The African clawed frog, Xenopus laevis, used at Boston University, is housed either centrally or in satellite facilities. The husbandry and care is performed either by LACF animal care staff in the centralized aquatic facility or by investigators or research staff in the satellite facilities. These guidelines describe minimum requirements for care.
I. WATER SYSTEM
Either static or recirculating water is acceptable.
II. SUPPLY TANK(S)
A. Supply water
With proper conditioning, either city tap water or distilled water is acceptable.
B. Conditioning of water
1. Dechlorinate city drinking water (tap water) by adding an aquarium dechlorination product according to manufacturer’s directions. Boston City water contains chloramines and must be conditioned by using Amquel or a similar product.(9) Chlorine as well as chloramines may be removed by using a Amquel+.(8) Distilled water does not require removal of any products; only addition of salts plus pH adjustment and temperature check as described below.
2. Add aquarium salts according to manufacturer’s directions.
3. Water hardness (Ca++ and Mg++ content) should be adjusted to 200-400 ppm (mg/l).(1)
4. Conductivity is recommended to be in the range of 500 – 2,000 microSiemens (µS).(1)
5. Check pH. It must be in the range 6.5 – 7.
a. If pH is too acidic add dilute NaHCO3. Recheck pH.
b. If pH is too basic add dilute sodium biphosphate. Recheck pH.
6. Check temperature. Room temperature is recommended. The temperature range must not fluctuate more than a few degrees Centigrade. Xenopus may be housed successfully at a constant temperature within the range of 16-22°C.
III. HOUSING TANK(S)
A. Housing tanks may be different sizes but must hold at least 1 gallon of water.
B. Maximum housing density is 4 liter (one gallon) of water per adult frog (over 100 g). For smaller frogs the housing density can be higher.
C. The recommended water depth is 4 -8 inches.
D. An air bubbler may be placed in the tank for oxygenation.
E. Housing of baby Xenopus (froglets ½ inch long) may be done in Tupperware type containers with about 1 quart (liter) of water.
F. Cover the top with a mesh cover or lids with air holes to keep frogs from jumping out.
G. Xenopus prefer a dark environment and are stressed by too much light. A darker area is better for the frogs and keeps them calm. The tanks may be kept in a darker area of the room and on a dark counter top.
H. Set the room light cycle to 12 hours light/12 hours dark. A timer is mandatory for housing animals.
I. Enrichment: One or two (or more, depending on size of tank) plexiglas tubes or PVC pipes with 3-4 inch diameter and about 5-6 inches long with polished edges may be placed in the tank to provide environmental enrichment and hiding places for the frogs. Clay flower pots with polished edges may also be used. Any environmental enrichment device must to be smooth on all edges to prevent abrasion of the skin of the animals and must be sanitizable as described in Section VIII.
IV. QUARANTINE TANK(S)
Quarantine tanks must be set up the same as housing tanks and new frogs are housed there for at least two (2) days after arrival for observation after receiving.
V. RECOVERY TANK(S)
Placing frogs in recovery tanks after procedures is strongly recommended. These should be set up well ahead of time prior to use. Newly used frogs are housed there for at least two days for observation after procedures. Alternatively, recently used frogs may be housed together with others provided that they are clearly identified and checked daily after the surgery or other experimental procedure. Frogs used for egg harvest must be housed in a designated tank separated from those used for oocyte surgical harvest or other experimental procedures after they have been administered hormones. See IACUC Policy for Xenopus laevis Hormone-induced Egg Collection.
A. Frogs may be fed commercially prepared chow, such as NASCO frog brittle.(3) Alternatively they may be fed 5LP3 Frog Diet (Scott’s Distributing).(4) Other feed may be considered as recommended by the vendor.(3,8) The amount of feed given should be estimated so that most of the feed is eaten during the first 15 minutes. If all feed is finished after 15 minutes, additional feed should be given. Frogs must be fed as much as they will eat during the first 30 minutes, taking care that they do not overstuff themselves, after which time any remaining food is removed.
B. The tank water is replaced 3-4 hours after feeding (time for the frogs to move their bowels) and no later than the morning of the next day if the frogs are fed in the afternoon or early evening.
C. Frogs may be fed two times or three times each week. Three times is recommended especially if oocytes are harvested regularly (every month) and the frogs are kept at room temperature (20 -22°C).(2,4,6) At this temperature their metabolism will be going at a higher rate and likewise, their ova production will be higher as well, so they will need more calories.
D. Feed time is a good time to observe the frogs for health checks, as sick frogs will not eat well or be interested in the food, whereas healthy frogs are very active feeders.
E. Feed Storage:
1. Chow must be refrigerated when not in use.
2. Freezing chow is recommended by the manufacturer for long-time preservation.
VII. CHANGE OF WATER
A. Water is changed each time after the frogs are fed.
B. The water may be changed as soon as two hours after feeding or as late as the morning of the following day, but not later than 16 hours after feeding. Changing the same day as frogs are fed is recommended to avoid bacterial contamination of the water. However, changing too soon may cause the frogs to regurgitate all of their stomach contents.
C. Changing the water may be accomplished one of two ways.
1. A new housing tank may be prepared and the frogs carefully transferred to the new tank using either a dedicated net or clean, smooth, powder-free gloves.
2. Any leftover food is removed from the housing tank with a small net, the housing tank is drained leaving a 1-2 inches of water and clean, conditioned water immediately siphoned into the tank.
VIII. CLEANING OF DIRTY TANKS AND INSERTS
A. Frequency of sanitation: Tanks may be sanitized after each feeding and not less than every two weeks. Various authorities differ on recommendations but frequent cleaning is recommended.(6,7,8)
B. Tanks may be sanitized by hand or put through dish washer or cage washer if available. If the tank is made of plastic be sure not to scratch the surface, since surface imperfections could harbor bacteria.
C. Use of detergents or soaps for cleaning tanks is NOT recommended for aquatic species including Xenopus. If needed, after cleaning with hot water, tanks may be disinfected using dilute bleach (1:32 dilution) followed by 70% alcohol followed by a thorough rinse.(7) UV light may also be used for sterilization after tank has been cleaned. Dilute white vinegar may be used as a descaler and to sanitize the tanks.
D. Use as hot water as possible and rinse thoroughly with distilled water. This thorough rinsing is necessary to assure that the frogs are not exposed to any residual sanitizing or descaling solution.
E. Disinfection of sanitized tanks with dilute bleach solution followed by very thorough rinsing is recommended on a regular basis, and at least between shipments, i.e. before using the tank for newly arrived frogs.
IX. DAILY HEALTH CHECKS AND HEALTH MONITORING
A. Check frogs every day. Note findings on the tank or room health sheet.
B. Note any sick or dead frogs on the sheet. Dead frogs must be removed from the tank immediately and reported to Supervisor or PI and LASC veterinary staff or LACF Director.
C. Check the water temperature.
D. Check pH BEFORE water is changed (low or high pH indicates that husbandry procedures are inadequate and must be changed since this may result in unhealthy frogs) – Also, BEFORE placing frogs in the new water check its pH as described under Section II.B.
X. LIGHT CYCLE
A. 12 hours ON 12 hours OFF is recommended. Satellite facilities must be equipped with a room light timer, so as to permit the cycle to be in effect even if no staff is present.
B. As required by the Guide for the Care and Use of Laboratory Animals,light timers must be monitored for accuracy and this procedure documented semi-annually.
XI. PROBLEM SOLVING –CARE OF SICK ANIMALS
A. A sudden change in any of the following parameters is detrimental to the frogs:
2. Salt concentration/ionic strength.
5. Increase in ammonia or nitrite. Rises in ammonia can be detected by measuring pH.
B. Consult with the LASC veterinary staff or the LACF Director.
C. If an infectious disease process is suspected, the following treatments may be prescribed: The LASC veterinary staff must be consulted prior to instituting any of these treatments.
1. Preparing a tank with higher salt concentration, 100 mM, and placing the affected frogs in it for 24 -48 hours may arrest the infection.
2. Cooling the water to 15-17°C.
3. High salt AND chilled water for a limited period of time (24 -48 hours).
4. Preparing a tank with oxytetracycline in the water may clear up an infection.
Conditioned tank water may be prepared as follows:
a) 100 mg/liter oxytetracycline for one hour.(5)
b) 125 mg/liter oxytetracycline for 12-24 hours and up to 3 days.(5)
XII. REQUIRED EQUIPMENT
A. Small nets are required. If a net is used for more than one tank, disinfection between tanks is advisable.
B. Clean, non-textured, powder-free gloves must be worn to protect the mucous layer of the frog skin when handled.
C. pH paper or a pH meter or liquid pH kit.
D. A kit to measure ammonia, nitrite and nitrate is recommended to have available if there are complications. Recirculating water systems may do this automatically using built-in equipment.
E. A temperature paper strip or thermometer to measure water temperature is required. Remember to disinfect the thermometer between tanks with 70% EtOH if used for more than one tank.
A. Boxes of newly arrived frogs are partially opened ASAP to assure safe arrival of the animals; however, they are left in packing material for 2 hours while the frogs are acclimating making sure that they cannot escape.
B. During this time the frogs are allowed to reach room temperature. They may be either colder or warmer on arrival.
C. Before placing frogs in the quarantine tank(s) they are rinsed with the conditioned tank water and inspected for overall health. Any problems are reported to the PI and LASC veterinary staff or LACF Director.
D. Frogs are fed the next day after arrival.
E. An acclimation period of 3 days, 72 hours is required before survival experimental use.
XIV. VERMIN CONTROL
A vermin control program must be in place. This may be done by the BU contractor responsible for vermin control.
A. A room sheet is required to document care of frogs.
1. Date of feeding, water change and daily health checks.
2. Conditioning of water and parameters measured.
3. Each entry must be signed or initialed to indicate who did the work and when.
B. Each tank or rack must be identified with PI and IACUC protocol number.
On a tag on each tank the following must be documented:
1. Number of frogs in each tank updated as frogs are added or removed
2. Vendor and date of arrival
1. 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.
2. Green, SL. Factors Affecting Oogenesis in the South African Clawed Frog (Xenopus laevis). Overview. August 2002. Comparative Medicine 52(4): 307-312.
4. MIT-DCM: Description of Husbandry Procedures for South African clawed frogs Updated 4/2007.
5. Formulary for Amphibians IN Use of Amphibians in the Laboratory, Research Classroom setting. 2007.ILAR Journal 48(3).
6. Thanks to Dr. Isabel Dominguez, Department of Medicine, and Dr. Karen Symes, Department of Biochemistry, BUMC, and Beverly Keniston, BU CRC, for review comments and expert advise.
7. AALAS Learning Library.
- Use of Amphibians in the Laboratory, Research or Classroom setting. 2007. In ILAR Journal 48(3).
- White-James, J. et al Alternative housing for Xenopus laevis. April, 2008, Lab Animal 37 (4):161-163.
- MIT-DCM: Care Guidelines for Frogs