Background
The following SOP provides a step-by-step guide for harvesting frog oocytes from Xenopus laevis (African clawed frogs). Frogs must be handled gently using clean nets or powder-free gloves that are moistened with frog water, to ensure that the slime coat and skin is not compromised. In this SOP, "YARC frog water" refers to the aquarium water that is available in the YARC frog facilities. Refer to the Policy on Aseptic Amphibian Survival Surgery for additional requirements.

Transport
1.	Frogs should be transported to the laboratory in a clean transport tank, provided in the frog rooms, with enough YARC frog water in the transport tank to completely cover the frog.
Anesthesia
2.	Ideally, frogs should not be fed on the day of surgery to avoid the rare complication of emesis while anesthetized. Frogs are fed twice weekly by YARC staff between 9-10 AM. Retrieving frogs before 9AM would ensure that a frog has not ingested food that same day. Alternatively, YARC staff could be advised not to feed a particular tank until after the frog has been transferred to the laboratory.
3.	Frogs should be anesthetized away from the surgical area. Anesthesia is induced by transferring the frogs (using a clean net) to deionized water containing 0.2% (2 g/L) tricaine methane sulfonate (MS222), buffered with 5mM Hepes (1.192 gm/L) and titrated to a pH of 7.5 with sodium bicarbonate or sodium hydroxide.
4.	A frog has reached a surgical plane of anesthesia when it does not respond to a toe pinch and/or has an absence of a righting reflex. Once an anesthetic depth has been achieved with MS222, continued anesthesia can be achieved by lowering the body temperature. The recommended method of achieving hypothermia is to transfer the frog to paper towels pre-wet with YARC frog water and then placing the towels on crushed ice that has been covered with aluminum foil or saran wrap. Never place the frog directly on crushed ice.
Surgical Technique
5.	Place the anesthetized frog in dorsal recumbency. Artificial slime, such as Shieldex (Aquatronic, Oxnard, CA) or YARC frog water should be applied to the frog's skin to keep it moist.
6.	The surgical technique requires asepsis, gentle tissue handling, minimal dissection of tissue, appropriate use of sterile instruments and correct use of suture material. The surgeon must scrub hands and arms with a disinfectant soap, don a mask and sterilized gloves and use sterile instruments.
7.	Make a diagonal incision in a lower quadrant of the abdomen, approximately 1cm long (sides should be alternated to allow maximum healing time). Frog skin is quite tough - the use of a pointed scalpel blade (ie. #11) is recommended. The skin and abdominal muscle incisions should be made in two stages. After incising the skin, lift the muscle layer with surgical forceps and make the incision in the tented muscle, to avoid inadvertently wounding any internal organs. Avoid transecting macroscopic glands, lymph hearts and blood vessels with the incisions.
8.	Grasp ovary with forceps and exteriorize the oocyte masses. The desired numbers of oocytes are excised, and the remainder carefully replaced in the coelomic cavity.
9.	The incision should be closed in 2 layers, with absorbable gut (5-0 chromic gut) or PDS for the muscle layer and nylon (non-wicking) suture preferable for the skin closure (5-0 or 6-0 monofilament nylon or PDS). A simple interrupted or horizontal mattress pattern of suturing, rather than continuous, is recommended for skin closure.
11.	Post-operative analgesics and antibiotics are not mandated by the Yale IACUC (see exemption statement in the Policy on the Use of Analgesics in Laboratory Animals). However, in the event that investigators wish to provide an analgesic, the following have proven efficacy in leopard frogs and would likely provide pain relief in Xenopus: A. xylazine (10 mg/kg intracoelomic every 12-24 hours) B. butorphanol (25 mg/kg every 12 hours) A single dose of xylazine may provide the greatest ease and efficacy, given its longer range of action. A suitable broad-spectrum antibiotic to prevent infections in frogs is enrofloxacin (BaytrilTM, 5 mg/kg, intracoelomic, 1 dose).> Note: the effect of these agents on oocyte production or RNA expression have not been investigated.
Post-operative Care
12.	Rinse off frog with YARC frog water.
13.	Place frog in a recovery tank, filled with room temperature YARC frog water.
14.	Recovery is monitored by watching for purposeful movements.
15.	Frogs must be returned to a recovery tank in the animal facility within 12 hours. After the frog rests in a recovery tank for 48 hours, it will be returned to its original tank by YARC staff following examination of the surgical site.
16.	The IACUC policy on Performance-Based Standard for Frog Oocyte Harvests must be followed. A minimum of a two-week rest period (four week period is optimal) between surgical procedures should be given to minimize stress. It is recommended that each laboratory keep several tanks of frogs and choose frogs from one tank only over a several week period for oocyte harvests, thus ensuring that all frogs get an appropriate amount of rest between sequential procedures.
Post op monitoring
17.	Check the frog daily for 3 consecutive days post-operatively to monitor for wound dehiscence and other signs of complications (i.e., lethargy, wound dehiscence, inflammation etc). Report any complications to VCS.
18.	The removal of sutures is not needed as they gradually dissolve over time and it is more stressful to the frog because it requires additional handling.
Record keeping
19.	Records must be kept of all survival surgical procedures in research animals. Refer to the IACUC Policy on Aseptic Amphibian Survival Surgery for the specifics that need to be recorded.
20.	The records may be kept in the animal room in a notebook through the recovery period and returned to your lab for storage afterwards.
References
O'Rouke et al., 2002. Biology and Diseases of Amphibians, In: Laboratory Animal Medicine. Eds. Fox et al. Academic Press, London Wright DM. 2001. Surgical Techniques. In: Amphibian Medicine and Captive Husbandry. Eds. Wright and Whitaker, Krieger Publishing Company, Malabar, Florida
Reviewed and approved by the IACUC: 3/17/04

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Last Modified: June 5, 2006 (jj)

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