosures with an overflow pipe to permit constant gentle flow of the water or the water should be changed periodically to prevent stagnation [see Chapter V, Section D.2.a(10~] . Fecal and food wastes must be re- moved within several hours after each feeding, either by draining the en- closure or by using an aspiration device. Though a liver diet has proved satisfactory for maintaining Xenopus, experience indicates that pieces of beef heart cut to resemble earthworms of a size appropriate to the size of the animals being fed is a superior diet. Chunked or diced meat is unsatisfactory because the poor surface-volume relationship does not allow adequate digestion. The pieces of beef heart should be soaked in a commercial vitamin mixture. Earthworms are a highly effective food. Feeding may be as infrequent as twice a week al- though more frequent feeding results in more rapid growth.

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go b. Bufo Bulo are more terrestrial than ranids (Blair, 1972~. Enclosures with shelves spaced appropriately to the size of the animals can accommodate large numbers. Such enclosures are described more fully in Chapter V (Sections C.4 and 6.a) and in Frazer (1966~. Water need be provided in only modest quantities; as a trickle on Me bottom of the cage or in a Petri dish or other shallow container. If the floor of the enclosure is moist, shelving or low tables covered with matting, such as neoprene, should be used to provide the animals a dry area. The food for Bulo may be highly varied. Crickets, sowbugs, earthworms, or any of the items mentioned in the diet for R. pipiens may be used. Meal- worms (Tenebrio) are appropriate for toads because they will not drown in the minimal amount of water in the containers. In addition, some toads will adapt to nonliving diets: B. marinus has been maintained on canned cat food or wet dog food. Force-feeding of liver and fish proteins has not proved satisfactory (Jakowska. 19721. c. Bombina orientalis These animals are climbers rather than jumpers and will escape if their containers are not carefully closed. Plastic or enamel pans, 50-76 mm (2-3 in.) deep, securely closed with nylon or metal screening are suitable for housing and are necessary if flying insects are used for food. The floor of the pan should be lined with neoprene matting on which shards of un- glazed flower pots are placed. The water need only cover the matting but should be flowing or be changed every second or third day to prevent the accumulation of toxins secreted by the animals. This toxin constitutes only minimal danger to laboratory personnel; the irritation produced on mucus surfaces simply guarantees that the worker will wash after handling them (see Chapter X, Section B.2~. The animals may be maintained throughout the year at laboratory tem- peratures. They show little preference for different types of food provided Me food is not too large: i.e., crickets, earthworms, sludge worms, sow- bugs, and flies [not maggots, see Section B.1.a(2) this chapter] . Crickets and sowbugs are recommended because they are available over a full series of graded sizes and can thus be selected appropriate to the size of the ani- mal. Fed and manipulated as described in Chapter VII, these animals can be induced to ovulate every second day, although in nature, they seem to mate only once a year.

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91 C. URODELA 1. Axolotis {Neoteric Larvae of Ambystoma mexicanum) lthe following description is modified from a privately distributed instruc- tion sheet that was prepared by R. R. Humphrey of Indiana University, Department of Zoology, Bloomington. a. Early Larvae Axolotl larvae do not require food immediately after hatching since at that time the stored yolk has not all been metabolized. Feeding should be in- itiated when the color of the intestine changes from white to a gray or gray-black, or shows a slightly greenish tinge from bile accumulation. Larvae Hat may have begun to float before feeding, because of air in the stomach, should at once be put in very shallow water and generously fed. Recently hatched brine shrimp (Artemia) are among the best food with which to begin feeding axolotls. Mosquito larvae are also excellent. Experi- ence at The University of Michigan Amphibian Facility demonstrates that mosquito larvae are readily produced, and because they may be harvested at any of their developmental stages, they are available in all sizes suitable for use as food throughout the lifetime of the axolotl larvae. Daphnia of small size or very small Tubifex or Enchytraeus may be used. These worms tend to "ball up" in masses but can be reduced to short lengths by cutting into the mass several times with scissors. Axolotl larvae at hatching are smaller than larvae of some other amphibian species; if the food first given is too large for them to handle, they may fail to start eating and die of starvation. Once they begin eating, they should receive only as much food as will be consumed within a few hours. Before feeding again, change the water to remove feces and uneaten food. Dishes require frequent scrubbing to keep them free of protozoa. Neither brine shrimp nor Enchytraeus alone constitute a suitable food for indefinite use. Larvae fed nothing else eventually show edema and/or ascites and hemorrhages in the limbs, skin, or wall of the stomach and soon die. Those that survive may How serious adhesions of the viscera. To pre- vent loss of animals from dietary insufficiency, add Daphnia or Tubifex to the diet, or earthworms cut into short pieces. Smaller amphibian larvae, embryos, or even infertile eggs (removed from membranes) may also be fed. Hand feeding with beef liver cut into very thin, narrow strips, while time-consuming, will save larvae from dietary insufficiencies if begun suf- ficiently early. Even when a variety of live food is available, the larvae

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92 should ultimately be hand fed with liver if they are to be brought to breeding condition when one year of age, unless maximal feeding by other methods is possible and has been found to be equally effective. Daily feed- ing for young larvae is advisable. After several months or as the animals approach maturity, this may be discontinued and food provided only on alternate days. It may be helpful for the welfare of the animal (growth and mortality levels) to add a small quantity of bone meal to the diet occasionally; sprinkle the meal over the sliced liver and stir to cause the meal to adhere to it. Young axolotls are seldom troubled by mold. If they become infected, however, they may be placed for several hours in a weak solution of Mer- curochrome (1: S00,000 or weaker). This treatment can be repeated if necessary. Larvae infected with a disklike protozoan (probably of the genus Trzch- odina) have been successfully treated by immersing them for a few sec- onds in a strong solution of Mercurochrome (a deep red color) and rinsing them under a low-pressure tap flow. Richardson (1937) reports successful treatment of similarly infested young trout by a 2-min immersion in 3 per- cent saline or a 15-see immersion in 1 :1,500 glacial acetic acid. b. Mature Larvae These larvae, which reproduce without undergoing metamorphosis, are totally aquatic and must be treated accordingly. (1) Enclosures Axolotls do not require running water or special provision for the aeration of water, provided the enclosures are of suitable size and are kept clean. The water should be changed after feeding and whenever it becomes fouled, and the containers should occasionally be washed with a detergent to keep them free of bacteria and protozoa. The siphoning device described for use with Xenopus can also be used to re- move debris from axolotl containers. Although several animals may be put together in a large aquarium, it is better to keep them separated, either in smaller aquaria or in glass or plastic bowls. This facilitates the breeding regimen and avoids the cannibalism that may occur if they are inadequately fed. Glass fish bowls of one gallon capacity are adequate, although larger ones may be provided for old animals of maximum size. Those of quart or 2-quart size are useful for smaller animals. Rectangular plastic containers instead of bowls are used in some laboratories and have the advantage of better stacking. Other enclosures are presented in Chap- ter V, Sections C.6.a and d. Axolotls must be provided with chlorine-free water (see Chapter V,

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93 Section B.2.a). Highly aerated water should be avoided since it will result in reduction of gill arborization and, while not a handicap to animals maintained under these conditions, it may result in difficulties if the ani- mals are later placed in poorly oxygenated environments as during ship- ping. Their water should be maintained at 20-22 C (68-72 F). Colder water will cause the animals to reject food or to regurgitate it if they have been recently fed under warmer conditions. Warmer water is detrimental to breeding. Particular care should be exercised in adjusting the tempera- ture of fresh water when the enclosures are cleaned. (~2) Feeding Adult axolotls can be fed earthworms or many types of insects. However, beef liver, lean beef, or beef heart may be used. Cut into pieces suitable for a single feeding, the meat can be frozen. In prepa- ration for feeding, the meat is cut into thin slices while frozen or partially thawed. These, in turn, are subdivided into narrower strips; earthworm size for large animals and toothpick size for smaller animals. Pieces cut to provide maximum surface area-volume ratios will facilitate digestion. The food should be handled with blunt forceps and offered to each animal as long as it is readily accepted. Animals heavily overfed or given large thick pieces are likely to regurgitate. Axolotls do not readily digest either fat or collagenous tissue (such as fascia or tendon); these should be trimmed away, as should parts of the very large blood vessels in liver. The animals seem to prefer liver to other food; beef and lamb liver are both suitable, but pig liver is frequently regurgitated. Since their food is digested rather slowly, adults need be fed only on alternate days or on a schedule of three feedings/week. 2. Other Urodeles Little is known about the laboratory management of the larval stages of other salamanders. Generally, they should be maintained in well-oxygen" ated, clean, chlorine~free water. Most are carnivorous and will eat live food such as mosquito larvae, Encytrea worms, brine shrimp, and other small crustaceans and annelids. Most species should be maintained at 10-20 C (50-68 F). Metamorphosed salamanders may be maintained in terraria or other containers that provide moist hiding areas and access to free water. The containers described above for adult R. pipiens are suitable. Live insects and earthworms are usually accepted as food. Necturus should be maintained in clean, filtered, and well-aerated water. Their containers should provide a gravel bottom and shelter, such as broken pot shards or flat stones, where the animals may hide with their backs

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HA ~ - against the overhead shelter. Normally, they will feed on earthworms and small crayfish and will sometimes accept small strips of raw meat or small dead fish after they have fed on live food while in captivity. Notophthalmus viridescens efts and adults may be maintained in a semi- aquatic~ terrarium or container similar to that described for R. pipiens and fed on earthworms, mealworms, crickets, and other small insects. The adult newt stage can also be maintained in aquaria or similar tanks, but a semi- terrestrial terrarium is recommended.