Regulations for Oocyte Surgery

The following guidelines were developed by the Veterinary staff here and outline regulations for non-mammalian surgery. Your institute's procedures with the frogs should be in accordance with the Guide for the Care and use of Laboratory Animals and the Animal Welfare Act, as well as conforming to contemporary standards of care and use of experimental animals. All non-mammalian surgery should be performed using aseptic technique and appropriate anesthetic and analgesic techniques to prevent pain and minimize distress to the animal.

Anesthesia in Frogs

Tricaine methane sulfonate (MS222) is the anesthetic of choice. Dissolve the powder in deionized water of an induction tank at a dose of 200-500 mg/L for tadpoles and 500mg-2g/L for adults. Concentrations of over 500 mg/L should be buffered using 10-25 mEq/L (420-1050 mg/L) NaHCO3. The unbuffered solution is acidic, predominately ionized, and poorly absorbed, resulting in a prolonged induction time. Prepare a fresh working solution of MS222 each day.

There is no evidence demonstrating appropriate analgesia with hypothermia in amphibians, although the animals become torpid. Thus ice or ice water is not permissible as the sole anesthetic agent.

Depth of anesthesia can be monitored through loss of the righting response and loss of response to painful stimuli. Respiratory movement will slow and then cease as anesthesia deepens, but alternate routes of gas exchange in amphibians (skin and buccopharyngeal cavity can suffice for short procedures). Once the animal is anesthetized, remove it from he solution. Keep the skin moist. Anesthesia will persist for 10-20 minutes, sufficient time for ovarian biopsy, host transfer, or other short procedures. For longer procedures, immerse the animal in a weaker solution of MS222, or drip dilute solution onto the skin.

Benzocaine, although sometimes cited as a possible anesthetic agent, should only be used for euthanasia.

Aseptic Technique for Oocyte Harvest in Frogs

Although a dedicated facility is not necessary, the frog surgical area must be a portion of the room that can be easily sanitized. The surface should be clean, uncluttered, and free from overhanging objects and chemicals. Surgery can be conducted in an uncluttered laminar flow hood. The area should be wiped down with a disinfectant (10% Clorox, Alcide, Lysol, Staphene, Cidex) before and after use.

Instruments must be sterilized before use in survival surgery. A sterile wrap must be provided as an aseptic surface on which to place instruments during the procedure. Acceptable sterilization includes steam (autoclave reaching 115 °C), gas sterilization (ethylene oxide), ionizing radiation, or soaking in the following solution for the appropriate length of time.

Glutaraldehyde (Cydex Plus): 10-30 minutes, less corrosive; rinse with sterile water after disinfecting.

The best skin prep: The endogenous antibacterial mucus layer over the skin should be disturbed as little as possible. Using a stream of Nolvasan gently blot only the surgical area.

Intraoperative and Postoperative Care

The skin should be kept moist during surgery. The skin is highly permeable and water loss will be significant if the skin dries out. The frog can be kept in a shallow dish of water, propped at one end, containing the maintenance concentration of anesthetic.

Skin closures should always be with non-absorbable sutures.

If the anesthetic agent was added to the water, the frog should be rinsed in fresh frog water for recovery. The water should be shallow or use a damp cloth during recovery, as frogs can drown before complete consciousness can be restored.

Xylazine hydrochloride at 10 mg/Kg injected intracoelomically provides analgesia for potentially painful procedures for up to 24 hours.

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