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Chapter Twenty-Seven Amphibians
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Amphibians Scaleless, smooth-skinned, ectothermic vertebrates Begin life in water as aquatic gill-breathing larvae. Some aquatic entire life, many metamorphose into air-breathing adults w/ lungs & appendages. Spend varying amounts of time in both aquatic & terrestrial environments. Laboratory use = frogs, toads & salamanders Used for genetic, physiological & neurology / endocrine studies. bullfrogs, leopard frogs, African clawed frog & tiger salamander (axolotl)
AXOLOTL (larval form of the Tiger Salamander) Mudpuppies
Handling & Restraint Slimy skin secretion a protective covering Handling w/ dry hands causes covering to rub off, allowing entrance of bacteria. Handle frogs & salamanders only as necessary. Pick up with wet gloves. Do not adapt well to handling & will struggle. Place fingers on each side & between legs to pick up frogs. Frog’s head will then face handler’s wrist. Axolotls can be injured if restrained in a net. A two-handed lift is preferred restraint.
Handling & Restraint II Marine toad & tropical frogs secrete toxic substances from skin glands. Wear protective gloves when handling. Handle amphibians gently & carefully to avoid injuring skin & delicate gills. All handling introduces a degree of stress. Separate newly arrived from established colonies. Rinse thoroughly with dechlorinated water as they are removed from shipping crates to remove feces accumulated in transit.
Dorsal lymph sac injection
Sexing & Breeding Most wild-caught or bred & reared by suppliers. Only way to determine sex is observe courtship. Males vocal sacs become prominent during breeding season. Bullfrogs - external tympanic membrane caudal to each eye. Same diameter as eye in female. In males, this membrane is twice as large as eye. Males also have heavy thumb pads. Xenopus female has larger ventral flaps & body. Male Xenopus have black surfaces on inner forelimbs, & larger digits during breeding season.
Behavior Skin more porous than most terrestrial vertebrate Sensitive to toxic substances in water. Temp change cause behavioral abnormalities, lethargy & loss of appetite, can trigger illnesses. Sensitive to sudden temp change of more than a few degrees at a time. Providing appropriate food may not be sufficient to ensure feeding. May stop eating & starve from stress. Feeding indicates acceptance of environment. Housing in low density & providing w/ adequate food can reduce or eliminate cannibalism.
Husbandry Require covers atop cages, hiding places, natural-spectrum lights, proper temp & humidity. Dry / wet composition of cage important; semi- aquatic, such as common leopard frogs, need to to leave water. Tank materials should not contain toxins that leach into water. Cleanliness of water & housing unit > importance. Small amount of detergents or disinfectants left in tank could poison. Manual cleaning followed by heat sterilization & rinsing is suggested when cleaning.
Maintaining clean, chlorine-free water is critical. Thin, porous skin susceptible to poisons & bacteria. Too many changes stressful; > chance of injury. Solution = house fewer animals together. < accumulation of fecal material, < # water changes Rinse cages after fed to eliminate uneaten food. Environmental Conditions Limit # of frogs / cage to # that allows all in or out of water without being on top of, or frequently bumping into one another.
Vary with species & stage of life cycle. Eliminate toxic materials or keep at safe levels. Water hardness levels less than 250 ppm, or mg/l Water CO 2 greater than 5 mg/l pH between Adjust new water temp. to old water temp. before placing the animals in it. sudden change can = animals > susceptible to disease Environmental Conditions II
Bullfrogs & leopard frogs, spend greater time submerged. Untreated tap water in most locations may be used. Low levels of chlorine (under 4 mg/l) may help inhibit bacteria growth. Chlorine levels vary with seasons & region. Harmful or lethal in excessive amounts. Check chlorine level before placing in tap water. Dissipate water in open containers for 24 hours or add sodium thiosulfate to water. Chloramine cannot be removed by aging 24 hrs. Check water for this before contact. Environmental Conditions III
O 2 enters at air-water interface. Maintain a large surface to volume ratio. Shallow tank has greater water surface / liter than a deep tank of = volume. Surface site of CO 2 dissipation greater surface area : volume & surface area : animal ratios minimize amount of CO 2 retained in water. Bubbling filtered air through water stirs surface. Increases direct O 2 contact with the water. Helps maintain proper levels of O 2 & CO 2 in tanks with greater population density. Environmental Conditions IV
Environmental Conditions V Slight aeration for newly hatched tadpoles Tadpole populations vary between 50 / liter of water when hatched to 5 / liter when metamorphosis begins. Adults of most species kept between 20 & 25°C, thrive better at cooler end of range. Rapid temperature change harmful for small larvae, eggs or embryos. Cooler temperature prevents early metamorphosis. At higher temperature, metamorphosis occurs more quickly, but adults may be smaller & less well- adapted for survival.
Environmental Conditions VI Little known about requirements for light. Natural photoperiod is probably best if no other requirements are known. For Xenopus, a 14:10 lighting cycle is recommended for optimum oocyte production. To identify individuals, the system of choice would be one that allows identification of individual animals at all stages of development. tattooing, toe clipping & use of drawings & photographs to record unique markings
Diet Larval amphibians are omnivorous, in nature consume algae or soft vegetable matter. Replace in captivity w/ boiled lettuce or canned low salt spinach, ground dog chow, rabbit pellets, liver or brewer’s yeast. Tadpoles of African clawed frog fed ground peas or split pea soup powder. Avoid overfeeding and remove uneaten food. Adults become carnivores, feed on insects. Terrestrial adults rely on movement to detect prey & to stimulate feeding response. Leave area once feeding is observed.
Diet II Some do not feed if below a certain temperature. Gradually raising temperature a few degrees before feeding may increase feeding activity. Diet of laboratory raised insects is preferable, likely to be cleaner than wild-caught. Crickets available from biological supply houses. easy to maintain & can be dusted prior to feeding w/ calcium / vitamin supplement powder Mealworms (beetle larvae) readily eaten by most. Feed to satiation, 1 - 3x / week. “Programmed” to eat all the prey they can get, in captivity limits must be set.
Diet III Sometimes a salamander does not metamorphose, remains in larval form w/ gills. neoteny Aquatic adult, larval & neotenic salamanders feed on aquatic invertebrates, such as isopods or crustaceans, & small fish. In captivity, eat brine shrimp. Live prey used with reluctant eaters. Salamanders & toads thrive on crickets, earthworms, insect larvae & non-flying insects Live mouse neonates & minnows will vary diet of larger salamanders.
Diet IV Frogs eat live insects, worms, crickets & slugs, but generally prefer flying insects. Leopard frogs & bullfrogs eat such a large amount that insect diet is impractical. Feed small mice, mouse neonates, crayfish or small fish. Xenopus will eat raw liver chunks or commercial frog food. Force-feeding done at risk of injury; trauma of handling often harms more than food benefits. It is extremely difficult to sustain an amphibian by force-feeding & can actually discourage natural feeding behavior.
Additional Reading DeNardo, D. “Amphibians as Laboratory Animals.” ILAR Journal, 37(4):173–181, Duellman, W.E., and T. Trube. Biology of Amphibians. McGraw-Hill Book Company, New York, NY Fowler, M.E. Zoo and Wild Animal Medicine. W.B. Saunders Company, Philadelphia, PA Rollin, Bernard E., and M. Lynne Kesel (ed.). The Experimental Animal in Biomedical Research. Volume II: Care, Husbandry and Well-Being — An Overview By Species. CRC Press, Boca Raton, FL