IACUC / LARC STANDARD PROCEDURES

OOCYTE COLLECTION FROM XENOPUS LAEVIS FROGS (Updated May 2007)

Oocytes can be removed from alternating ovaries in the same female frog, but each collection procedure must be done at least 4 weeks between surgeries. Oocytes may be collected up to 6 harvestings and the last or 6th removal must be a terminal procedure followed by euthanasia. Surgery and post-procedural care may only be performed by trained personnel. Animals must be adequately anesthetized throughout each surgical procedure.

Pre-procedural preparation and anesthesia:

•  Clean gloves moistened with water should be worn at all times when handling frogs. All surgical procedures must be recorded and records checked to assure that the frog is an appropriate surgical candidate.
•  Choose a female frog that is large and active. Transport frogs in water from their home tank to and from the lab. Collect the frog with a net that is dedicated to the tank.
•  Anesthetize the frog with a fresh mixture of Tricaine methane sulfonate (MS222): 500mg-2g/liter dissolved in deionized water. The solution must be buffered.
•  Submerge the frog in the MS222 and wait for the onset of anesthesia - it usually takes approximately 15-20 minutes.
•  A surgical level of anesthesia is confirmed by gently pinching the fleshy part of both rear feet with hemostats or forceps and ensuring that the frog is non-responsive to painful stimuli.
•  Place the frog on a clean surface.
•  Remove contaminants on the skin by gently swabbing* or spraying dilute surgical disinfection solution (chlorhexidine, povidone) only on the portion of the skin where the incision will be made.

Use cotton for swabbing as gauze sponges are too rough in texture and may abrade the frog's skin.

Oocyte Collection:

•  The use of sterile instruments is standard practice for survival collection surgeries. Instruments should be sterilized by autoclaving or bead sterilization. 'Cold' sterilization fluids (such as cidex or zephrin) should be avoided, as they may introduce potentially toxic chemicals into the surgical site or onto permeable amphibian skin.
•  Make a small (1-2cm horizontal or vertical) incision on the abdomen above the groin and in between the midline and the lateral aspect of the abdomen.
•  Scissors are used to dissect through the fascia and muscle to visualize the oocytes. Oocyte strands are then gently externalized and cut.
•  The incision is closed by suturing both the fascia and skin layer - skin should be closed using an interrupted suture pattern to prevent dehiscence.
•  Recover the frog in tank water with head elevated (to prevent drowning) and the rest of the body submerged. Recovery takes up to 1 hour. MONITOR FROG FREQUENTLY.

Post-collection Care:

•  You may wish to return the frog to a separate recovery tank in standard housing when the animal is able to swim normally to be monitored more easily. Alternately post-operative animals can be returned to a home tank with a small enough population so that they can be easily observed. NOTE: LARC needs a minimum of 24 hours notice to set up a new recovery tank.
•  All post-surgical animals must be appropriately identified. If there are multiple post-op animals in the same tank, then each animal must be individually identified (no-absorbable suture placed in webbing, web notching, etc.).
•  Monitor frogs daily for a minimum of 48 hours for wound dehiscence or infection and then every 2-3 days for two weeks. Contact the LARC Veterinary Services group if there any health concerns.

Documentation:

All procedures and care must be documented. This includes:

•  Identification method used and location on the animal.
•  Date of surgery, and applicable comments (e.g. frog recovered normally from anesthesia).
•  Post-op observations days 1 & 2 after surgery (e.g activity level, condition of incision, signs of infection).
•  Date of suture removal.
•  Euthanasia.

Literature search words required:

Literature search was performed for refinement of this Standard Procedure on April 9, 2007

Key Words	Search Site	Years Covered
Xenopus, frog, oocyte collection, harvest, guideline	Pubmed, CompMed	2000-present

Agents:

This procedure requires an anesthetic agent.

Adverse Effects:

Procedure, Agent or Phenotype	Potential Adverse Effects	Management
infection of surgical site	Dehiscence, decreased activity and appetite	Consult with the LARC Veterinary staff

Monitoring Parameters:

Monitoring Parameters	Frequency	PI/Lab will Document
General appearance/activity level	Days 1 & 2 post-op, then every 2-3 days for 2 weeks	Yes
Condition of incision	Days 1 & 2 post-op, then every 2-3 days for 2 weeks	Yes

Describe the conditions, complications, and criteria (e.g. uncontrolled infection, loss of more than 15% body weight, etc.) that would lead to removal of an animal from the study, and describe how this will be accomplished (e.g. stopping treatment, euthanasia).

If an animal develops any problem at the surgical site or has any evidence of post-operative complications (inactivity, not eating), the LARC Veterinary Services group will be called.

For all investigators housing animals with tumor formation, skin lesions, neurological deficits, or that are in Category E, list the expected characteristics/clinical presentations and endpoints of the animal model and the criteria for euthanasia. Note: The IACUC also requires such lists to be posted in the respective animal rooms and monitored by the IACUC compliance staff and LARC, to assure PI adherence to the endpoints listed.

N/A.