Xenopus Hormone Induced Egg Collection

Introduction

Boston University is committed to observing Federal Guidelines and AAALAC International Guidelines for Humane Care and Use of Animals.

Xenopus eggs are used for studies in molecular biology, embryology and biochemistry and for a variety of experiments; however, the quality of the eggs can vary greatly from animal to animal. Thus, the replication of experiments can be improved if the eggs are from a consistently good producer. Please see IACUC Policy for Minimum Requirements for Housing Xenopus laevis. Females can be induced to lay eggs using hormone injections. This policy describes approved methods for obtaining eggs from the African Clawed Frog (Xenopus laevis).

I. POLICIES FOR XENOPUS EGG HARVEST

A. Personnel performing procedures must be properly trained.

B. A system to identify frogs/tanks must be in place.

C. As long as frogs remain healthy they may be used repeatedly.

D. Frogs can be induced to lay eggs repeatedly for several years, but rest periods of at least four (4) months between ovulations are required.

E. Postprocedure frogs must be monitored daily.

F. Records must be maintained to document 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 a clean pair of non-textured gloves to remove the frog from the tank.

C. Inject the hormone and return the frog to a designated tank for egg laying.

III. HORMONE ADMINISTRATION AND EGG COLLECTION

A. Preparation of hormones
Hormones are diluted in double distilled sterilized water at a concentration of 1-2 U/microliter. Recommended injection volumes are 0.25 – 0.5 ml into the dorsal lymph sac.(4)

B. Females can be induced to lay eggs by dorsal lymph sac injection of 100 IU of pregnant mare serum gonadotropin (PMSG) followed 24 -48 hours later by 500 – 800 IU of human chorionic gonadotropin (HCG).

C. Alternatively females are injected with HCG, 500 – 800 IU into the dorsal lymph sac. Twelve hours later females should begin laying eggs.

D. Eggs are obtained either by allowing the female to lay the eggs in the tank water or by gently massaging the abdomen and sides of the frogs. This procedure is also called “egg stripping”. “When properly done by experienced personnel female Xenopus are not harmed by egg stripping and can be used again after a recovery period of 4-6 months.”(1,4)

E. Anesthesia is not required when female either lays the eggs in the tank or for egg stripping.

F. Post-procedural monitoring of frogs after hormone induced-egg collection include note of swimming, behavior, loss of skin, redness or white skin, appetite and any whole body edema as may occur due to ovarian hyperstimulation.(2,4)

IV. IDENTIFICATION OF FROGS AT THE TIME OF EGG COLLECTION.

Acceptable methods of identification include tagging the frog with colored beads or tattooing the animal. Microchips are acceptable as well. Alternatively, females may be identified by housing them in a designated tank.

V. POST-PROCEDURAL CARE

A. Have a recovery tank prepared before the start of the procedure.

B. Place the frogs in this designated tank after the procedure so that it/they can be monitored more easily.

C. Monitor frogs daily for a minimum of 48 hours for well-being and appetite and then every weekday for two weeks. Contact the Supervisor or PI or LASC/LACF Veterinary Services if there are any health concerns.

VI . DOCUMENTATION AND RECORDS

All procedures and care must be documented. Records must include all treatments (including doses and routes); animal ID or tank number, protocol number, post-procedure monitoring information, complications, date and method of euthanasia.

This also includes:

A. Date and time of hormone injection

B. Date and time of egg collection and applicable findings.

C. Post-procedure observations days 1 & 2 after procedure (e.g. activity level, condition).

D. 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. For preparation of MS-222 see IACUC Policy for Xenopus Surgical Oocyte Harvest.

B. Euthanize the animal by injecting 1 ml of 5% buffered MS-222 into the dorsal lymph sac 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 -20°C freezer.(3)

References

1. Guidelines for Egg and Oocyte Harvesting in Xenopus laevis. NIH. ARAC. 2005.

2. Green, SL et al. Ovarian Hyperstimulation Syndrome in Gonadotrophin-treated Laboratory South African Clawed Frogs (Xenopus laevis). May 2007. JAALAS 46(3):64-67.

3. AVMA Panel on Euthanasia.

4.Thanks to Dr. Karen Symes, BUMC, Department of Biochemistry, and Dr. Isabel Dominguez, Department of Medicine, for review comments and expert advise.

Additional Resources

  • 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.
  • Green, SL. Factors Affecting Oogenesis in the South African Clawed Frog (Xenopus laevis). Overview. August 2002. Comparative Medicine 52(4): 307-312.
  • Guidelines for harvesting frog oocytes. Johns Hopkins University IACUC Guidelines.
  • Collection of Xenopus Oocytes. University of California at San Francisco IACUC Guidelines.
  • Use of Amphibians in the Laboratory, Research or Classroom setting. 2007. In ILAR Journal 48(3).
BU IACUC Approved September 2009