Amphibians are scaleless, smooth-skinned, ectothermic (cold-blooded) vertebrates, most of which are closely associated with aquatic or very moist environments. Virtually all amphibians begin their lives in the water as fully aquatic gill-breathing larvae. Some remain aquatic their entire lives and many metamorphose into air-breathing adults with lungs and appendages. Depending on the species these animals spend varying amounts of time in both aquatic environments and terrestrial environments.
Frogs, toads and salamanders are the types of amphibians commonly seen in the lab. Various species have been used for genetic, physiological and neurology/endocrine studies.
Cage cards should be used for identification. Individuals can be identified by visible implant elastomers, PIT transponders, soft visible implant alphanumeric tags, and the use of drawings and photographs to record unique markings.
Handling and Restraint
The slimy skin secretion of amphibians is a protective covering that is similar to that of fishes. Handling these animals with dry hands can cause this skin covering to rub off, allowing the entrance of bacteria. Frogs and salamanders should be picked up with gloves that are wet. Amphibians do not adapt well to handling. When picked up they almost always struggle. Frogs should be picked up by placing fingers on each side and between the legs. The frog’s head will then face the handler’s wrist. (Figure 7) The marine toad and other tropical frogs secrete toxic substances from skin glands. When handling these species one should wear protective gloves.
Newly arrived amphibians should be kept separate from established colonies. They should be disturbed as little as possible. They should be rinsed thoroughly with dechlorinated water as they are removed from shipping crates to remove feces accumulated in transit.
Sexing and Breeding
Most amphibians are wild caught or bred and reared by suppliers. There are many variations in sexual characteristics among amphibian species. Among most, the only absolute way to determine sex is to observe the actual courtship to see the difference in morphology and behavior. During the breeding season the male’s vocal sacs become more prominent. Xenopus laevis females have larger ventral flaps and a larger body than males. Male Xenopus have black surfaces on the inner aspect of the forelimbs, and they have larger digits during the breeding season. In bullfrogs the tympanic membrane (the ear) is approximately the same diameter as the eye in the female and in males it is twice a large as the eye.
Xenopus are mature at about 14 weeks and frogs produce eggs every 3 to 4 months. Amphibians are “cold-blooded”. Frogs live about 15 years, maximum. Heart rate is about 8 beats per minute at 2° C and 40 to 60 per minute at 25° C. Rana pipiens are around 2.5 to 4 inches long and represent a great diversity of types. The skin of frogs contains pigmented cells called chromatophores which can change colors against different backgrounds. Teeth are present only in the upper jaw and are used for gripping food. Frogs have a 3-chambered heart and no diaphragm. Large fat bodies attached to each kidney provide energy during winter hibernation and spring reproduction. Amphibians have breathing structures adapted for their aquatic (water) and terrestrial (land) stages.
Amphibians are particularly sensitive to toxic substances in the water since their skin is very porous. Slight temperature may cause behavioral abnormalities such as lethargy and loss of appetite and can trigger illnesses.
Simply providing amphibians with the appropriate food may not be sufficient to ensure feeding. If amphibians become stressed from overhandling or too many disturbances they may stop eating and starve to death. Establishment and maintenance of feeding behavior is an indicator that the amphibian has accepted its environment.
Many amphibians tend to be cannibalistic. Large tadpoles may eat small ones, and large adults may eat tadpoles or smaller adults. Cannibalism among larvae is largely a result of overcrowding. Housing larvae and adult amphibians in a low population density and providing them with adequate food can help reduce or eliminate cannibalism.
An amphibian’s environmental requirements include screens or other types of covers atop cages or aquariums, hiding places, natural-spectrum lights and proper temperature and humidity controls. Many semi-aquatic amphibians benefit by being able to occasionally leave the water. Stainless steel, plastic or glass tanks or aquariums are commonly used for housing amphibians. Tank materials should not contain any potential toxins that could leach into the water. Sanitation of tanks with detergents or disinfectants is discouraged unless rinsed very well since a small amount left in the tank could poison the amphibians.
Maintaining clean, chlorine-free water is critical. It is recommended that cages be routinely rinsed or flushed a few hours after the animals have been fed in order to eliminate uneaten food and associated bacteria. The number of frogs should be limited to the number that allows all to be in or out of the water at the same time without being on top of one another or frequently bumping into one another.
Water hardness should measure below 250 ppm or mg/l; the carbon dioxide content of the water should measure less than 5 mg/l; and the pH should fall between 6.5 and 8.5. The temperature of new water should be adjusted to that of the old water before placing the animals in it, as a sudden change of more than a few degrees can make the animals more susceptible to disease.
For bullfrogs and leopard frogs untreated tap water in most locations may be used. Caution should be exercised with chlorinated water, however, because chlorine levels vary tremendously with seasons and geographic region and can be harmful or lethal in excessive amounts. Water can be held in open containers for 24 hours; this permits most of the chlorine to dissipate. Another alternative is to add sodium thiosulfate to the water. Chloramine, another additive to municipal water supplies, cannot be removed by holding and aging for 24 hours.
Oxygen, which enters the water at the air-water interface, must be maintained at an adequate level. This may be accomplished by maintaining a large surface to volume ratio; a shallow tank has a larger amount of water surface per liter of water than a deep tank of the same volume capacity. Carbon dioxide will be dissipated by at the water surface. Bubbling filtered air through the water stirs the surface and increases direct oxygen contact with the water, which helps maintain the proper levels of oxygen and carbon dioxide in tanks with a high population density.
For newly hatched tadpoles, slight aeration is best. Tadpole populations should vary between 50 per liter of water when newly hatched to 5 per liter when metamorphosis begins. Adults of most common laboratory species can be kept between 20°C and 25°C, but generally thrive better at the cooler end of the range. Rapid temperature changes can be particularly harmful for small larvae, eggs or embryos. Cooler temperature prevents early metamorphosis in tadpoles. A natural photoperiod is probably best if no other requirements are known. For Xenopus sp. a 14:10 (light:dark) lighting cycle is recommended for optimum oocyte production.
Larval amphibians are generally omnivorous and most in nature consume algae or other soft vegetable matter. In the lab this can be replaced with boiled lettuce or canned low salt spinach, ground dog chow, rabbit pellets, liver or brewer’s yeast. Tadpoles of the African clawed frog can be fed ground peas or split pea soup powder. One should avoid overfeeding and promptly remove uneaten food.
Adult amphibians are carnivores and most species eat insects. Terrestrial adults rely on movement of prey for detection and stimulation of appetite. In most cases live food is needed for these species. Although some amphibians can withstand a cooler environment some do not feed if kept below a certain temperature. Sometimes raising the temperature a few degrees before feeding can increase feeding activity if the room is usually kept cooler. Crickets are easy to obtain from lab supply houses and can be dusted with vitamins which should contain calcium.
Dusting the insect colony’s food with vitamin-mineral supplement is an alternate method of supplementing the diet of amphibians. Mealworms are also readily eaten by most amphibians. Feeding quantity and frequency varies greatly with size and species. Generally feeding one to three times a week is good. Overfeeding can occur and should be avoided.
Salamanders feed on brine shrimp in captivity. Frozen foods are preferable to live foods as they reduce the risk of parasite ingestion and simplify the acquisition and storage of foods. Salamanders and toads thrive on crickets, earthworms, insect larvae and non-flying adult insects. Live mouse neonates and minnows may be used to vary the diet of larger salamander species.
Frogs eat a variety of live insects, worms, crickets and slugs. They generally show a preference to flying insects. Larger species such as leopard and bull frogs can be fed small mice, mouse neonates, crayfish or small live fishes as long as mice remain out of the water or fish in the water long enough for their movement to be detected. Xenopus will readily eat raw liver chunks or commercially prepared frog food.
Injections can be given into the dorsal lymph sac, which is approached with a long, thin needle inserted into the middle of the back and directed toward the tip of the urostyle. The thigh muscles can be used for intramuscular injections, and some anesthetic substances can simply be placed in the water for cutaneous absorption. Blood samples can be obtained from a foot web clip or cardiac puncture. Frogs can be stomach tubed.