Guidelines for Major Invasive Surgery (Oocyte Harvest) in Frogs
Adopted by the University Committee on Animal Resources
The following guidelines were developed by UCAR and
the veterinary staff to assist investigators in using frogs in research
in accordance with Guide for the Care and Use of Laboratory Animals
as well as maximize the quality of oocytes harvested via the surgical
approach. AAALAC cites three references listed below describing
the importance of aseptic technique for major invasive surgery (e.g.
oocyte harvest) in frogs.
1. Multiple survival laparotomies
for oocyte harvesting in frogs must be scientifically justified
in the UCAR protocol.
2. Frogs experiencing laparotomies
must be appropriately anesthetized. The use of hypothermia as an
anesthetic is not approved. Transcutaneous anesthesia via immersion
in a buffered solution of tricaine methane sulfonate (MS-222) is
a common and acceptable anesthetic method in frogs.
MS-222 Anesthetic Protocol:
- 0.5-2 g/liter MS-222 should be buffered with sodium bicarbonate
at 0.42 -1.05 g/liter. (Unbuffered MS-222 solution is irritating
to frog skin and poorly absorbed resulting in a prolonged induction
time).
- Surgical anesthesia is achieved within 10-15 minutes. Depth
of anesthesia is monitored by lack of a righting reflex, slowed
to ceased respiration and loss of response to stimuli.
- After removal of the frog from the anesthetic solution, maintenance
of anesthesia can be achieved by dripping
MS-222 anesthetic solution onto the skin.
- Frogs can be recovered by rinsing with fresh dechlorinated
water and/or placement in container of shallow water.
Signs of recovery should be evident within 15-30 minutes.
3. Survival surgeries must be performed
using modified aseptic technique. This requires the use of
a mask, sterile gloves, sterile instruments and materials (e.g.,
suture) and sterile surgical technique. A sterile prep of the surgical
site is usually not indicated for frogs but may be helpful to remove
gross surface debris. An appropriate sterile prep for frogs
consists of wiping the surgical site with dilute 0.75% chlorhexidine
solution or 0.5% povidone iodine solution. The use of soaps
or scrubs may be toxic to frogs and is not recommended.
4. Frogs experiencing multiple surgeries
must be identified. This can be accomplished by group housing frogs
that have experienced an identical number of procedures, and clearly
labeling of the housing enclosure. Pattern marking is an alternative
identification method which involves recording characteristic skin
patterns on each animal.
5. UCAR allows for a maximum of three
survival laparotomies with euthanasia at the fourth harvest. A maximum
of two surgeries per side is permitted. There should be a period
of at least one month between surgeries. Any exemption must be scientifically
justified and discussed by the Committee.
References:
DeNardo, D. Amphibians as Laboratory Animals. ILAR Journal
1985; 37(4): 173-181.
* Elsner, H-A., Honck, H-H., Willmann, F., Kreienkamp,
H-J., Iglauer, F. Poor Quality of Oocytes from Xenopus laevis Used
in Laboratory Experiments: Prevention by Use of Antiseptic Surgical
technique and Antibiotic Supplementation. Comparative Medicine 2000
April; 50(2): 206-211.
* Green, S.L. Overview: Factors Affecting Oogenesis
in the South African Clawed Frog (Xenopus laevis). Comparative Medicine
2002 August; 52 (4): 307-312.
O’Rourke, D.P., Schultz, W.S. (2002) Biology and Diseases of
Amphibians. In J.G. Fox, B.J. Cohen, F. M. Loew (eds.), Laboratory
Animal Medicine (793-826). New York: Academic Press.
Schaeffer, D. (1997) Anesthesia and Analgesia in Nontraditional Laboratory
Animal Species. In D. F. Kohn, S. K. Wixson, W.J. White,G.J.
Benson, (eds), Laboratory Animal Medicine (337-378). New
York: Academic Press.
* Schultz, T.W., Dawson, D.A. Housing and Husbandry of Xenopus
for Oocyte Production. Lab Animal 2003 February; 32(2): 34-39.
* AAALAC cited
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