National Bio-Resource Project Tropical Clawed Frog

Rearing Methods for Xenopus tropicalis

Keiko Kashiwagi & Akihiko Kashiwagi
Institute for Amphibian Biology, Graduate School of ScienceHiroshima University
2015.3.6

Rearing methods for Xenopus tropicalis
Keiko Kashiwagi and Akihiko Kashiwagi
2015.3.6

In order to prevent bacterial and other types of contamination, always wash both hands
thoroughly with soap and keep them clean before starting care. The optimum watertemperature is 25–27°C for tadpoles and 24–26°C for frogs; therefore control of room
temperature is also required to maintain water temperature. If it is not possible tocontrol room temperature, you can use any commercially available heating system towarm the water. Aeration is not required for either tadpoles or frogs.
Below is a general description of the basic of methods. Of course local water quality,temperature, and hours of daylight may vary considerably, so you will need to develop
your own specific protocol.
[Preparation](1) Rearing water
Attention needs to be paid to water quality when tap water is used, because chlorine containedin the water may have an adverse impact on tadpoles and frogs. To avoid such adverse impact,
put tap water in a polyethylene bucket and ① leave it for 2–3 days at room temperature,or ② add one sodium thiosulfate crystal (one grain / 20 L water). For ②, the water can be used
as rearing water immediately after the crystal has dissolved and disappeared. If the temperatureof the water is below optimum, adjust it by adding amounts of boiling water. Pay carefulattention to the temperature because both tadpoles and adults of X. tropicalis are extremely
vulnerable to low temperatures.
(2) Tools① Polyethylene bucket② Rearing container (glass or plastic)
There is a great chance of contamination with chemical substances used during themanufacturing processes when new commercially available buckets and containers are used;attention needs to be paid to this issue. First fill the buckets and containers with tap water andleave them for two whole days and nights. Discard the water. Briefly wash the inner surface

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of the buckets and containers with hot water, and then wash the containers and lids with tap
water thoroughly before use.

③ Glass Petri dishes
Soak in tap water for a whole day and night and wash with running water before use.

④ Plastic beaker with a handle
Wash with running water before use.

⑤ Pure coarse sand, 10 kg (PL-10), GEX 50203 (GEX, Japan).
Wash thoroughly with water before use.

⑥ Sponge scrubbers (ones that do not scratch the containers)⑦ Net screened with gauze (hand made)⑧ Droppers or pipettes(3) Gonadotropin
Dissolve commercially available hCG (Chorionic gonadotropin, human, SIGMA, USA) into
Ringer's solution for adult frogs. Gonadotropin for animals (ASKA Pharmaceutical, Japan) andPuberogen for animals (Nippon Zenyaku Kogyo, Japan) may also be used.
(4) Feed
For frogs: Product name, “Floating feed for eels, for finishing” (Marubeni Nisshin Feed, Japan;
distributor, Hiroshima Laboratory Animal Center, Japan) or salmon pellets (Oriental Kobo,Tokyo; distributor, Hiroshima Laboratory Animal Center, Japan) or crickets (IAB)
For young frogs: Feed for frogs, XL-2 (Oriental Yeast; distributor, Hiroshima Laboratory
Animal Center, Japan)
For tadpoles: SERA Micron powder food (Sera Heinsberg, Germany; distributor, Hiroshima
Laboratory Animal Center, Japan)[Rearing methods](1) Tadpoles① Fertilized eggs are obtained by injecting human chorionic gonadotropin (hCG) into thedorsal lymph sac. First 10–20 U of hCG is injected to adult males and females (priming),
followed by 100 U 20–24 hours after the first injection (boosting). After boosting, place one
male ―female pair in a container containing 25°C dechlorinated water to a depth of 5–10
cm. Fertilized eggs will be available within 4–6 hours. The use of a false bottom isrecommended to allow eggs to sink beyond the reach of the adult frogs. Aggregation of eggs
can be avoided by adding salt to the water at a concentration of 0.05%. After completion ofegg laying, frogs are removed from the container, and change to new rearing water at this2

point is recommended . If frogs do not proceed with mating behavior, this may often beinitiated by injecting an additional 100 U of hCG into both male and female. Developmental
stages are as described in Nieuwkoop & Faber (1956).

② When tadpoles developed from the fertilized eggs reach the end of the hatchling stage,transfer only tadpoles with normal morphology. Give a small amount of SERA Micron forfood when feeding behavior is exhibited.

③ If tadpoles have a large appetite, feed them every morning and evening.
④ Water changes every other day should be fine. When changing water, use a dropper for
young tadpoles or a net screened with gauze for larger tadpoles.

⑤ Size differences will gradually become obvious when tadpoles develop. Avoid overcrowded
conditions in the containers. Select larger tadpoles and transfer them into separate containers(2–3 tadpoles/L). At one month after fertilization some of the larger tadpoles will havealready undergone metamorphosis.
(2) Frogs① 2 or 3 times a week should be adequate for feeding and changing water.
② Begin with 5 grains of feed per frog. Increase the amount of feed by several grains as frogs
grow in size and devour all food within 30 minutes.

③ Change water 2~3 hours after each feeding. Discard old water and add new water using abeaker with handle.
Note ──1) Container bottoms, sides, and corners tend to become dirty over time. Remove slimyaccumulations with the sponge scrubber.
2) After changing water, discard any left over water remaining in the bucket and prepare newwater for the next water change.
3) For frogs, two or three double handfuls of pure coarse sand at the bottom of rearing
containers is helpful for water clarification.
4) Any unhealthy frogs should be immediately transferred to a separate water container with
0.3% salt added to the rearing water. In the case of tadpoles, remove and discard any sickly
individuals immediately upon discovery to avoid adverse effects on healthy tadpoles.

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Manipulation of Xenopus tropicalis eggs and embryos
──In vitro fertilization of eggs and manipulation of embryos──Keiko Kashiwagi, Hideki Hanada and Akihiko Kashiwagi
Reagents for in vitro fertilization and dejellying

・Crystal of sodium thiosulfate (Daito Chemical Co., Ltd., Japan)
・Human chorionic gonadotropin (Sigma)
・Ringer’s solution (amphibian adults: NaCl 0.65g, KCl 0.025g, CaCl2 0.03g, NaHCO3 0.02g
per 100ml distilled water)・MS222 (Sigma)
・1×MMR (Marc’ Modified Ringer’s) solution : 100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 2mM CaCl2 and 5 mM Na-HEPES, pH 7.5

・Leibovitz 15 medium (Sigma)
・Cysteine (Sigma)
・Ficoll (Sigma)
・Gentamycin (Sigma)
Maintenance equipment

・Plastic vessels
・Glass vessels
・Glass Petri dishes (20 ~25 centimeters in diameter)
・Ophthalmologists’ scissors
・Forceps
・Glass Pasteur pipettes with rubber caps
When purchasing plastic or glass vessels to be used in raising frogs, tadpoles and embryos, theymust be washed thoroughly with a soft sponge and soap to remove any chemical substancesused in the manufacturing process adhering to inside surfaces. Endocrine disrupting substances
will have detrimental effects on development and growth. Ophthalmologists’ scissors, forceps,
glass Pasteur pipettes and rubber caps should also be washed before each use.

・Disposable syringes (10 cc) with needles (23 Gauge) for drawing up Ringer’s solution andsyringes (1 cc) with needles (27 Gauge) for hypodermic injection

・9.0 cm plastic Petri dishes for use in artificial fertilization

4・6.0 cm plastic Petri dishes (Falcon’s cell incubation dishes) for use in dejellying
Food for raising tadpoles or frogs

・SERA Micron (Sera Heinsberg, Germany)・Salmon pellets (Oriental Kobo, Tokyo)
・Two-spotted crickets Gryllus bimaculatus

Preparation for in vitro fertilization
X. tropicalis adults are raised in 24℃ to 26℃ chlorine (Cl)-free tap water (one crystal ofsodium thiosulfate/20 L water).
Ovulation and spermiation are induced by two separate injections of human chorionicgonadotropin into the dorsal lymph sac. Adult female and male frogs initially receive 10 units ofHCG in Ringer’s solution, followed approximately 20 hours later by a second injection of 100
units in the same Ringer’s solution. Injected frogs must be maintained in a dark quiet
environment by covering trays with a black cloth. Frogs should not be kept in temperature units
because electrical vibration will be detrimental to their egg laying.
Approximately 3～4 hours after the second injection, the egg mass from the uterus of theovulating female is stripped into the 9.0 cm plastic Petri dishes and immediately mixed with afresh sperm suspension using a wide-bore glass Pasteur pipette. The sperm suspension isprepared as follows. Males are deeply anesthetized in 0.15% MS222 in rearing water. The testes
are surgically removed, and fat bodies and blood are removed by rolling on a fresh paper towel.
Next the testes are placed in Leibovitz (L)15 medium and crushed into small pieces with forceps,and mobility is ascertained before use. When they are not immediately used, the testes can be
stored in L15 ＋ 10% calf serum for a few days at 14℃.
Five minutes after being mixed with the sperm suspension, the eggs in the Petri dish areflooded with 0.05 × MMR. Successful fertilization is observed when eggs are oriented with thedark animal pole side up, or so called ‘cortical rotation’. Eggs are maintained at 24～26℃ andobserved at close intervals at the time of the first and second cleavages.
Viable hatched tadpoles at Nieuwkoop and Faber (1956) stage 35～36 are drawn into awide-bore pipette and transferred to glass rearing vessels containing Cl-free water. Temperature
is maintained at 25～27℃ throughout the larval period. Tadpoles are raised on SERA Micron,and frogs on salmon pellets or two-spotted crickets Gryllus bimaculatus. Food levels areadjusted to allow normal tadpole and frog development and maximum growth. Water ischanged every other day.
5Removing jelly coating from eggs
Immediately after the beginning of cortical rotation, eggs are transferred into the 6.0 cm
Falcon’s plastic Petri dishes and can be dejellied using 1.5 % cysteine in 0.1 × MMR, pH 8.0,
and then rinsed carefully 3～5 times with 0.1×MMR. Following this the eggs are rinsed 3 times
with 5% Ficoll and 50 mM gentamycin in 0.1× MMR and loaded into Petri dishes with bottoms
coated in a layer of agarose containing the above solution to be used for transgenic or knockout
microinjection.
REFERENCES
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Kashiwagi, K., Kashiwagi, A., Kurabayashi, A., Hanada, H., Nakajima, K., Okada, M., Takase,
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