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National study Council (US) Subcommittee ~ above Amphibian Standards. Amphibians: Guidelines because that the breeding, care, and management of laboratory animals. Washington (DC): national Academies push (US); 1974.


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A. General Comments

Care that amphibians is necessarily complex because of the different environmental requirements at different stages in your life cycles. In your pre-metamorphic or larval stages, castle live completely in one aquatic environment; ~ above metamorphosis they become partially or completely terrestrial, v the exemption of the newt which undergoes a 2nd change and also returns to water. Thus, any institution developing or keeping laboratory colonies of amphibians must style housing infrastructure to administer for this twin requirement that aquatic and also terrestrial environments.

Aquatic infrastructure require much more care than terrestrial environments (we thing V). Sanitation and waste removed are an important as the materials frequently are soluble and also may directly affect exposed amphibians. Thus, huge volumes the water because that flushing are required or, if the water is to be recycled, treatment facilities must be installed and also maintained. Water quality have to be rigidly regulated as suddenly physical or chemical alters may severely stress the animals. It is often important to build special feeding formulations since conventional feed might rapidly deteriorate and contaminate the atmosphere when included to the water.

The duration of revolution from one aquatic come a terrestrial mode of living requires both aquatic and terrestrial environments. Some varieties continue to feeding in the aquatic environment; others end up being terrestrial in feeding habits; and also still others may feed in one of two people environment. Nutritional habits and requirements readjust as the pet moves indigenous an aquatic to a terrestrial environment. Various methods of an illness control should be developed depending on the kind of environment. Housing, surgical methods, humane disposal, shipping and also receiving, quarantining, and protection native injury must all be handled in different way for pets in the two environments.

Finally, extreme treatment must be worked out in offering optimal environmental conditions during two vulnerable stages in amphibian life cycles, i.e., throughout embryonic breakthrough and metamorphosis. Quick physiological changes occur at both periods and also sudden environmental transforms may prove detrimental or lethal.

Even though numerous problems in amphibian society still exist, enough progress has actually been made come warrant description of society techniques for several varieties (see likewise Boterenbrood, 1966; Frazer, 1966). Procedures for R. Pipiens and R. Catesbeiana will certainly be explained as prototypes. Where well-known variations exist between these and other species, these will be noted. These approaches do job-related as attested come by current success in maintaining nests of amphibians. Nevertheless, there is lot room for innovation and existing methods will be boosted and brand-new techniques will be arisen as our expertise of the optimum conditions for rearing amphibians increases.

Most that the methods defined here space designed for infrastructure maintaining less than 10,000 animals. Although an ext animals might be preserved with this methods, commercial certified dealer cannot financially afford to perform so in ~ present. However, incorporation of few of the husbandry principles can improve commercial share at little extra cost, and also attention to the target of stock advancement would certainly result in the advancement of enhanced techniques. A better quality animal commands a higher price. As soon as these animals and also prices end up being established, commercial certified dealer should improve their infrastructure to include larger number of high-quality amphibians.


a. R. Pipiens(1)

Premetamorphosis

(a)

Enclosures watch Chapter V, sections C. 1,2,3, and 6.

(b)

Environmental Controls watch Chapter V, section B.

(c)

General care Until feeding stages space reached, embryos are hosted at low densities in shallow pans. To carry out a preferably surface-volume ratio, the medium should not exceed a depth the 15–20 mm (0.6–0.75 in.). Dead embryos have to be removed routinely to prevent contamination, and the medium changed at the very least every 3rd day or more frequently if it becomes turbid. Before hatching, 200–300 eggs/liter can construct successfully.

After hatching and also when feeding begins, larvae must be thinned come 50 or less/liter; overcrowding will stunt growth (Richards, 1958, 1962; Rose and also Rose, 1965; Gromko et al., 1973). Together the tadpoles grow, they must be continuous thinned or given more medium so that near metamorphosis there room only four to six tadpoles/liter. The embryos and also tadpoles must be housed in a container that gives a huge surface area because that gas exchange. If the volume-to-surface-area ratio is no adequate, the use of an air fine come oxygenate the water is advisable.

During this duration the usage of fabricated medium is recommended . The formulations the Steinberg"s and Barth"s media may be uncovered in Johnson and Volpe (1973).

At this and also all other stages, great care have to be worked out for the cleanliness of instruments used in manipulating the embryos and also larvae. Air pollution with formalin, etc., have to be rigorously avoided. Indeed, that is great practice come reserve set of instruments for each clutch that animals.

For bigger numbers of later larval stages, the tadpoles may be housed in any type of of several varieties of enclosures (see chapter V, sections C.2 and 6). Preference need to be offered to those species requiring minimum day-to-day attention and also minimal dealing with of the stock in stimulate to mitigate stress come the animals and the possibilities for error in labeling or in mixing tadpoles from various clutches.

(d)

Food supply Ranid larvae have to be fed once strands that fecal material appear in the water some days ~ they hatch from your jelly. Young larvae often tend to it is in vegetarians and the larger tadpoles are omnivorous. Because larvae might be raised on a range of foods items (Hamburger, 1960; Rugh, 1965; Di Berardino, 1967), the selection should be identified by the kind of tadpole enclosure. Without special care, the food will float to the surface and also produce a scum that inhibits gas exchange (see chapter V, section C.6.c). If the animals are housed in a water bottle device (see thing V, section C.6.a), food must not be of together fine grain regarding settle in the dividing display screen or be shed as a an outcome of the constant water flow. The food should not be enabled to disintegrate and also decay in the medium—a problem that is an especially applicable to static water enclosures. For nests with laboratory-reared or laboratory-bred animals, food that uniform quality must be easily accessible throughout the year.

Spinach should be avoided due to the fact that it causes kidney stones in a number of amphibian varieties (Bems, 1965). Romaine or escarole lettuces have been found most suitable though their nutritional adequacy remains uncertain. Life lettuce will not it is in eaten, but it may be easily softened in a pressure cooker and may it is in stored in a frozen state and also dispensed to the pet containers v ease. Some caution in feeding is required due to the fact that tadpoles are killed if their containers space overloaded with wilted lettuce. Since large tadpoles eat enormous quantities, it might be important to it is provided the containers through lettuce twice a day. Even in flowing-water bottle solution uneaten lettuce must be removed to avoid fouling that the water. Protein complement is listed by feeding cubes of life or boiled liver two or 3 times a week.

Tadpoles will additionally eat pulverized rabbit chow, ""dry"" dogfood, or a selection of various other commercially accessible animal feeds. They will likewise survive ~ above a range of various other diets, consisting of powdered or hard-boiled egg yolk, life liver, or liverwurst. This diets, however, are usually inappropriate because the foods items rapidly disintegrate and also decay in water. Thus not only is the water top quality reduced however the product may be caught in the dividing display screen if the flowing-water bottle mechanism is used.

Tadpole feeds that incorporate advertisement feeds and also binders come prevent fragmentation have been developed. These feeds prevent the challenges encountered with the feeds explained above. Together a feeding was explained by Hirschfeld, Richards, and Nace (1970). It is all set by including 250 g that pulverized chow, 20 g granular agar, and 14 g unflavored Knox gelatin/liter that water; the mixture is carried to about 100 °C (212 °F), ~ which it is enabled to solidify in level pans. It deserve to then it is in sliced for use or stored at 20 °C (-4 °F). It might be stored indefinitely at this temperature or for as much as 14 days in ~ refrigerator temperatures but should not be held at room temperature. No agar no one gelatin alone is satisfactory together a matrix. Rabbit chow included in such binders has actually now been offered for part years. That has good holding characteristics in the flowing-water bottle mechanism at the temperatures used for increasing R. Pipiens.

Experience has indicated that freshly hatched tadpoles should an initial be fed wilted lettuce in the "embryo enclosures" for number of days; then they should receive both lettuce and also the agar-gelatin diets for several extr days, after i m sorry the agar-gelatin diet alone is sufficient. This diet does no cloud the water and also requires minimal attention. A complete 1- come 2-day ration have the right to be administered at one time in the it is not extreme for a single feeding, and also no debris continues to be for hands-on removal.

Various diets have to be tested on the larvae grown in every facility. Variations may exist between pets from various original sources. Further

more, postmetamorphic survival and maturation performance are greatly improved by adequate larval nutrition.

(2)

Postmetamorphosis

(a)

Enclosures check out Chapter V, Sections. C.3,4,5, and 6.

(b)

Environmental Controls view Chapter V, ar B.

(c)

Feeding Juveniles The most successful diet because that juvenile R. Pipiens has been sowbugs or crickets, selected in sizes proper to the animals to be fed. (See below for details on handling these arthropods.)

Another suitable diet is mosquitos. Kawamura the the Hiroshima laboratory for Amphibian Biology has actually routinely introduced mosquito tumblers right into the containers with metamorphosed ranids. ~ above eclosion, they room readily consumed by also the the smallest froglets. The mosquito is great nutrient source and have the right to be easily retained within the containers. In ~ the university of Michigan Amphibian basic Culex pipiens, a bird mosquito the is easily raised follow to standard procedures (Keppler et al., 1965) v Japanese quail as blood donor, has actually been offered successfully.

Other dietary regimens in ~ this stage need to be perceived with caution. Force-feeding of small, freshly metamorphosed frogs has been found to be nutritionally inadequate and also time-consuming. Live insects should be provided. Mealwoms are frequently recommended, but separation of big numbers of tiny mealworms from their culture medium is onerous and they conveniently die when put in a moist environment. Furthermore, mealworms have been reported as nutritionally insufficient for R. Pipiens (Cairns et al., 1967). If mealworms room used, the frogs must be moved to dried containers during the feeding period. This handling is deleterious and time-consuming. The low biomass of Drosophila, their inability to survive near water, and the challenge in containing them does not support the referral they generally receive together frog food.

(d)

Management of active Adults The activities management and also care of energetic adult R. Piplens call for both aquatic and terrestrial settings as comprise by the description of enclosures (see chapter V, sections C.4 and 6). All pets should originally be treated as defined in chapter IV, section C.2, pertinent information should be videotaped (Chapter VIII), and also special attention need to be given to the opportunity of condition (Chapter IX). Those pets to be provided within a few days for any type of objective various other than egg production may be hosted for this short period in a crate or covered aquarium at room temperature. The enclosure must contain enough water to allow the animals to submerge completely. It additionally should be listed with a table or shelf around 10 mm (0.5 in.) above water level or the enclosure have to be sufficiently slanted to administer both aquatic and terrestrial areas.

Northern frogs received between October and also late March and to be supplied as energetic frogs might be save in hibernation together described listed below (see thing V, part C.5 and 6). Northern frogs to be used for egg production and received throughout these very same months have to be stored in hibernation. North frogs obtained in other months can not be offered for egg manufacturing without provision for feeding and management design to support oogenesis. Attempts have to not it is in made to force northern "summer" frogs right into hibernation uneven the chilling is component of a specific, knowledgeable speculative design. They must be hosted as described over if they are to be used within a couple of days and also as described below if they room to be hosted for much longer periods.

Southern frogs have to never be refrigerated or they will certainly die. Attempts have to not it is in made to achieve eggs native them until more is learned worrying their reproductive cycle. Southerly frogs at any season of the year may be held temporarily as defined above; if they are to be organized for longer periods, they need to be regulated as described below for active frogs.

Active adult that are to be retained for an ext than a few days are best contained wherein the aquatic and terrestrial locations can be adjusted in accordance with the frog"s well-known behavior. Such containers are described more completely in thing V, part C.4 and 6.

Water in the aquatic part of the adult enclosures have to be saturated deep to permit the animals to rise in their typical position v eyes and nostrils over water, legs dangling and also relaxed. Together a water depth should be noted even for primarily terrestrial animals; in the confinement of one enclosure, they attain needed exercise by swimming fairly than by jumping. This aquatic area likewise provides a suitable place for them come rest. Water must flow-through the enclosure tenderness (two to three transforms per day) but in adequate volume to maintain bacterial counts below tolerance levels . Contrary to the circumstances for larvae, chlorinated water (4–6 ppm) because that the palliation of bacter counts may be used.

Although not as preferable as other species of enclosure (Chapter V, ar C.6), plastic vegetable crispers may be used. Because that these it is an overwhelming to arrange flow water and, if revolution water is used, caution is required because of the toxicity of some generally used products . A smaller opaque crisper with one finish removed should be inverted inside a larger one to administer a terrestrial area; this constitutes a feeding platform on top and also a hiding ar below.

Matting do from a solid neoprene mesh should be put on exposed surfaces. Not only does this save the frog native direct call with the plastic surface but also permits desirable air circulation about the animal.

Adult R. Pipiens, specifically from certain collection sites in the northern states, are very susceptible to the Lucké renal adenocarcinoma. Whether together a result of tension or other factors, the breakthrough of this tumor is promoted when the animals are held for long periods at room temperature under crowded conditions (Rafferty, 1962). Thus, experience suggests that 12 frogs room an suitable number for a vegetable crisper measuring around 0.36 × 0.25 × 0. 12 in (14 × 10 × 4.5 in.).

Aquaria or terraria can likewise be used. If the frogs room not in to run water, the water need to be adjusted frequently to avoid it from ending up being fouled. Because frogs are usually secretive, numerous shards that unglazed clay flower pot placed in the container carry out places to hide. Extr pieces of pottery in the water, however extending above it, administer a cool respite. Northern R. Pipiens eat well and also are energetic at 22–24 °C (72–75 °F); those native Mexico do ideal if organized at greater temperatures, e.g., 25–27 °C (77–80 °F).

Optimal management ethics to support and synchronize oogenesis in laboratory-reared and laboratory-bred R. Pipiens are right now under investigation and as however cannot it is in specified. However, a far-reaching proportion of tires females will develop eggs if kept as described over and fed as defined below. No criteria have yet been created to recognize once eggs in living females are mature; however, tires eggs may be found 3–4 months adhering to a ahead ovulation. Together eggs will certainly be resorbed in ~ a couple of days of their maturity if the females space not artificially ovulated. This is additionally true of wild-caught gravid females if not returned to hibernation immediately. Colours from together resorbed egg is stored in the liver, i beg your pardon becomes strongly black together 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 transition from an omnivorous come a carnivorous diet made up of food that should be moving. They different from R. Caresbeiana and R. Clamitans in that they execute not or cannot take it food while submerged. Reports in the literary works that "frogs and toads" will certainly take food native mechanical gadgets such together "lazy susans" (Kaess and also Kaess, 1960) execute not apply to R. Pipiens. A few individuals will certainly strike in ~ food gift in this manner, and an even smaller number will certainly swallow it. Attempts are in progression to find out the criteria for motion, taste, and texture that have to be met to current prepared foods items to R. Pipiens. In ~ present, results carry out not warrant consist of of a protocol because that prepared foods items in this document.

Adult frogs deserve to survive for expanded periods (3–4 weeks) there is no feeding if your quarters are clean, yet long-term survival requires feeding the tantamount of 10–12 full-grown crickets two to 3 times a week. Frogs have the right to be force-fed raw liver or hamburger, but not just is this time-consuming but quantity and also timing are hard to judge and also growth prices are poor. Therefore, as soon as possible, usage of live food is recommended. Definitive researches of R. Pipiens dietary requirements have not to be done since of the dependence on live food or force-feeding. Should gadgets for the presentation of all set diets be effectively developed, such research studies will it is in possible. At the university of Michigan Amphibian Facility, bone real-formations the resulted from diet insufficiencies have been observed. These have actually been alleviated, at the very least partially, by dusting the arthropod dietary items v commercial vitamin and protein supplements.

Some adult amphibians do not conveniently accept live food. These animals must it is in force-fed through special food mixture from a syringe through a stomach catheter. Cautious insertion that the catheter and a slow administration of the food will aid prevent regurgitation. One ration that has actually been used efficiently is all set by homogenizing beef liver, eggs, and also lettuce (all boiled) in a blender in a ratio of 4: 1: 1. A tiny amount the bone meal, one drop of ko liver oil/ml mixture, and commercial vitamin supplements may be added. Antibiotics may also be included in proper quantities (see thing IX, section B), and also the mixture frozen for later on use. Canned dog food mixed with heat water and homogenized in a blender was another adequate force-feeding diet; when supplied for Necturus, ko liver oil was included (Kaplan and Glaczenski, 1965).

Food items from the wild, such as countless arthropods and snails, can be used. One reliable collection method is "light trapping" because that 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 thing III, sections B.3 and also 4), such together crickets, sowbugs, earthworms, beetles, flies, caterpillers, and also moths. Even newborn or young mice will certainly be readily taken. A native of caution need to be included concerning the usage of fly maggots: since maggots of plenty of fly varieties are not killed in the digestive street of the frog, such maggots may destroy the frog. The trouble of supplying a life diet, perhaps, has been the greatest solitary deterrent to occurring laboratory-maintained societies of R. Pipiens; by proper an option of food types and standardization that food culture, however, this is not a prohibitively challenging problem and becomes significantly feasible together the swarm enlarges.

Many potential food items may be obtained commercially. Thus, return crickets and also earthworms might be cultured, castle may likewise be purchased indigenous bait dealers and also arrangements have the right to be do for distribution on a consistent basis. Sowbugs, which have the right to be uncovered in nature under old boards and dry leaves, are conveniently raised. They room recommended because they frequently appear on checklists of wild R. Pipiens stomach contents, room "clean," and also do not drown in the aquatic section of the frog container. Indeed, lock crawl out of the water onto the flower pots or up the political parties of the container. Sowbugs may be raised at temperatures in between 25 and 30 °C (77–86 °F) in roaster pans v a half inch of damp peat moss. The peat moss must be covered with a moist rectangle of corrugated cardboard and with a plastic lid indigenous a vegetables crisper ~ above the cardboard to reduce loss of moisture by evaporation. Sowbugs feeding on crumbled rabbit chow and also crushed blackboard chalk, which must be inserted in a dry corner to retard molding. Both the rabbit chow and the peat moss bedding need to be warm treated to kill organisms such together weevils. (The weevils no only complete with the sowbug colony but may actually damage it.) Under this conditions, a colony of sowbugs will dual its number about every 40 days.

Some frogs will certainly kill themselves by overeating (e.g., the Japanese frog R. Nigromaculata), however this go not happen with R. Pipiens. Nevertheless, extremely fed activities R. Pipiens do build muscle glycogen levels appreciably greater than those found in nature (Smith-Farrar, 1972).

(f)

Hibernation is necessary for holding northern gravid females in the gravid state and is useful in the winter months for low-cost holding of north adults come be offered for other purposes. Gravid females, if held under correct conditions, may be ovulated together late together July, 3 month after the moment of typical ovulation. If exactly conditions, however, space not met, the energy reserves in the eggs room mobilized and also the eggs regress. Even a few days the improper problems will lead to diminished fertility.

Summer frogs forcibly submerged without suitable preparation because that hibernation will drown. ~ entering hibernation, however, they stay submerged below the ice. Throughout hibernation a respiratory transition from aerobic come anaerobic may happen to part extent, back cutaneous respiration does occur. V these factors in mind describe Chapter V, sections C.5 and 6 for descriptions that hibernation quarters. Note that because that wild-caught north R. Pipiens water temperature need to be between 3 and also 4 °C (37–39 °F) in October and also November, 1.5–2 °C (34–36 °F) from December, and 3–4 °C (37–39 °F) because that 2–4 weeks prior to removal indigenous hibernation, unless the frogs are being preserved at room temperature only long sufficient to have actually ovulation induced.

Southern frogs have to never be inserted under hibernation conditions.

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Criteria to recognize once an animal is in hibernation as distinctive from cold torpor have not to be devised. Such information is critically needed. Also, criteria because that recognizing true emergence from hibernation are missing.