Office of Research Compliance

Institutional Animal Care and Use Committee

Xenopus Surgical Oocyte Harvest

Introduction

Boston University is committed to observing Federal Guidelines and AAALAC International Guidelines for Humane Care and Use of Animals.
Amphibian oocytes are used for studies in molecular biology, cellular biology and biochemistry and for a variety of experiments; however, the quality of the oocytes can vary greatly from animal to animal. Thus, the replication of experiments can be improved if the oocytes are from a consistently good producer. Please see Guidelines for Minimum Requirements for Housing Xenopus laevis.
The surgical procedure is quick and is well tolerated with a short recovery period. There are rarely any post procedural complications; therefore, multiple harvests are permitted. This policy describes approved methods for obtaining oocytes stages I though VI from the African Clawed Frog (Xenopus laevis).

I. POLICIES FOR XENOPUS OOCYTE HARVEST

A. Personnel performing surgeries and other procedures must be properly trained.
B. All surgeries must be performed under general anesthesia and using aseptic technique.
C. Tricaine methane sulfonate (MS-222) is the anesthetic of choice.
D. Hypothermia is not recommended as adjunct to anesthesia.
E. Frogs must not be fed on the day of surgery to avoid emesis while anesthetized.
F. The administration of perioperative analgesia is required.
G. Up to three (3) recovery surgeries (the 4th would be terminal) per animal are acceptable.
H. Investigators must alternate surgical oocyte collection between left and right ovaries and rotate frogs so that the recovery period between surgeries in any individual is maximized.
I. A minimum of one month recovery period is required between surgical collections.
J. The protocol must state a method used to identify animals after oocyte collection.
K. Animals must be monitored for at least two days after surgery and then the incision monitored three (3) times/week until suture removal.
L. The incision site must be monitored closely for signs of necrosis, dehiscence, inflammation, or fungal infection.
M. Skin sutures and wound clips, if non-absorbable, and not removed by frog, must be removed 10-14 days after surgery.
N. Records must be maintained to ensure proper animal care.

PROCEDURES

II. HANDLING THE FROG

A. Clean, non-textured, powder-free gloves moistened with tank water or deionized water must be worn at all times when handling frogs.
B. 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 or use clean gloves to remove the frog from the tank.
C. All surgical procedures must be recorded and records checked to assure that the frog is an appropriate surgical candidate.

III. ANESTHESIA.

A. Preparation of MS-222
1. Prepare a fresh mixture of Tricaine methane sulfonate (MS-222) in fume hood:
200-500mg mg/liter1 dissolved in deionized water or prepare a 0.15% solution by dissolving 1.5 g/l.8
2. Buffer the solution to pH 7.0 using sodium bicarbonate, NaHCO3, assuring that the pH is neutral with a pH meter or pH paper. Buffering the MS-222 is required as the Tricaine solution is really acidic and can harm the frogs. In addition, it makes it more bioavailable and better absorbed resulting in a smoother induction.9
3. Check the temperature assuring that the temp is the same as that of the tank.
4. Alternatively, concentrated solutions (5%) of MS-222 may be prepared and buffered and kept frozen until use either as is for euthanasia (see III.J.) or diluted for anesthesia. Steps 2-3 must be observed when preparing the anesthesia bath.
B. Anesthetizing the frog
Submerge the body of the frog keeping the head above water in the MS-222 and wait for the onset of anesthesia - it usually takes approximately 15-20 minutes.
C. Perioperative analgesia
The administration of analgesia is required and may be administered either before surgery when the frog has been anesthetized or during recovery. The following agents and doses are recommended.10
1. Bupivacaine, (0.25% may be swabbed onto the skin )8 or may be administered locally infiltrated at the incision site and allowed time to absorb prior to surgery.
2. In addition or alternatively the following analgesics are recommended
• Banamine (flunixin meglumine) 1 mg/kg SC
• Meloxicam 0.1 mg/kg SC
• Butorphanol 1-3 mg/kg SC
D. Anesthesia Monitoring
Comment: COOLING AND HYPOTHERMIA IS NOT RECOMMENDED AS AN ADJUNCT TO MS-222 ANESTHESIA.9
1. According to Dr. O’Rourke “I don’t recommend hypothermia for anesthesia or as an adjunct for oocyte harvest. In fact, it will prolong the recovery time. When I did oocyte harvest, I had ample time (over 30 minutes) of anesthesia to collect and suture. So did our PIs and grad students. In fact, you can drip MS-222 onto the skin if you feel you need to supplement. I would be worried about compromising the immune system and causing tissue damage with ice/ice water. That would only prolong healing”.9
2. A surgical level of anesthesia is confirmed by gently pinching the fleshy part of
both rear feet with gloved hands (metals can scrape the mucous layer) and ensuring that the frog is non-responsive to painful stimuli.
3. Place the frog on a clean moist surface. Paper towels wetted with MS-222 solution from the anesthesia tank are recommended.
4. Keep the skin moistened during surgery.
5. Anesthesia is maintained by sprinkling MS-222 solution on the frog as needed.
6. Remove contaminants on the skin by gently swabbing. Use cotton for swabbing as gauze sponges are too rough in texture and may abrade the frog’s skin.
7. The use of disinfection solution (chlorhexidine, povidone iodine) remains controversial and if used must be used in dilution and sparingly and only on the portion of the skin where the incision will be made.
8. Draping the surgical site may be done using moistened drapes and taking care not to abrade the skin.

IV. OOCYTE COLLECTION

A. The use of sterile instruments is standard practice for survival collection surgeries. Instruments must 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.
B. Make a small 1-2 cm incision on the abdomen above the groin and in between the midline and the lateral aspect of the abdomen.
C. Scissors are used to dissect through the fascia and muscle to visualize the
oocytes. Both should be picked up with forceps before cutting to avoid cutting the liver (that sometimes is just underneath). Oocyte strands are then gently externalized and cut.
D. A section of the ovary containing several dozen oocytes is removed. One stitch using absorbable suture may be taken in the ovary to control bleeding.
E. The incision is closed by suturing both the fascia and skin layer in two layers. Absorbable 3-0 or 4-0 PDS sutures is recommended for the fascia. Nonabsorbable monofilament nylon 3-0 or 4-0 is recommended for skin closure.6 The skin should be closed using an interrupted suture pattern to prevent dehiscence.
F. During the surgical procedure also make sure to identify the frog, if this has not been done previously.
G. Identification of frogs after oocyte harvest.
Acceptable methods of identification include tagging the frog with colored beads, tattooing the animal or placing the incision such that animal can be differentiated based on the appearance of the incision. Microchips are acceptable as well.
H. Recovering the frog from anesthesia
Recover the frog in shallow tank water with head elevated (to prevent drowning)
and the rest of the body submerged. Recovery may take up to 1 hour. MONITOR FROG FREQUENTLY.

V. POST-PROCEDURAL CARE

A. Have a recovery tank prepared before you start the surgery.
B. You may wish to return the frog to a separate recovery tank when the animal is able to swim normally and where one or more postop frogs can be monitored more easily. Alternately post-operative animals may be returned to a home tank with a small enough population density so that they can be easily observed.
C. 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.
D. Monitor postop frogs daily for a minimum of 48 hours for wound dehiscence or
infection and then every weekday for two weeks. Contact the Supervisor or PI or LASC/LACF Veterinary Services if there any health concerns.

VI. DOCUMENTATION AND RECORDS

All procedures and care must be documented. Records must include all treatments (doses and routes of administration) including anesthesia and analgesia, animal ID, protocol number, post-procedure monitoring information, complications, date and method of euthanasia.
This includes:
A. Identification method used and location on the animal.
B. Date of surgery and applicable comments (e.g. frog recovered normally from anesthesia).
C. Post-op observations days 1 & 2 after surgery (e.g. activity level, condition of incision, signs of infection)
D. Date of suture removal if the sutures have not fallen out on their own.
E. All entries must be dated and initialed.

VII. EUTHANASIA

Acceptable methods of euthanasia for Xenopus are:
A. Anesthetize the animal in a buffered solution of MS-222 followed by a secondary method.
B. Euthanize the animal by injecting 1 ml of 5% buffered MS-222 into the dorsal lymph sac or SC or intracoelomically followed by a secondary method.
C. To assure death of the frog the following secondary methods are acceptable:
1. Decapitation.
2. Remove the heart followed by freezing the carcass.
3. Double pith the animal. This is done by first severing the spinal cord at the highest cervical vertebrae with a sharp knife or scalpel and then using a wire (an unbent big paper clip works well) insert the wire first cranially and then caudally to destroy the nerves.
4. Place the euthanized frog in a -20o C freezer.12

References

1. Guidelines for Egg and Oocyte Harvesting in Xenopus laevis. NIH. ARAC. 2005.
2. Godfrey, EW and Sanders, GE. Effect of Water Hardness on Oocyte Quality and Embryo Development in the African Clawed Frog (Xenopus laevis). April 2004. Comparative Medicine 54(2):170-175.
3. Green, SL. Factors Affecting Oogenesis in the South African Clawed Frog (Xenopus laevis). Overview. August 2002. Comparative Medicine 52(4): 307-312.
4. Elsner, H-A. et al. Poor Quality of Oocytes from Xenopus laevis Used in Laboratory Experiments: Prevention by Use of Antiseptic Surgical Technique and Antibiotic Supplementation. April 2000. Comparative Medicine 50(2):206-211.
6. Tuttle AD et al. Evaluation of the Gross and Histologic Reactions to Five Commonly Used Suture Materials in the Skin of the Clawed Frog (Xenopus laevis). Nov 2006. JAALAS 45(6): 22-26.
7. Guidelines for harvesting frog oocytes. Johns Hopkins University IACUC Guidelines.
8. Collection of Xenopus Oocytes. University of California at San Francisco IACUC Guidelines.
9. O’Rourke, Dorcas, DVM, DACLAM, Amphibian Expert. Personal communication.
10. Use of Amphibians in the Laboratory, Research or Classroom setting. 2007. In ILAR Journal 48(3).
11. AVMA Panel on Euthanasia.
12. Thanks to Dr. Karen Symes, BUMC, Department of Biochemistry, and Dr. Isabel