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XENOPUS FROG SURGERY FOR OOCYTE REMOVAL
Purpose
Amphibian oocytes are used for studies in molecular biology, embryology and
biochemistry. Stage I-VI oocytes are obtained by surgical laparotomy. Multiple
surgeries on a single animal may be justified considering the reduction in the total
number of animals used over the long term. However, the total number of animals
used must be considered relative to the pain or distress experienced by an individual
animal. The total number of laparotomies should be limited and will depend on the condition
of the animal and quality of the oocytes as well as the life span of the animal and the
duration of egg production. Up to five recovery surgeries (Sixth surgery is terminal) per animal are acceptable. Adequate recovery time must be allowed between laparotomies. The investigator
can alternate oocyte collection between left and right ovaries and consider rotation
of frogs so that the interval between surgeries in any individual is maximized.
Recovery time of less than one month should have prior IACUC.
Guidelines for xenopus Oocyte Collection
1. Surgery Area
• Although a dedicated facility is not necessary, the surgical area should be a dedicated space that is easily sanitized, uncluttered and away from the flow of traffic to minimize contamination from other laboratory activities.
• The work surface must be thoroughly cleaned with an approved hard surface disinfectant (e.g. Clidox®, 10% sodium hypochlorite,70% alcohol) prior to and after each procedure.
2. Preparation of the surgical instruments:
• Sterilization methods include steam, ethylene oxide, chemical sterilants and dry heat. For a small number of instruments a glass bead sterilizer can be used.
• If 70% alcohol is used to disinfect instruments between animals, they must be immersed for at least 30 minutes and thoroughly rinsed with sterile water or air dried.
3. Preparation of the animal:
• Frogs are anaesthetized by immersion in a 0.15% solution of tricaine methane sulfonate (MS222).
• Sodium bicarbonate (NaHCO3) must be used to buffer the solution to a neutral pH of 7.0 because the unbuffered MS222 is acidic and poorly absorbed, resulting in a prolonged induction time.
• A fresh solution of MS222 must be made daily.
4. Preparation of the surgical site:
• Surgical scrub solutions are not appropriate for use in amphibians due to the nature of their semi permeable skin.
5. Preparation of the surgeon:
• Wash hands prior to procedure with a surgical scrub solution .
• Wear sterile gloves. Change gloves between animals.
• Recommended attire is a clean, designated labcoat.
6. Surgical procedure:
• Place the frog in dorsal recumbency.
• A 1-1.5 cm incision is made in the abdomen, lateral to the midline.
• A piece of ovary containing several dozen oocytes is resected.
• A single absorbable suture is used to appose the ovarian tissue and control any haemorrhaging.
• The skin must be kept moist throughout the surgery using sterile physiologic saline.
• After ensuring that there is no haemorrhaging from the surgical site, the abdominal muscles and skin layers can be sutured together as a single layer or in two separate layers. • Skin sutures must be non absorbable and removed 10-14 days post operatively.
• Recovery from the anaesthesia can take up to an hour. To hasten the recovery process, rinse the frog with fresh water, placing it in shallow water as it will drown if unconscious. During this time the frogs are checked for general appearance, muscle tone and mobility every 15 minutes.
• Thereafter, the frogs are inspected twice a week and any frogs with poorly healing incisions are euthanized.
7.
Intra-operative monitoring:
• Anaesthesia will usually last between 10-20 minutes. Anaesthetized frogs lose muscle tone and become inactive. • Monitor the animal regularly, at least every 10 minutes during the procedure and until the animal is fully ambulatory. • Anaesthetic depth parameters:
Respiratory rate: regular and relaxed
Reflexes: absent withdrawal reflex
External stimuli: no response
• Analgesic depth parameter:
Withdrawal reflex: absent. Reassess every 10-15 minutes.
• For longer procedures, immerse the frog in a weaker solution of MS222 or drip the dilute solution directly onto the skin. Hypothermia alone is inadequate for maintaining a surgical plane of anaesthesia.
Template
Frogs are anesthetized by immersion in .15% MS222 sodium bicarbonate buffered solution.The frog is placed in dorsal recumbency. A 1-1.5 cm incision is made in the abdomen, lateral to the midline. A piece of ovary containing several dozen oocytes is resected. A single absorbable suture is used to appose the ovarian tissue and control any haemorrhaging. The skin is kept moist throughout the surgery using sterile physiologic saline. After ensuring that there is no haemorrhaging from the surgical site, the abdominal muscles and skin layers are sutured together as a single layer using non absorbable sutures. Sutures are removed 10-14 days post operatively. Animals are recovered in a separate tank. The frong is rinsed with fresh water and placed in shallow water to hasten the recovery process. During the recovery period frogs are checked for general appearance, muscle tone and mobility every 15 minutes.
References
Report of the Subcommittee on Amphibian Standards. ILAR, National Research council. National Academy of Sciences , Washington D.C., 1974.
Hitt, Nancy D.: "Basics of Amphibian Medicine". Presented at 44th Annual AALAS Meeting, Nashville, TN., Nov. 18, 1993.
Goodwin, Bradford S.: "Amphibians in the Lab". Presented at 44th Annual AALAS Meeting, Nashville, Tenn., Nov. 18, 1993.
McGill University Research Grants Office Standard Operating Procedures: http://www.mcgill.ca/rgo/animal/procedures
Office of Animal Care and Use ARAC Guidelines: http://oacu.od.nih.gov/ARAC/index.htm |
