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 DOI: 10.1258/002367795780740276

1995 29: 152Lab AnimG. Hilken, J. Dimigen and F. Iglauer

under different laboratory rearing conditions.Xenopus laevisGrowth of   

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- Apr 1, 1995Version of Record >>

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Growth of Xenopus laevis under differentlaboratory rearing conditions.

G. Hilken, J. Dimigen & F. Iglauerlaboratory Animal Facilities of the University Hospital Eppendorf. Hamburg. Germany

SummarySince the European frogs (Ran a spp.) have fallen under the German endangered speciesregulation, Xenopus laevis (South African Clawed Frog) is being used increasingly inanimal research and education. Optimal growth rates and homogeneity of groups havenot necessarily been attained as little statistical analysis of growth data has beenavailable. Following metamorphosis, an as yet not understood variability of growth isexhibited by X. laevis. In this study the effect of environmental factors on this variabilitywas determined. Feeding, population density, background colouring, water temperature,the availability of hiding places, water level and water care were each examinedseparately _Development of body weight and body length were recorded. A definitecorrelation between the feeding programme, population density, cover and water care onthe one hand and growth on the other were seen. Of lesser importance were watertemperature, water level and background colouring. The observed variability of growth isassumed to also be of ethological origin.

Keywords Xenopus laevisj growth developmenti maintenancei feedingi population densitYicoverj water carei water levelj water temperaturej background colouring

Since placement on the German list ofendangered species (Bundesarten-schutzverordnung 1989, BundesgesetzblattNr.44) European frogs (Rana spp.) areincreasingly being replaced by the SouthAfrican Clawed Frog (Xenopus laevisDautinj in research and education.Clawed Frogs are aquatic animals,surviving on land no more than 3 h IOchse1948). An aquarium without a landcomponent is adequate, making their careless costly than that of terrestrial-aquaticRanidae. This, together with the easy, non-seasonal breeding, makes the South AfricanClawed Frog suitable for use as laboratoryanimal.Years ago, Xenopus laevis played a

special role in pregnancy diagnosis (Shapiro& Zwarenstein 1934). Later, these animalswere used in pharmacological tolerance

Correspondence to: Dr F. Iglauer

Accepted 31 May 1994

testing and for the demonstration of growthregulating factors (Grimm 1951, Grimm1952/53). The embryo toxic effects ofenvironmental pollutants have been testedusing the tadpoles (Dumpert 1983). TodayX. laevis oocytes are used in expressioncloning (Jentsch et a1. 1990). Clawed Frogsare also important as indicators ofradioactive environmental contamination(Giannetti et a1. 1990). During recent yearsthese animals have increasingly been usedfor school instruction (Raether 1978b,c).Kiihler (1987) has shown that Ranidae canbe replaced by Clawed Frogs in manybiomedical experiments.To date, generally accepted guidelines for

the care of Clawed Frogs are rare (Elkan1970, Verhoeff de Fremeryl & Griffin1987). Wu & Gerhart 11991) deplored thefact that no comparative studies have yetbeen published, and that only anecdotalreports exist. Similarly, little statistical

Laboratory Animals (1995) 29, 152-162

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Laboratory rearing of Xenopus Jaevis

evaluation of growth data is available.Therefore adequate optimization of growthrate and group homogeneity was notpossible. Early studies do mention theinfluence of environmental factors on thegrowth of Xenopus tadpoles IGasche 1943,Ochse 1948, Nieuwkoop & Faber 1975).The great variability of growth, especiallyalso following metamorphosis, was onlypointed out by Parker et 01. (1947).This study attempts to analyse some of

the factors responsible for variability indevelopment. Different environmentalconditions were tested in order to optimizedevelopment. The development of weightand length was recorded.

Materials and methods

BreedingAccording to Gasche (1943) and Ochse(1948) egg laying was induced by treatmentwith human chorionic gonadotropin(HCG). In our study breeding animals wereseparated according to sex and given nofood for 3 days prior to hormonaltreatment. On day one, 200 ill HCG(Sigma, FRG) were injected into the dorsallymph sac of the male animals. On day 2,female animals were given the first HCGinjection (200 IU). Six hours later, a secondinjection 1600ill) was given to the female,concomitant with the second injection ofthe males (200 ill).Pairs of animals were then placed in

breeding cages and kept under low lightconditions. Makrolon type IV cages withgrid flooring were used to protect the eggsfrom the parent animals. The eggs

153

(500-2000) were generally passed duringthe night.Larvae were raised using standardized

algae powder (Dose Inc. Mikrozell® J asfood. Only those frogs which completedmetamorphosis early (5 to 7 weeksfollowing egg laying) were studied. For theevaluation of environmental factors youngsiblings at the same stage of the breedingcycle were used to each factor (Table 1).Animals of different weight and length hadto be used. However, these weredistributed equally amongst the groups tobe compared, so that no differences inaverage length or weight existed initially.Ages at the beginning and length ofobservation periods are listed in Table 1.

Environmental conditionsEight frogs (4 males and 4 females) werekept in each tank [Table 2). The tanksmeasured 60.5 x 41 cm at the bottom andwere made of white polyethylene (RBBInc., Item nr. 647034-30). For each frog300 cm2 of bottom surface area wereavailable. Hiding places were not provided.With a water level of 10 cm and a totaltank water volume of approx. 25 I, 3.11 ofwater were allowed for each frog. The pHof the water was approx. 7 1±0.2). Thewater temperature was maintained withthermostatic heaters (Jager Inc. 100 watt) ata constant 20°C (± l°q. The frogs werefed exclusively with Tubifex sp. Thisoligochaete (Annelida) is commonly used aslive food in aquatic cultures. Food wasavailable ad lib to the frogs. Previous trialshad shown this to be the most acceptablefood. Once daily half the water wassiphoned off to remove faecal material and

Table 1 Age of frogs and length of observation periods

Length ofEnvironmental factors Ova obtained Starting observation (month)

Water temperature Dec. 1990 28.3.91 5Water level Dec. 1990 2.3.91 8Background colouring Dec. 1990 23.4.91 6Cover (hiding place) Apr. 1991 1.12.91 7Population density Jan. 1991 1.6.91 9Water care Apr. 1991 15.12.91 6Feeding Dec. 1990 2.3.91 9

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154 Hilken, Dimigen & Iglauer

Table 2 Rearing conditions

Test groupsl Water Background PopulationValue temperature Water level colouring (over density Water care Feeding

Water 19°( 10cm White Not available 8 Daily Tubifex sp.temperature 22°( 10cm White Not available 8 Daily Tubifex sp.

24°( 10cm White Not available 8 Daily Tubifex sp.

Water level 200( 5cm White Not available 8 Daily Tubifex sp.200( 10cm White Not available 8 Daily Tubifex sp.200( 20cm White Not available 8 Daily Tubifex sp.

Background 200( 10cm White Not available 8 Daily Tubifex sp.colouring 200( 10 cm Grey Not available 8 Daily Tubifex sp.

200( 10 cm Black Not available 8 Daily Tubifex sp.

(over 200( 10cm White Available 8 Daily Tubifex sp.200( 10cm White Not available 8 Daily Tubifex sp.

Population 200( 20cm Green Not available 22 Daily Tubifex sp.density 200e 20cm Green Not available 37 Daily Tubifex sp.

Water care 200( 10cm White Not available 8 Daily Tubifex sp.200( 10cm White Not available 8 Weekly Tubifex sp.200( 10cm White Not available 8 Filtration Tubifex sp.

Feeding 200( 10cm White Not available 8 Daily Bovine heart200( 10cm White Not available 8 Daily Xenopus-food200( 10cm White Not available 8 Daily Mixed feeding200e 10cm White Not available 8 Daily Tubifex sp.

left-over food. The water level wasrestored again with tap water that hadbeen allowed to stand for 3 days. To studythe effects of different environmentalparameters the management describedabove was varied as described under 2.3 a-g(Table 2).

Environmental factors studieda) Water temperature: Frogs were kept at3 different temperatures. One tankwas maintained at 19°C (± 1°C), oneat noc (± 1°C) and one at 24°C(±1°C).

b) Water level: Water levels wereadjusted to 5 cm, 10 cm and 20 cmheight. The water volume available toeach frog was 1.551 in the first, 3.11in the second and 6.21 in the thirdtank.

c) Background colouring: The influenceof background colouring was evaluatedusing white, grey and black tanks.

dJ Cover: In one tank frogs were keptwithout any hiding place. The tank of

the second group contained 3 halvesof earthenware pipe [radius of4 cm). These covered approximatelyone-third of the bottom surfacearea.

e) Population density: Growth developmentwas observed in 2 larger tanks(200 cm x 80 cm x 20 cm) stocked withdifferent numbers of frogs. The firsttank contained 22 frogs (13 females,9 males), the second tank 37 frogs (21females, 16 males). For each frog inthe lower stocked tank 730 cm2 ofbottom surface and approximately 141of water were available. In the tankwith higher population density foreach frog 430 cm2 of bottom surfaceand 8.61 of water were available.During the first 8 months onlyaverage body weights could bedetermined. However, after the 9thmonth all frogs were measured andweighed individually. Only the datafrom this last month could beanalysed biometrically.

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Laboratory rearing of Xenopus laevis 155

Fig 1a-b Water temperature and growth ofXenopus laevis. a} Development of body weight.b} Development of body length.

o 19'C

• 22'C• 24'C

o 19'C

• 22'C

• 24'C

o 5 em• 10 em• 20 em

[month]

5 [month]

4

43

32

2

o

50

40

30

20

10

[g) 30

25

20

15

10

5

00

[em) 8

7

6

5

4

3

2

00

[g] 60

f) Water care: One tank was connectedto a high power filter system IEheimInc; Eheim-Filter, 320 lIh). Filterswere changed as necessary,approximately once a month. Water inthe other 2 tanks was not filtered. Inone of these tanks approximately one-half of the water was changed dailyand food remnants and faecal materialwere siphoned off. In the other tankwater was changed only weekly. Bythat time the water appeared muddy.

g) Feeding: Three groups of frogs wereexclusively fed with only one type offood. The first received non-floatingXenopus pellets (Kahler Inc.j F00200).The second group received bovineheart, minced to a particle size of notgreater than 2 mm. The third groupwas fed only Tubifex sp. Frogs in thefourth group were offered in turnXenopus pellets (3 times weekly),bovine heart (2 times weekly) andTubifex sp. (2 times weekly).To document growth development

all the frogs were measured oncemonthly (tip of mouth to the anusl, aswell as being weighed. The data arepresented as means ± SD. Comparisonsbetween groups were made by onefactor ANOVA. Calculations of pairwisecomparisons were made by Fisher'sPLSD test and Scheffe F-test. Allstatistical tests were carried out usingthe StatView 512+ software package.

Resultso 2 4 8 8 (month]

Fig 2a-b Water level and growth of Xenopuslaevis. a) Development of body weight.b} Development of body length.

a) Water temperature: The development ofbody weight and body length was irregularin this experiment. Until the 4th month ofexperiment, frogs kept in water of 24°Cshowed the greatest increases in weight(Fig. la). This trend was not evident forbody length (Fig. Ib). Frogs kept in waterof 19°C grew slower than the others forthe first four months. However, during thelast month of observation these frogssurpassed those kept in warmer water inbody weight and length. In no month werethe differences between the 3 groupsstatistically significant.

[eml 10

987

6

5

4

3

2

1

00

o 5em• 10 em• 20 em

2 3 4 5 8 7 8 [monthl

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156 Hilken, Dimigen & Iglauer

[g] 40 b) Water level: Significant differences ino white growth between groups were not evidentII grey

30 • bleckduring the 8-month observation period (Fig.2a-b). A difference in the development of

20body length could only be seen after thefirst month. During the following months

10growth was very homogeneous between, aswell as within, the groups.c) Background colouring: Significant

D differences were not seen during the 60 2 3 4 5 6 [month]

months of observation. After the firstmonth, frogs kept in the black tank

[em]developed in body length faster than those

1 D kept in the white tank (Fig. 3b). Frogs kept9 0 white

• grey in the grey tank also grew better than8 • black7 those kept in the white tank during the6 first monthsj however, the differences were5 only small. Variability in body weight and4 length was greatest within the group of3 frogs kept in the white tank and smallest2

within the group kept in the black oneD [Table 3).

0 2 3 4 5 6 [month] d) Cover: During the first month of

Fig 3a-b Background colour and growth of observation frogs kept in the tank withXenopus laevis. a} Development of body weight. hiding places grew better (weight andb) Development of body length length). Starting during the second month

Table 3 Body weight and length in last months of experiment

Bodyweight Bodylength

Environmentalfactor Value Mean [g] SO Min.Max.Coef. Var. Mean [cm] SO Min.Max.Coef. Var.

Water temperature 19°C 16.9 4.4 11.9 23.8 25.9 5.4 0.44.4 6.1 8.222°C 15.4 5 8.7 25.5 32.4 5.3 0.54.5 6.3 10.324°C 16.6 7.7 9.5 30.1 46.1 5.3 0.84.3 6.5 14.8

Water level 5cm 37.4 13.5 15.5 51.7 36.1 6.8 0.95.1 7.7 13.710cm 37.9 13.316 54 35.2 6.8 0.95.1 7.8 13.320cm 34.8 11.616.749.3 33.4 6.7 0.95.2 7.7 13.7

BackgroundcolouringWhite 25.1 9.8 8.8 37.8 39 6 0.84.6 6.8 12.8Grey 26.4 7.3 16.3 36.2 27.5 6.2 0.65.4 6.8 8.9Black 26.7 6.4 19.4 40.2 23.9 6.3 0.45.6 7 6.7

Cover (hiding place) Available 28.4 3.3 23.8 32.3 11.5 6.4 0.36 6.7 4.4Not available 36.3 12.127.3 56.6 33.3 6.7 0.76 7.9 11

Population density 22 per tank 40.6 12.621.9 60.6 30.9 7.3 0.95.8 8.5 12.237 per tank 25.3 6.714 39.9 26.627 6.2 0.64.9 7.4 9.9

Water care Daily 34.9 9.926.3 50.3 28.4 6.8 0.76.2 8 10Weekly 49.7 12.2 36.1 74.3 24.5 7.4 0.96 8.5 11.6Filtration 59.8 19.4 37.8 92.7 32.4 8.2 0.96.8 9.5 11

Feeding Bovineheart 5 1.4 2.8 6.5 28 3.5 0.33 3.8 8.8Xenopus pellets 5.9 2.2 3.5 8.8 37 3.8 0.53.2 4.6 13.8Mixedfeeding 18.8 5.513.2 28 29 5.6 0.55 6.4 8.1Tubifex sp. 31.4 10.7 16.2 47.5 34 6.2 0.75.2 7.1 10.9

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Laboratory rearing of Xenopus laevis 157

o 1 2 3 ••. 5 6 7 e 9 {month}

[al 50

• cove, 0 37 Froga/lank

o no cover so r • 22 Frog&ltlnk

40

30

20

10

7 [month]2o

20

10

30

40

[g] 50

Fig 4a-b Availability of cover and growth ofXenopus laevis. a) Development of body weight.b) Development of body length.

[em] 10

9

8

7

8

5

4

2

1

00

• covero no cover

4 5 7 [month]

Fig 5 Population density and development ofbody weight

o dally• weakly• 1l1tr.tlon

(month]

frogs kept in the tank without any covershowed better growth of weight (Fig. 4a-b).First significant differences were seen inbody weight between the groups from the7th month of observation onwards(ANaVA: P~O.OOOli Fisher's PLSD test:P~O.05i ScheHe F-test: P~O.051.Differences in body length between thegroups were not significant.eJ Population density: During the first

month the frogs kept in the tank with ahigher population density grew faster. Thischanged with the 3rd month of observation(Fig. 51. Differences in growth between thegroups increased with each month. Afterthe 9th month the following differenceswere found: Frogs from the group with 22frogs were significantly heavier and longerthan these from the group with 37 frogsper tank (weight: ANaVA: P~O,OOOliFisher's PLSD test: P~O,Oli Scheffe F-test:P~OJOlj length: ANaVA: P~O,OOOlJFisher's PLSD test: P~OJOli Scheffe F-test:P~O,Oll·f) Water care: The group of frogs kept in

filtered water grew by far the fastest,

o dally• waakly• filtration

8 [month]

Fig 6a-b Water care and growth of Xenopuslaevis. a) Development of body weight.b) Development of body length

followed by the group kept in waterchanged weekly (Fig. 6a-bl. Significantdifferences in increases in body lengthbetween the 3 groups were seen alreadyafter the 2nd month. Clawed Frogs kept infiltered water showed a significantly fasterincrease in body length than frogs kept inwater cleaned daily (ANOVA: P~O,OliFisher's PLSD: P~O.Olj Scheffe F-test:P~O.Ol). Significant differences in thedevelopment of body weight were also seenfrom the 2nd month onwards only between

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158

animals kept in filtered water and thosekept in water cleaned daily (in the lastmonth: ANOVA P~O.Olj Fisher's PLSDtest: P~O.05) (Fig.6a). The variabilitywithin a group was at first highest forfrogs kept in water cleaned daily. Thevariation coefficient was 1/3 higher thanfor the other 2 groups. Later, the variationcoefficient for frogs kept in filteredwater increased and finally surpassedthat of the group kept in unfiltered water(Table 3).g) Feeding: At the end of the first month

of observation differences already existed inthe development of weight and lengthbetween the groups. Clawed Frogs fed onlyTubifex sp. were already significantlylonger and heavier than the others(ANOVA:P~O.005i Fisher's PLSD test:P~O.Olj Scheffe F-test: P~O.OIJ. Thisdifference remained and had increased bythe end of the 9 month observation period[Fig. la-b).

1111 50

Hilken, Dimigen & Iglauer

From the 3rd month, frogs receivingmixed feeding grew significantly betterthan those fed only bovine heart [ANOVA:P~O.OOOlj Fisher's PLSD test: P~O.005iScheffe F-test: P~O.05J, From the 5th monththey also grew better than those fed onlyXenopus pellets (ANOVA:P~O.OOOlj Fisher'sPLSD test: P~O.05j Scheffe F-test: P~O.05J.Between the 5th and the 9th month,

frogs receiving Xenopus pellets were larger(length and weightl than those fed bovineheart, but differences were not significant.Three frogs in the group fed bovine heart

died, one each in the 3rd, 5th and 6thmonth of observation. Two frogs in thegroup fed Xenopus pellets died during the 9months.From the 3rd month onwards the

variation coefficient was lowest in thegroup receiving mixed feeding. Thevariation coefficient of the group fedTubifex sp. was highest (Table 3).

Discussion

Fig 7a-b Feeding and growth of Xenopus laevis.a) Development of body weight. b) Developmentof body length

oo • 5

(monlh)

Factors studiedIn this study of X. laevis, 4 of 7environmental factors examined were seento affect the development of body lengthand weight.a) Water temperature: It was expected

that water temperature would affect thegrowth of poikilothermic (ectothermic)anurans. It was therefore surprising that nodifferences in growth development wereobserved between Clawed Frogs kept at19°C, 22°C or 24°C during the 5-monthobservation period.Delays in growth in tadpoles of X. laevis

kept at low temperature have beendescribed (Ochse 1948). The developmentof tadpoles is arrested at watertemperatures below 15°C. The adults arealso considered to be sensitive to coldwater [Ochse, 1948). This has beenconfirmed by Wu &. Gerhart (1991). Theyfound animals to be stressed attemperatures below 14°C and above 26°C.Our observations showed the vitality ofanimals not to be reduced. We were able toobserve, for example, that animals

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laboratory rearing of Xenopus laevis

continued feeding after a temperature dropto 8°C. Lerch (1948) also describedmaintenance of these frogs at 2-3°Cwithout mortality. He states the optimaltemperature for the species to be 21°C.Andres et al. (1948) found 23°C to beoptimal. Other authors, as for exampleRaether (1978aJand Parker et al. (1947Lalso give water temperatures in this range.How these optimum values were derived,however, is never explained.b) Water level: It was expected that a

higher water level would increase theenergy required for reaching the watersurface, leading in turn to slower growthrates. This hypothesis could not beconfirmed. The surprising degree ofuniformity (weight and length) between thethree groups demonstrates that waterdepth, at least for the range studied(5-20 em), does not affect growth.Until now water level had only been

examined for its effect on the rearing oftadpoles (Ochse 1948). A tank with asmaller surface area and greater waterdepth was shown to be more suitable thana tank holding the same volume of waterbut with a larger surface area and lesswater depth. Ochse (19481was unable toexplain this phenomenon. Parker et al.(1947) described frogs to be restless at lowwater depths. Escape attempts arementioned. This behaviour could beinterpreted as an adaptation to life inseasonal ponds.c) Background colouring: By shifting

chromatophores, X. laevis is capable ofmaking its body colour conform to itsbackground (Bagnara 1976). By the end ofthe 6-month observation period no tankcolour had proved to be significantly morebeneficial than the others. However, fromthe end of the first month onwards, frogskept in the black tank grew fastest, frogskept in the grey tank intermediately, andfrogs kept in the white tank grew slowest.This placing never changed. It can thereforebe postulated that a dark tank colour isbetter than a light one. Grimm (1952) alsomentioned that frogs avoid lightness. Forthe raising of larvae, tank colour should beof no importance [Gasche 1943).

159

d) Cover: It was postulated that theavailability of hiding places would reducestress and promote growth. This hypothesiswas not confirmed.More movement could, indeed, be

observed in the tank without coverage.However these frogs lost their shyness veryquickly and reacted to disturbances laterwith less panic than frogs kept in the tankwith hiding places. Their longer periods ofswimming probably led to a highermetabolic rate. These frogs regularlyconsumed a greater amount of Tubifex sp.than the frogs kept with cover. The largerfood intake might be due to the greateramount of swimming, that lead to anincreased frequency of contact with food. Itis also possible that the frogs without coverwere hungrier, because of their highswimming activity.In the tank with cover, frogs left their

hiding place to feed only after a long time.These frogs never lost their shyness. Whenconsidering the poorer growth of this groupit appears questionable that every Xenopustank should have cover, as recommendedby Grimm (1952).e) Population density: Frogs with more

area available per animal grew significantlyfaster than animals kept in the tank with ahigher population density. Without doubtpopulation density is one of the mostimportant factors affecting the growth of X.laevis.f) Water care: Opinions diverge regarding

the necessity of water changes for themaintenance of X. laevis. Most authorsconsider water changes necessary at a rateof one to 3 times weekly (Parker et al.1947, Andres et 01. 1948, Raether 1978a).Arson [cit. in Parker et al. 1947), on theother hand, considers water changes 3 to 4times yearly sufficient. Goldin (19921describes a continuous inflow of water ashindering development and leading tosymptoms of red-leg disease.Water quality is not high in the natural

habitats of x. laevis. These frogs apparentlylive frequently in sewage treatment pools(Deuchar 1975). We therefore expected thatfiltration of tank water would not have apositive effect on the growth of Clawed Frogs.

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160

The slower development of frogs kept inthe tank with daily water replacementdemonstrates that the short period of dailydisturbance at least outweighs any positiveeffects due to better water quality. Frogsgrew better in the tank with water replacedonly once weekly. Water in this tank wasso contaminated after 7 days that the frogswere hardly visible. According to Deuchar(1975) only little oxygen is absorbed by X.laevis through its skin. The vitality ofthese frogs may, therefore, be hardlyimpaired by highly contaminated waterwith a low oxygen concentration.In contrast, cleaning of the water via a

continuous filtering system affected thegrowth of the frogs definitely positively.We would therefore recommend thismethod of water care.g) Feeding: Avila & Fryre (1978) have

studied the feeding behaviour of X. laevisand showed it to be adapted to aquaticlife, differing from that of terrestrialanurans. For example, hypobranchialpump movements, producing a watercurrent into the oral cavity, are performedby X. laevis. This behaviour has beendescribed by Grimm (1952) as 'sucking-snapping'.Feeding 1 to 2 times weekly is

considered sufficient by some authors(Parker et al. 1947, Ochse 1948, Raether1978a, Wu & Gerhart 1991). HoweverParker et al. (1947) mentioned thenecessity of feeding at least the juvenilefrogs daily to prevent cannibalisticbehaviour. X. laevis takes in any types offood it is capable of ingesting. In captivitymost are fed earthworms, slices of liverand heart, Daphnia sp. and also drypelleted food.In the wild X. laevis lives in seasonable

ponds. The dry periods are spent buriedinto the mud at the pond bottom. Thesefrogs are therefore adapted to long periodsof starvation [Merkle 1990). Nevertheless,from the results obtained in this study,feeding appears to be the factor mostdecisively influencing the growth of thesefrogs. The greatest differences in growthfound in this study were seen between the4 groups with different feeding. The only

Hilken, Dimigen & Iglauer

deaths occurred in 2 of these groups.Several frogs fed exclusively bovine heartor Xenopus pellets died, indicating thesefeeds to be unsuitable as the sole food forgrowing Clawed Frogs. These feeds aloneshould be used only for adult frogs. Inaddition, acceptance of these 2 types offood was poor and a high degree of watercontamination occurred. With these 2 foodtypes a filtering system or some other kindof daily water exchange is necessary.Goldin (1992) reports that newly capturedwild Clawed Frogs were unable to adjust topelleted food and died of starvation. Frogsraised in captivity, on the other hand, haveapparently no problems with this type offood. In contrast, it is our experience thatnewly captured wild frogs feed even betteron pelleted food than ones raised incaptivity.Clawed Frogs appeared to favour feeding

on Tubifex sp., probably because this is amoving, living food. The water wascontaminated macroscopically much lessthan with the other types of food offered inthis study. Nevertheless Tubifex sp. doesnot appear to be suitable as a sale food.Resulting nutritional deficiencies cannot beruled out. Furthermore, it is suspected thatmany parasites and bacteria, especiallyAeIomonas hydIophila, are introduced tothe large tank in large number along withTubifex sp. The large variation coefficientin the group fed Tubifex sp. might beexplained by this. A. hydrophila is thoughtto be responsible for red-leg disease.Finally, Tubifex sp. is very expensive,making the raising of Clawed Frogs on italone expensive. We recommend mixedfeeding, alternating heart, Xenopus pelletsand Tubifex sp. Nutritional deficiencieswere not observed with this feedingprogramme and the introduction of bacteriawas kept at a low level. In addition thevariation coefficient was very low.

Variability of dataIt has been shown that some of the factorsexamined in this study influence growthof X. laevis. An unexplained, andfrequently large, variance remainedwithin the individual groups. A possible

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Laboratory rearing of Xenopus laevis

individual-ethological component should beconsidered as the cause and could be thesubject of future studies.According to our observations, frogs with

the least amount of fear, appearing first atthe place of feeding, showed the bestgrowth development. A little later, allanimals would be swimming aboutexcitedly and snap at everything their frontlimbs contacted, including other frogs. Onecan assume that feeding under theseconditions is an important stress factor forthe animals. We therefore postulate thatnot only genetical and physiological, butalso ethological factors could influence thegrowth rate of these frogs.Chemical growth inhibitors secreted by

members of the same species are said toplaya role in some amphibians. Goldin(1992.) considers an epinephrine-likesubstance as mediator. Increasing the tankvolume would dilute such a chemicalinhibitor. Clawed Frogs with more wateravailable should therefore grow faster. Ourobservations of frogs kept in tanks withdifferent water volumes per frog do notsupport this theory. Chemical inhibitors donot appear to playa significant role in thegrowth regulation of X. laevis. Studiesby others also show that Clawed Frogs dowell in small water volumes. Raether(1978a) and Ochse (1948) consider 11 perfrog as adequate. This value is similar tothe 3.11 arrived at in this study. Wu &Gerhart (1991) successfully kept 2000-3000animals in tanks with a water volume of8001. Schneider (1956), on the otherhand, advocated 15-201 of water peranimal.

Acknowledgments The authors would like tothank Dr Anette Weinmar-Daniels for her advice ontranslation of the manuscript into English and DiplBioi Inez Gamenick for critical reviewing.

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