IACUC Learning Module - Xenopus laevis

PreviousNext

Injection Sites and Other Techniques

Needle gauge for most frogs should be 25-27 with a 3/4 inch length.

Anurans have subcutaneous lymph spaces that connect with the vascular system via the anterior and posterior lymph hearts. Drugs administered via lateral or dorsal lymph sacs should be rapidly absorbed into the blood. Therefore, intravenous injections are normally given to frogs via the dorsal lymph sac. This is also the common site for injection of hormones to stimulate breeding in Xenopus.

The thigh muscles can be used for intramuscular injections.

Intraperitoneal injections should be given with the frog on its back in your hand and its head directed downward. A needle can then be inserted into the groin area with less likelihood of puncturing any internal organs.

Frogs can be gavaged by stomach tube, but the stress caused to the animal often outweighs the end result.

Blood collection in amphibians is often difficult because of low body weight and poor access to vessels. Unfortunately, it may be necessary to euthanize animals in order to maximize sample volume.

Acceptable blood withdrawal methods are toe web clipping, cardiac puncture, and venous cutdowns of ventral abdominal veins. Toe clipping can be done with a sharp fingernail clipper to obtain a few drops to 0.5 ml of blood. The blood flow usually stops spontaneously, but silver nitrate sticks may be needed to cauterize the wound. Cardiac puncture can be performed on anesthetized animals by locating the heart via palpation and using a small needle (25-27 gauge).

Amphibian blood contains nucleated erythrocytes, leukocytes and thrombocytes. Amphibian erythrocytes are biconvex, oval and the largest of all vertebrate erythrocytes. Accurate RBC counts are obtained by means of hemocytometer and standard counting procedures. The use of automated white cell counters is contraindicated.

Anesthesia

The absorption and excretion of anesthetic agents are directly affected by environmental temperatures. Therefore, amphibian anesthetic dosages are frequently listed with environmental temperatures.

Tricaine methanesulfonate (MS-222) is the most useful anesthetic for amphibians and can be administered in a bath or by injection. (One drawback of injecting MS-222 is that MS-222 is a powder that must be dissolved in water. Solutions for injection are no longer sterile once the powder container has been opened.) Bath concentrations range from 0.1-0.5% for adults to slightly less for tadpoles. Once the animal has been immersed in the bath, sedation should occur within five minutes. Recovery occurs 15-30 minutes after removal from the anesthetic solution. Anesthesia may be extended by keeping part of the amphibian in a shallow dish of anesthetic solution or by additional injections. Benzocaine (ethyl p-aminobenzoate) has a similar effect to MS-222 and may be considered superior by some investigators, but it requires dissolution in alcohol first.

Ketamine or Telazol (tiletamine/zolazepam) can be used for minor procedures such as radiography. Animals anesthetized with these drugs remain sensitive to pain, even at high doses. The surgical use of these drugs is therefore limited to preanesthetic use.

Table 1 Anesthetic Doses for Amphibians

Reproduced from Zoo and Wild Animal Medicine Current Therapy 3, edited by M.E. Fowler, Publisher W.B. Saunders Co., 1993.

Important Link: IACUC Approved Analgesics, Anesthetics & Sedatives

Euthanasia

Body condition may be evaluated by looking for prominence of the skeleton and palpation of musculature and abdominal contents. Weight is highly variable and depends on the state of hydration; amphibians can lose up to 50% body weight in fluids before death.

Skin scrapings and gill biopsies are useful to detect fungal, bacterial and parasitic infections. Focal and nodular lesions should be biopsied, Gram's and acid-fast stained and cultured. Hematology has limited value because of the lack of normal data, plus wide variations due to sex, season, and hydration levels. A fecal exam can detect protozoan and metazoan parasites, but significance depends upon the type and extent of infestation with presence of clinical signs. Radiology can be useful to find skeletal deformities, gastrointestinal impactions, foreign bodies or pneumonia. Fiberoptics can be used to view the stomach, biopsy internal organs or for sex identification. Transillumination of small, thin skinned amphibians by means of a cool fiberoptic source can be used to view the lungs, ova and other coelomic contents.

Back to Training Modules