Guidance on the housing andcare of theAfrican clawed frog
Xenopus laevis

Barney T Reed
Research Animals Department - RSPCAGuidelines for the housing and care of the African clawed frog (Xenopus laevis)

May 2005

Guidance on the housing and care of the African clawed frog Xenopus laevis)

Acknowledgements

The author would like to sincerely thank the following people for their helpful
and constructive comments during the preparation of this report:
Mr. D. Anderson - Home Office, Animals Scientific Procedures Division
Mr. M. Brown - MRC Laboratory of Molecular Biology, Cambridge
Dr. G. Griffin - Canadian Council for Animal Care (CCAC)
Dr. M. Guille - School of Biological Sciences, University of Portsmouth
Dr. P. Hawkins - Research Animals Department, RSPCA
Dr. R. Hubrecht - Universities Federation for Animal Welfare (UFAW)
Dr. M. Jennings - Research Animals Department, RSPCA
Dr. K. Mathers - MRC National Institute for Medical Research
Dr. G. Sanders - School of Medicine, University of Washington
Prof. R. Tinsley - School of Biological Sciences, University of Bristol

Special thanks also to those organisations whose establishments I visited andwhose scientific and animal care staff provided valuable input and advice.

Note:
The views expressed in this document are those of the author, and may notnecessarily represent those of the persons named above or their affiliatedorganisations.

About the author

Barney Reed studied psychology and biology at the University of Exeter
before obtaining a MSc in applied animal behaviour and animal welfare fromthe University of Edinburgh. He worked in the Animals Scientific Procedures
Division of the Home Office before joining the research animals department of
the RSPCA as a scientific officer.

[Front cover photo: Xenbase]2

Guidance on the housing and care of the African clawed frog Xenopus laevis)

RSPCA policy
The prime objectives of the Royal Society for the Prevention of Cruelty to
Animals (RSPCA) are to promote kindness and prevent or suppress cruelty toanimals. With regard to laboratory animals, the RSPCA is opposed to all
experiments or procedures that cause pain, suffering or distress and believes
the necessity and justification for the use of animals must be criticallychallenged on a case by case basis. As long as animals continue to be used,
the Society believes every possible effort should be made to prevent suffering
at every stage of the animals’ lives, not just during experiments but also
throughout their acquisition, transport, housing, husbandry and care.
The Society adopts a constructive, practical approach to this issue, supportingand promoting the development and adoption of techniques that will result in
the replacement, reduction or refinement of animal experiments. This report ispart of the Society's work on refinement.

For further information regarding this publication please contact:

Research Animals Department
RSPCA
Wilberforce Way
Southwater, Horsham
West Sussex
RH13 9RS
www.rspca.org.uk
Or email: research_animals@rspca.org.uk

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Guidance on the housing and care of the African clawed frog Xenopus laevis)

CONTENTS

page

1

Introduction1

1.1

Aims of the report2

2

Background information on Xenopus laevis

4

2.1

Natural geographic range and habitat

4

2.2

Species characteristics

5

2.3

Use in research and testing
2.3.1 Past and present use
2.3.2 Numbers of Xenopus laevis frogs used

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3

Supply and transport

10

3.1

Wild-caught versus purpose-bred

10

3.2

Transport considerations
3.2.1 Packing and insulation
3.2.2 Travel delays
3.2.3 Grouping animals for travel together
3.2.4 Arrival

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3.3

Quarantine

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4

Housing and care

15

4.1

Lighting provision
4.1.1 Photoperiod
4.1.2 Spectrum
4.1.3 Intensity
4.1.4 Nocturnal nature

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4.2

Humidity

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4.3

Water provision
4.3.1 Quantity
i)
Depth
ii)
Volume4.3.2 Temperaturei)
Adverse effects associated with temperatureii)
Optimal temperature

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Guidance on the housing and care of the African clawed frog Xenopus laevis)

4.3.3 Water quality
i)
pH
ii)
General hardnessiii)
Other water quality parameters
4.3.4 Cleaningi)Standing water tanksii)
Drip-through water systems
iii)
Minimising temperature variation
iv)
Removing dissolved gasesv)
Careful use of cleaning agents

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4.4

Tank housing
4.4.1 Labelling
4.4.2 Tank material
4.4.3 Colour and transparency
4.4.4 Lids
4.4.5 A specialised design
4.4.6 Dimensions

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4.5

Identification and marking techniques
4.5.1 Criteria for an acceptable method
4.5.2 Assessment of methods

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4.6

Group housing

32

4.7

Handling
4.7.1 Capture
4.7.2 Use of hands
4.7.3 Use of gloves
4.7.4 Use of nets
4.7.5 Use of plastic containers
4.7.6 Restraint
4.7.7 Other considerations

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4.8

Food type and feeding regime
4.8.1 Natural behaviour in the wild
4.8.2 Feeding requirements of African clawed frogs
4.8.3 What food should be given?
4.8.4 How often should food be provided?
4.8.5 Regurgitation of food

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4.9

Environmental enrichment
4.9.1 What is it?
4.9.2 Why is it important?
4.9.3 Hides and refuges
4.9.4 Additional environmental complexity

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4.10

Assessment of health and disease prevention
4.10.1 Diagnosis of ill health
4.10.2 What does a healthy frog look like?

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Guidance on the housing and care of the African clawed frog Xenopus laevis)

4.10.3 Some key signs of ill health in Xenopus laevis frogs
4.10.4 Common diseases
4.10.5 Injury to skin4.10.6 Responses to acute noxious stimuli
4.10.7 Disturbance caused by noise

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5

Scientific procedures

45

5.1

Egg harvesting
5.1.1 Natural mating
5.1.2 Induction of ovulation and mating behaviour
5.1.3 Squeezing/milking of frog for eggs
5.1.4 Egg quality
5.1.5 Frequency of egg collection
5.1.6 Age of females

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5.2

Oocyte collection
5.2.1 Surgery to obtain oocytesi)Post-operative care
5.2.2 Oocyte quality

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5.3

Blood collection

52

5.4

Injections

53

5.5

Analgesia and anaesthesia
5.5.1 Analgesia
5.5.2 Anaesthesia

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5.6

Euthanasia
5.6.1 The principle
5.6.2 Methods for euthanasia of Xenopus laevis frogs
5.6.3 Unacceptable methods

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6

Training of animal care staff and users

60

7Conclusions

61

8

Summary of recommendations

62

9

Future areas for focus

66

10

References

67

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Guidance on the housing and care of the African clawed frog Xenopus laevis)

1

Introduction

The refinement of all aspects of the husbandry, care and use of laboratory
animals is important for many reasons - legal, ethical, scientific and not least
animal welfare. The latter two are inextricably linked, since it is increasingly
being demonstrated that good animal welfare is essential to produce goodscientific results (e.g. Poole, 1997).
Refinement encompasses both the reduction of suffering (i.e. negative effects
on physical or psychological wellbeing) and the improvement of animal
welfare. This can be achieved through the development and subsequent
application of comprehensive guidelines for every stage of animal care anduse, always striving to define not just appropriate standards and protocols, but
optimal ones. Such guidelines should be based on the findings of behavioural,
health and welfare research, and are best developed through inclusive
discussions with all relevant interested parties.
Amphibians have been a relatively neglected group in this respect, and are
frequently given only the briefest, if any, mention in handbooks for the careand management of laboratory animals. In addition, there is a widely held
belief that Xenopus laevis frogs are hardy (Wolfensohn & Lloyd, 2003) androbust (The Wellcome Trust, 2003), capable of tolerating a range ofenvironmental conditions and experimental procedures. There has historically
been little investigation into the capacity of these animals to suffer and failure
to appreciate that they are at times housed in sub-optimal environments.
The information that is available on Xenopus laevis in the scientific andcommercial literature tends to deal with the intricate issues at the molecular or
cellular level that are associated with studies utilising Xenopus eggs or
oocytes, in the fields of biochemistry, developmental biology, genetic
technologies or physiology. There is relatively little information with regard to
how the animals from whom the raw materials for such studies are obtained,should optimally be cared for, both so that the oocytes and eggs produced areof consistent high quality and so that each individual animal’s welfare issafeguarded.
Where information relating to the care and use of Xenopus laevis can befound, it is often inconsistent or incomplete. Thus, with respect to Xenopus
laevis frogs specifically there is: an absence of thorough ecological studies of population biology, interaction
within groups, generation times and growth rates derived from long-term
research of Xenopus laevis in the field (Tinsley & Kobel,1996);

 little systematic research to ascertain conditions for: optimal growth rate
and group homogeneity (Hilken at al, 1995); housing requirements (The
Berlin Workshop, 1994; O’Rourke, 2002); or the environmental conditions,water quality and nutritional regime necessary to optimise health, egg /oocyte production (Green, 2002) and welfare.
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Guidance on the housing and care of the African clawed frog Xenopus laevis)

It is therefore difficult for establishments to decide upon the standards andconditions necessary to achieve the highest standards of animal wellbeing,
and most appear to act on the principle of, "if what they are doing seems to beworking, then there is no need to change". This can have serious implications
for scientific studies as well as animal welfare since egg quality may beaffected by the environmental conditions experienced by the female frog. Egg
quantity can also be affected by poor environmental conditions leading to an
unnecessary increase in animal use.
There is a lot of interest in Xenopus welfare within the animal care and use
community, and a clear demand for a better understanding of the behaviours
and requirements of these animals, together with much more comprehensive
and consistent guidance in this respect. This report aims to assist in achieving
this goal. It is based on information obtained from: a) comprehensive searchesof the relevant UK, European and other legislation; b) the scientific literature;c) material produced by commercial organisations; d) information from ethics
committees around the world; e) observations from visits to researchestablishments; and f) from discussions with animal research regulators,
animal users and animal care staff including veterinarians.

1.1

Aims of the report

The purpose of this report is: to draw together previous research undertaken and recommendations
made, in order to illustrate areas of both consensus and inconsistency;

 to highlight potential welfare and ethical concerns relating to the supply,
housing and care of adult Xenopus laevis frogs;

 to facilitate understanding of Xenopus laevis behaviour and a subsequent
improved appreciation of their requirements;

 to arrive, where possible, at consensus based on available data and soundscientific argument for optimal environmental and care conditions forkeeping Xenopus laevis frogs;

 to provide some basic recommendations for improving health, welfare andegg quality, for reducing the potential for stress and suffering and possibly,
the total number of animals required; in areas where current knowledge is sparse or inconclusive, to stimulate
discussion that will enable movement towards identifying what should be
regarded as ‘best practice’ [see text in boxes].

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Guidance on the housing and care of the African clawed frog Xenopus laevis)

The care of eggs and tadpoles will not be addressed here, nor will the
intricacies of all the various experimental techniques associated with research
involving Xenopus laevis eggs and oocytes. However, a number of the morecommon procedures involving adult frogs of this species will be discussed1.

1

Xenopus tropicalis is used as an alternative amphibian species for some areas of study (Siveet al 2000). Xenopus tropicalis has a shorter generation time (around 5 months) and is diploidin genetic disposition as opposed to the tetraploid Xenopus laevis. It is unlikely however, thatuse of this species will widely replace that of Xenopus laevis because its eggs are smaller andare produced in clumps rather than singly, so are perceived as being trickier to do experimentswith (The Wellcome Trust 2003). Differences in physiological characteristics and natural
habitat mean the optimal conditions for keeping Xenopus tropicalis are likely to be different tothat for Xenopus laevis and so similar future stringent consideration must also be given to how
best to care for Xenopus tropicalis.
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Guidance on the housing and care of the African clawed frog Xenopus laevis)

2

Background information on Xenopus laevis

Understanding the behavioural biology of those species kept and used inlaboratories is crucial to improving both animal welfare and the quality ofscientific research (Olsson et al, 2003). The following section therefore
includes details of the anatomy, physiology and behaviour of wild Xenopus
laevis frogs. These are some of the characteristics that need to be taken into
consideration by animal care staff and others when designing andimplementing laboratory housing and husbandry regimes for these animals.

2.1

Natural geographic range and habitat

Xenopus laevis, also known as the African clawed frog (or toad) or thecommon platanna, is comprised of a series of sub-species that may be foundranging from the Cape of South Africa, to Sudan to the north-east and Nigeria
to the north-west. The sub-species studied in most laboratories is Xenopus
laevis laevis (though for the purposes of this report the generic term Xenopus
laevis will be used). Although members of this sub-species occur throughout
South Africa and parts of Zimbabwe, virtually all the individuals used inresearch actually originate from the Cape of South Africa (Tinsley,pers.comm), or are probably the descendants of such individuals. Aided by thepast actions of humans who released them after use in laboratories or as
unwanted pets, they can now be found in established populations, as aninvasive species, in areas of freshwater elsewhere in the world, such as
California, USA (Gampper, 2002) and in Britain (Measey, 1998; 2001;
Mattison, 1998).
Xenopus laevis can typically be found in the stagnant water (Deuchar, 1975)of pools, ponds, lakes or ditches, usually based on a substrate of deep mud.
Under normal circumstances these frogs are totally aquatic (Mattison, 1998,
Tyler 1999) and typically never leave the water, although they have been
known to migrate between connected water bodies or overland (Wilson et al,2002), or to aestivate2 in a burrowed hole with an opening for air (Garvey,
2000; Deuchar, 1975) if their home pond dries up. Migration is limited bydistance, and time of year, because once out of water and exposed, they aresusceptible to rapid dehydration (van Zutphen et al, 2001) and possible death(Kelley, n.d.).

2

To aestivate, means to pass the summer or periods of drought, in a state of dormancy or
torpor4

Guidance on the housing and care of the African clawed frog Xenopus laevis)

2.2
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Species characteristics

Xenopus laevis is one of 17 named species in the Xenopus genus (Tinsley &Kobel, 1996) included in the Pipidae family.
Xenopus are naturally nocturnal (Elepfandt, 1996a).
They have a head smaller than their flat body, with small, round, upward-gazing
eyes with convex cornea (Schultz & Dawson, 2003) that lack eyelids (USGS,
2003). The anatomy and positioning of the eyes on top of the head (Elepfandt,
1996a) are suited for atmospheric rather than underwater vision (Schultz &Dawson, 2003). These features allow the free-living animals a better chance ofdetecting prey or reacting to attacks from above by potential predators such as
birds, when they are near to, or at, the water’s surface.
Vision is not the main sense used for gathering information from the dark andmurky aquatic environment that these animals typically inhabit. Blind individuals
have been held without observed disadvantage in the same tank as sighted
individuals with no apparent impairment of the animal's ability to locate movementof, and within, water (Elepfandt, 1996a). It is suggested that vision has a more
important role where these frogs attempt to catch terrestrial prey (Measey, 1998).
The ears are not visible externally, but these animals have a highly developed
auditory system facilitating complex underwater acoustic communication between
individuals (Elepfandt, 1996b). The full repertoire of this communication in theform of ‘clicks’ may be difficult for humans to perceive due to the reflection ofsound at the water’s surface.
It is also thought probable that pheromones are involved in Xenopus
communication, though this has yet to be proven (Elepfandt, 2004).
Nostrils are positioned on the top of the head on the snout, and the presence of
two nasal cavities (the vomeronasal and the rostral) affords the ability to smell inboth air and water (Elepfandt, 1996a). Animals are aroused by the odour of foodnearby and swim around in search of it until they touch it, often only making adecision about its palatability after the object reaches the mouth (Elepfandt,
1996a). A highly developed olfactory sense affords this species the rare ability
amongst frogs of locating and consuming non-living food items (Beck, 1994).
The front limbs, used to direct food towards the mouth (Phillips, 1979; O’Rourke,2002), are short and the four fingers are not webbed. The hind legs are larger andprimarily used to powerfully propel the frog through the water. The five toes on thehind legs are webbed and the inner three have small claws at their end. These
claws are useful for digging out insects and other food items from the mud, andfor shredding larger prey into more manageable-sized portions (Garvey, 2000).
These frogs lack a moveable 'fleshy' tongue.
When Xenopus do have to occasionally traverse land surfaces, they are capableof moving reasonably fast by a series of lolloping leaps, landing flat on their belly
each time (Deuchar, 1975).
African clawed frogs elevate their urea levels in periods of drought as an adaptive
strategy that may help them to survive (Wray & Wilkie, 1995).
Like fish, these frogs have a lateral line of specialised sensory organs (around
180 individual organs in an adult – Elepfandt, 1996a) that run in a circle aroundthe back of the body, the head and around the eyes. This highly sensitive system,
retained to adulthood only by some amphibian species (usually those who are
predominantly aquatic) is utilised to locate prey and disturbances in theenvironment via the perception of water movement and velocity. The lateral line isvisible with the naked eye and is seen as a symmetrical pattern of elongatedgreyish dots or white lines, each 2-3 mm in length (van Zutphen et al, 2001).
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Guidance on the housing and care of the African clawed frog Xenopus laevis)

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Cutaneous respiration is not well developed in this genus (Deuchar, 1975) sothese animals mainly rely on well-developed lungs for gaseous exchange while
the animal is at the water’s surface (Garvey, 2000). A clawed frog will drown if it isprevented from breathing air as the intake of oxygen via the skin alone will beinsufficient (van Zutphen et al, 2001).
Xenopus laevis have dorsal skin colouring in various shades of olive grey withlarge darkened blotches (although albino strains have been purposely maintained
for use in some laboratories). The undersides of Xenopus laevis frogs areyellowish-white (Deuchar, 1975). The skin contains chromatophores (Phillips,
1979), which are cells containing pigment, used for camouflage. This mechanism
is under hormonal control.
The skin is damp and slippery. Mucous glands secrete a slimy protective layer,
which helps prevent mechanical damage to the skin and provides a barrier
against pathogens. Serous glands, which are typically present on the head andshoulders, synthesise and secrete a variety of compounds that help protect
against predators and chemicals, or that have antibacterial or anti-fungal
properties (Schultz & Dawson, 2003).
Females are larger than males, growing up to 15cms from snout to vent compared
to 10cms in the male (University of Arizona, 2001).
Cloacal flaps are only visible in the females, while the males develop darkened
nuptial pads on inner forearms and fingers to hold on to the females during mating(Sive et al, 2000). Whilst performing amplexus (a mating embrace where the malegrasps the female from behind with his forearms) both frogs will perform a seriesof somersaults in the water, with hundreds of eggs – usually between 500 and1000 (Deuchar, 1975) though possibly as many as 5,000 (Dawson et al, 1992) –being released and fertilised. The eggs are scattered over the area and as there isno further parental investment, they are left unprotected. Environmental conditions
experienced by the adult female prior to egg-laying, and subsequently by thefertilised eggs, will largely dictate the ratio developing successfully into tadpoles.
There is much field information on wild Xenopus laevis frogs from areas within its
range in South Africa, to suggest that breeding is seasonal - notably September(early spring) to March (late summer) (Tinsley, pers.comm).
Gravid females are recorded as containing from 1,000 to 27,000 eggs with larger
females producing larger clutches. In the wild, they will produce multiple clutches
in a season under favourable conditions (Measey, 2004).
The tadpoles are herbivorous, eating very small plant particles. They adopt acarnivorous diet after metamorphosis into a froglet, which depending on
conditions, is completed by between two and three months (University of Arizona,2001). Male frogs typically reach sexual maturity in around 10 to 12 months, whilefemales can, again depending on environmental conditions, take up to two years(Kelly n.d.). Optimal laboratory rearing conditions can shorten this time to one
year for females (Gurdon, 1996) or even 8 months (Tinsley & Kobel, 1996).
Individuals regularly live for 10-15 years in captivity. It has been suggested thatthey can survive for up to 25 years in the laboratory (Wolfensohn and Lloyd, 2003)
or even have a life span as long as 30 years (Kelly, n.d.). No comprehensive data
has been collected on the longevity of wild individuals in South Africa though there
has been a recording of at least 18 years for an individual living in the wild in the
UK (Tinsley, unpublished).
Normal behaviour for these frogs is to spend most of their time lying motionlessbelow the surface of the water (University of Arizona, 2001) with outstretchedarms, waiting for food to pass by (Mattison, 1998).

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Guidance on the housing and care of the African clawed frog Xenopus laevis)••

The unique structure of the sliding pelvis allows these frogs to attempt to avoid
predators by diving backwards from the water surface (Videler & Jorna, 1985) andis also a useful adaptation which helps this animal to burrow in mud (Tinsley,pers.comm).
It was believed that Xenopus laevis did not hold physical territory in the samemanner some of the more terrestrial species might. However, studies are now
revealing that interactions within populations include the maintenance of territories
in underwater habitats (Elepfandt and Yager, in Tinsley and Kobel, 1996).

2.3

Use of Xenopus laevis in research and testing

2.3.1 Past and present use
There is a long tradition of using this species in scientific study (Halliday,
1998) and it is certainly the most widely used and easily recognised amphibian
research animal (Schultz & Dawson, 2003). During the 1940s, before thedevelopment of more efficient endocrinological techniques, female Xenopus
laevis were commonly housed and used in hospital laboratories as a means ofdetecting human pregnancy. Upon injection of urine from a human female, thefrogs start to produce eggs if the hormones associated with human pregnancy(primarily, human chorionic gonadotrophin) are present (Mattison, 1998).
Later on, Xenopus laevis was the first vertebrate animal to be cloned (Gurdon
et al, 1975), and recent years have seen their continued use with the advent offurther developments in genetic technologies. They are now one of the mostwidely used vertebrate species in developmental, cell and molecularbiology research (Gurdon, 1996).
Developmental biologists, embryologists or geneticists use Xenopus laevis
because the eggs and early embryos of this animal are large and experimental
manipulations are thus relatively easy (Woodland, 2003). Their growth outsideof the uterus means that embryos can be observed throughout development(Kelley, n.d.) which is extremely rapid3. Manipulations of the cells within
fertilised developing eggs, or of the environment in which the eggs aremaintained are often undertaken to discover how subsequent growth anddevelopment is affected. A researcher may manipulate embryos directly
following fertilisation, allow the embryos to develop in a simple saline solution
for a couple of days, and then examine the tadpoles to determine whether andhow the experimental intervention influences development. The role of genes
in development can for example, be assayed by injecting a small amount ofmRNA encoding the gene of interest into an early embryo, then allowing theembryo to grow into a tadpole (Xenbase, 2003).
Xenopus display a circannual cycle in relation to reproduction and this initiallylimited their use. Seasonal effects impact on breeding frequency and successand in addition, for over five months of the year, no breeding usually takes
place in wild individuals (Tinsley, pers.comm). Once this was overcome with
3

Cell division proceeds soon after fertilisation and within a few hours the blastula stage isreached. Hatching occurs after two to three days whereupon the transparent, filter-feeding
tadpoles measure around 4mm in length (Phillips, 1979).
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Guidance on the housing and care of the African clawed frog Xenopus laevis)

the inducement of egg laying behaviour through hormonal injection (seeSection 5.1), demand for these frogs for research purposes increased.
This species, like most frogs, has a high degree of physiological sensitivity totheir surrounding environment. For this reason, in some countries, they areused in the safety assessment testing regimes of many potential
environmental pollutants and other toxicological substances (Dumpert,
1983, in Hilken et al, 1995). Since normal Xenopus laevis development was
described (initially by Nieuwkoop and Faber in 1956), the use of this frog has
become common for many in vitro development toxicology investigations.
Indeed, the ‘Frog Embryo Teratogenesis Assay: Xenopus’ (FETAX) system
has become standard protocol (University of Arizona, 2001) and has beenused to evaluate hundreds of substances since its inception (Wright &Whitaker, 2001). The test is based on assessing the toxicity of a substance on
its capacity to induce embryonic mortality, malformation or growth inhibition(Altafaj et al, 2004).
These frogs are also kept and used for educational purposes in a small
number of schools and universities (e.g. dissection classes). However, this useof amphibians, and animals in general, appears to be declining markedly(Halliday, 1999) due, in particular, to ethical concerns voiced by students,
parents, teachers, animal welfare organisations and others, and due tofinancial constraints.
The wide-ranging use of this species is further illustrated by the fact that they
have been used in research studies aboard various space-shuttle missions(The Wellcome Trust, 2003).
2.3.2 Numbers of Xenopus laevis used
It is extremely difficult to gain an accurate assessment of the current use of
Xenopus laevis for scientific research and testing purposes. In the UK for
example, the Home Office publishes details of the numbers of animals used
under the Animals (Scientific Procedures) Act 1986 each year. These figures
show 9,288 amphibians were used in 14,985 procedures in 2003 (HomeOffice, 2004). However, although it is acknowledged that the main amphibian
species used is Xenopus laevis (Mrozek et al, 1995), the figures for eachamphibian species are not provided separately, so it is not possible todetermine the exact number.
A further obstacle to discovering the full extent of the numbers maintained in
UK laboratories is that there are some practices routinely carried out on
Xenopus laevis that do not require reporting in the annual Home Officestatistics. These include: the euthanasia by an approved (Schedule 1) method of frogs for tissues,organs, or oocytes (if oocytes are collected only once);

 the euthanasia by an approved (Schedule 1) method of those frogs bredbut not then required for experimental purposes.
8Guidance on the housing and care of the African clawed frog Xenopus laevis)

Current UK figures are therefore likely to be a significant underestimate of theactual numbers kept in research and testing establishments, which are likely to
run into many thousands.
The most recent European wide figures, produced for 2002 (EC, 2005) fromdata provided by the 15 member states, are also likely to be an underestimateof the total numbers kept in laboratories, for similar reasons. These list 59,689
amphibians having being used in ‘scientific procedures with the potential tocause pain, suffering, distress or lasting harm’. This was predominantly for
fundamental biological research (28,891), but also for education and training
purposes (14,057), and a smaller number for toxicological and other safety
evaluation studies (5,535), and for research and development of products anddevices for human or veterinary medicine (5,411).
Elsewhere it is even harder to estimate the use of this species. During 2001, in
Canada, 49,350 ‘amphibians’ were used in research (CCAC, 2004). In New
Zealand for 2002, this figure was 409 (NAEAC, 2003). It is unknown how
many of these animals were Xenopus laevis. The United States of America
does not even categorise the total number of ‘amphibians’, let alone thenumbers used of this particular frog species (e.g. USDA, 2002).
Amphibian species are becoming better understood and scientists areclaiming an increasing number of advantages and applications for using theseanimals in research (AWIC, 2001). Many view Xenopus laevis in particular asan ‘ideal model organism’ (The Wellcome Trust, 2003). In addition,
researchers in many countries are looking to shift where possible to thosespecies perceived to have less capacity for experiencing pain and distress andfor which there tends to be less emotive appeal and public concern.
Furthermore, in Germany, the European frogs (Rana spp.) now fall under
endangered species regulations, and so their use is often replaced where
possible and appropriate by Xenopus laevis (Hilken et al, 1995). As a result of
all these factors, many establishments now house Xenopus frogs as routinely
as rodent species (O’Rourke, 2002) and it is likely that Xenopus use will
increase further in the future (Green, 2002). The need to define factors that
affect their welfare and seek ways of refining all aspects of their husbandry,care and use is therefore of paramount importance.

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Guidance on the housing and care of the African clawed frog Xenopus laevis)

3

Supply and transport

For many years, large numbers of Xenopus laevis frogs have been taken
directly from the wild in southern Africa for the purposes of breeding and/or
supply to research and testing establishments around the world. Morerecently, an increasing number of captive frogs have been purpose-bred forscientific use. The source of animals is an important consideration for bothanimal welfare and scientific reasons, as are other factors relating to supply
such as transport and quarantine.

3.1

Wild-caught versus purpose-bred

An estimated third of Xenopus laevis frogs used are taken from the wild(anon., pers.comm.). The impact of large-scale capture is not currently a direct
threat to their existence, as this species is not endangered. However, foranimal health and welfare, scientific reliability, and consequently ethicalreasons, it is better that animals obtained are from captive colonies bred
specifically for the purpose.
Xenopus laevis can be bred and reared easily under laboratory conditions(Council of Europe, 2003) and many establishments currently already routinely
breed their own frogs. Xenopus laevis frogs of either sex and at any stage ofdevelopment can also usually be readily obtained from commercial suppliers(Kelly, n.d).
Commercial organisations may supply: wild-caught animals; wild-caught
animals that have been held in captivity for a period of time; or captive-bredanimals. Some companies selling purpose-bred animals reared specifically for
research state that the animals they are selling are a minimum of 5
generations removed from wild-caught stock (e.g. NASCO, 2003).
A comparison of the perceived benefits of using either captive-bred or wildcaught Xenopus laevis frogs is given in Figure 1.

10

Guidance on the housing and care of the African clawed frog Xenopus laevis)

Figure 1: Comparison of perceived benefits of using either captive-bred or wildcaught Xenopus laevis frogs
Perceived benefits of using captivebred frogs

Perceived benefits of using wildcaught frogs

Laboratory or captive-bred individuals have aknown life history, age and diet, and thepotential for introducing unwanted disease orparasites to an existing colony can be
reduced.
Captive-bred animals have experience ofartificial rearing, housing and husbandry
conditions similar to those they are likely toexperience for the rest of their lives. Events
such as handling by humans, tank and watercleaning practices are likely to cause lessstress, health and welfare problems than innaïve wild-caught animals. It is noted that lab
bred Xenopus may remain completely‘unmoved’ by sudden sharp loud noise andcan habituate to people moving frequently inthe room (Council of Europe, 2003).
Lab-reared frogs inbred over a number ofgenerations will be more genetically similar
which could be beneficial to the reliability of
results in many studies as it reduces theeffects of potential variables.
Amphibians are an important part of theecological balance of many habitats. By nottaking these frogs from the wild, the balance ofthe local ecosystem remains less disturbed

Wild caught animals are usually slightly lower
in price to buy than their captive-bredconspecifics.

Wild-caught Xenopus are considered bysome to be more fecund than lab-bredcounterparts, resulting in scientists using
fewer animals to obtain the amount of
biological material that is required (Council ofEurope, 2003). Wild-caught animals may begenerally larger and therefore may produce
more eggs (Xenopus Express, 2003) though
this size and productivity differential may be
age related.

It allows genetic diversity to be maintained ina colony if this is required.

Comprehensive data on the comparative health of wild-caught
versus purpose-bred Xenopus laevis frogs throughout the durationof their use in a laboratory setting should be recorded andcritically evaluated. Currently, many of the comparisons maderegarding egg production and quality, and health, are anecdotal.
Purpose-bred frogs should be used in preference to animals takenfrom the wild (Council of Europe, 2004) - a researcher must be able
to provide specific justification for the need to use wild-caught
individuals.

3.2

Transport considerations

Animals bred off-site, or wild-caught and transported to the place of use, may
have to endure a long journey, often to another country or continent. For
example, many Xenopus laevis frogs are imported into Europe from the UnitedStates of America, South America or South Africa. Long journeys could pose
potential health and welfare problems to animals, so suppliers often go to
great lengths to ensure the health of the frogs in transit. When all measures
are taken to safeguard welfare, long-distance flights should not pose serious
dangers to Xenopus laevis and in most instances these animals seem to travel
11

Guidance on the housing and care of the African clawed frog Xenopus laevis)

well and appear healthy on arrival. However there are major problems thatneed to be avoided.
The factors to consider when transporting Xenopus laevis are the same as
those relating to the movement of other species. Relevant codes of practicesuch as the European Convention on the Protection of Animals during
International Transport (1968: CETS 65) should be adhered to. Companies
should also follow the recommendations for adequate animal transportation of
the International Air Transport Association and the Animal Air Transport
Association (Council of Europe, 2003).
More information on general principles for the transport of laboratory animals
can be found in the report of the LASA Transport Working Group (2005).
The biggest problem for Xenopus laevis frogs in transit, whether it is by air,road or other means is exposure to excessive heat. Even a few hours attemperatures above 25ºC can damage egg quality in the females (Sive et al,2000) though the frogs themselves may appear unharmed. Where egg quality
is affected by such heat exposure (potentially a problem in the warm summer
months), more females may need to be supplied and overall numbers required
would rise. It has also been illustrated that exposure to temperatures above
25ºC may lead to skin cell disintegration in Xenopus (Callaghan, 1953, inDeuchar, 1975).
It is preferable for frogs to be bred at the establishment where they
will be used - this will avoid any potential risks to health or welfare
associated with their transport.
3.2.1 Packing and insulation
When transporting animals, the potential for causing them injury or stress must
be minimised. Some suppliers use a primary transport box made of waterresistant cardboard or polystyrene, and pack the frogs with damp sphagnum
moss (e.g. NASCO) while others prefer to use damp foam cubes (e.g.
Xenopus Express), into which the frogs can burrow. Primary transport boxes
placed together in a larger container, are usually then surrounded byprotective polystyrene or styrofoam insulation packing chips.
Traditionally, shipments were often prevented during the winter or summer
months due to the frogs’ sensitivity to temperature. More recently, it has
become regular practice for commercial suppliers to add ice packets or
chemical heat packets to the containers (making sure that they do not comeinto direct contact with the animals) as necessary, depending on the time ofyear and climate. Weather conditions thus now have less impact on delivery
reliability, whatever the time of year.

12

Guidance on the housing and care of the African clawed frog Xenopus laevis)

Transport containers must have an adequate air supply.
During transport frogs must be kept within acceptable temperature
limits (see Section 4.3.2) and measures should be taken to prevent
desiccation.
Transportation of frogs should be postponed if extreme weather
conditions (e.g. extreme cold or heat) could threaten the health or
welfare of the animals.
3.2.2 Travel delays
The second biggest consideration regarding the transport of Xenopus laevis
frogs and a potential welfare concern, are delays in transit.
Journey times should be the minimum practicable.
Adverse weather, mechanical problems or industrial action cancause flight delays or cancellations and it is important to checkand plan for such eventualities.
3.2.3 Grouping animals together during travel
It is unlikely that Xenopus frogs travelling together will cause each other injury.
Nevertheless, given their carnivorous nature and observed instances ofcannibalism of younger and smaller individuals (McCoid and Fritts, 1980 inTinsley and Kobel, 1996), frogs travelling in the same box should be of similar
size and weight.
Frogs being transported together in the same box should be at asimilar stage of development, size and weight, and should not be
overcrowded.
3.2.4 Arrival
Sufficient preparation and communication between supplier and buyer
regarding transport itinerary and conditions, breeding history of frogs, dietary
background and health status should take place before shipment. This should
also occur within the establishment receiving the animals, to ensure thatshould the primary people involved in the transfer be absent, other, suitablytrained and informed animal care staff are on hand to receive the shipment,
checking the health and welfare of each individual animal upon arrival.
Care for the first two weeks following arrival after transport is particularly
critical for amphibian survival (University of California, 2000). Animals which
arrive in ill health and do not have a chance to recover, should be euthanased
13

Guidance on the housing and care of the African clawed frog Xenopus laevis)

immediately and the sender of the frogs should be informed (Council ofEurope, 2003).
An appropriately trained and competent person must check thehealth and welfare of frogs on arrival. Animals should then be
closely monitored and, to avoid introducing disease, should bequarantined away from any existing resident colonies.
To help prevent the potential spread of disease or bacteria, when
animals are transferred from shipping containers to the newholding tanks, the sphagnum moss and foam cubes with which
they were transported should not be transferred with them.

3.3

Quarantine

The principle of quarantining newly arrived frogs away from those alreadypresent in an establishment (in order to reduce the opportunity for infections or
disease to be passed between the two groups) is widely agreed, however, theperiod of quarantine is debated. The Xenopus laevis supply company
Xenopus Express (2003) recommend that all new amphibians should bequarantined for 7 to 10 days post-arrival, the University of California (2002)
guidelines suggest two weeks, while DeNardo (1995) suggests a minimum
period of 30 days. Wright & Whitaker (2001) suggest wild-caught amphibians,
whether obtained directly or indirectly, should be held for an extended
quarantine of 90 days or more. O’Rourke (2002) states that captive-bredanimals coming from sources of known health status can generally spend lesstime in quarantine than wild caught animals but does not provide subsequentguidance on how long these relative periods should be, simply stating thatrecommendations that have been made vary, from weeks to months.
The lifecycles of many of the major parasites of Xenopus laevis are aroundfour to six weeks (Sanders, 2004a). It would therefore seem wise for thequarantine period to be at least of this length. During this time frogs should be
examined daily for signs of ill health, with regard to activity level, skindiscoloration, ulceration, petechial (pinpoint) haemorrhages on the legs,
coelomic swelling or any other unusual changes (Xenopus Express, 2003).
Frogs can also be given prophylactic treatments for pathogens during this time(see Section 4.10)
A minimum quarantine period of 30 days is advised for all new
animals entering an establishment with strong consideration given
to extending this period to 90 days for those animals obtained
directly from the wild.
Routine husbandry procedures for frogs in quarantine should beundertaken after the resident colony has been attended to andmust be carried out with separate dedicated equipment.
Frogs should be observed daily for signs of ill health.

14

Guidance on the housing and care of the African clawed frog Xenopus laevis)

4

Housing and care

It is important that frogs are kept under optimal rather than tolerable conditions
both to maintain a healthy colony and in order to stimulate good quality egg
production throughout the year. This section highlights and assesses current
practice, guidance and research in relation to the range of environmental
parameters that need to be considered, with the aim of helping develop
consensus on best practice.

4.1

Lighting provision

4.1.1 Photoperiod
African clawed frogs can grow and reproduce over a fairly broad range oflighting regimes (Major & Wassersug, 1998). However, continuous light, light
deprivation, and inappropriate photoperiods can cause varying symptoms
ranging from lethargy to sterility and even death (Hayes et al, 1998).
A number of laboratories house Xenopus species under natural lighting, but
the majority now employ artificial sources and block out all outdoor lighting
cues. A 14-hour light and 10-hour dark period has been recommended (e.g.
Council of Europe, 2003) as roughly corresponding to nature, though this ispresumably seasonally-affected. Many establishments choose to use aconstant regime of 12 hours light, 12 hours dark (Major & Wassersug,1998).
Either of these protocols is suitable, as frogs appear to respond well to lighting
regimes of between 12-14 hours of light and 12-10 hours of dark (Sive et al,2000).
Gradual dimming/brightening of the lights is more akin to natural conditions
and allows animals to adapt to the change. This measure also removes apotential stressor from the environment if lights had previously been abruptly
turned on or off. Although vision is not the primary sense utilised by thesefrogs, it is worth giving these animals the benefit of the doubt with regard topotential sensitivity to such rapid changes in environmental lighting levels.
Continuous 24-hour lighting should not be used.
A lighting regime of between 12 and 14 hours light, and 12 and 10
hours dark is recommended.
Where artificial lighting is used, a gradual brightening/dimming
period of around 20-30 minutes in the morning/evening should beincorporated.

15

Guidance on the housing and care of the African clawed frog Xenopus laevis)

4.1.2 Spectrum
When frogs are exposed to sunlight-equivalent light levels, proper vitamin Dlevels and correct calcium/phosphorous balance can be maintained (Universityof Arizona, 2001) and many researchers believe egg quality improves (Sive etal, 2000). It is therefore important to consider providing full spectrum lighting,including ultraviolet (UV) range4 (University of California, 2000; NASCO, 2003;
Wolfensohn and Lloyd, 2003; Tyler, 1999), particularly for animals kept over along period such as six months or more.
Any artificial lighting used should simulate outdoor sunlight levels,with consideration given to including the ultraviolet (UV) part of thespectrum.
4.1.2 Intensity
Frogs should not be subjected to bright light, though illumination in the animal
house must be of an adequate level to allow observation of the animals andfor routine housing and husbandry procedures to be carried out. Many
laboratories employ standard lighting that may be particularly aversive to frogs
in tanks in the highest position of tank racking i.e. those closest to the lightsource. Frogs should have access to places where they can avoid light(Xenopus Express, 2003) as the stress caused by inappropriate lighting will be
exacerbated where no hiding refuges are provided.
There appears to be a lack of conclusive evidence as to whether albino
Xenopus frogs suffer from light-induced retinal damage in the same way albino
mammals can. However, a precautionary principle should be followed andsuch frogs should not be exposed to bright lighting.
Tanks should not be openly exposed to direct sunlight or very
bright artificial light and frogs should have access to darkened
refuges (see Section 4.9).
4.1.3 Nocturnal nature
Xenopus laevis are naturally nocturnal animals (Elepfandt, 1996a). Few
establishments appear to have developed husbandry protocols that takeaccount of this behaviour. Therefore, there may be potential, particularly for
the welfare of wild-caught animals, to be compromised through disturbancecaused by human activity or an inappropriate lighting regime. The Council ofEurope (2003) suggests frogs bred and reared under laboratory conditions arecommonly active by day and night, but literature and information is extremely
hard to find regarding their behaviour in laboratories during the dark phase.
4

Animal care staff and laboratory personnel who enter the animal house and who areexposed to UV light should wear protective eye goggles and long-sleeved garments.
16

Guidance on the housing and care of the African clawed frog Xenopus laevis)

Establishments may wish to review current lighting, feeding andtank cleaning protocols to determine the practicality of alternative
regimes that may better cater for the nocturnal nature of theseanimals.

4.2

Humidity

Under normal circumstances Xenopus laevis frogs are totally aquatic (i.e.
equivalent of 100% humidity). Controlling the humidity levels in the room
holding these animals in tanks is therefore less important than for thosespecies that naturally spend large periods of time on land. In any case, it isdifficult to control environmental humidity in rooms holding open water tanks
as the humidity at the water's surface is likely to be different from thatelsewhere in the room.

4.3

Water provision

4.3.1 Quantity
There is inconsistency in the literature and between establishment practices
regarding the amount of water to be provided per animal. In some cases thisvariation may be related to differences in other husbandry routines such asgroup size, feeding or water changing regime. However, there does not seemto be a clear and consistent basis for the decisions made.
Potential constraints imposed on frogs (and their behaviour) by enclosure size
and the amount of water provided may be overlooked because of a generalbelief that amphibians are insensitive to changes in the size of the captive
enclosure (Hayes et al, 1998). However, little research has been undertakenon the capacity of this species to perceive changes in the spatial environment.
It is generally accepted that supplying suitable depth and volume is very
important, but that water quality is the most important factor (DeNardo, 1995;
British Xenopus Group, 2001; Wolfensohn and Lloyd, 2003).
The dimensions of the tank and the volume of water must be great enough toallow frogs:to move or swim around;to lie fully submerged well away from the surface of the water;
to avoid contact with other animals if desired;to turn fully in any direction without impediment either from tank walls orother animals.

There should also be sufficient space within the tank to allow the addition ofsuitable environmental enrichment (see Section 3.4).

17

Guidance on the housing and care of the African clawed frog Xenopus laevis)

i)

Depth

It has been shown that Xenopus can ‘live’ in standing or slowly moving waterof any depth (Elepfandt, 1996b) but there is no evidence with respect to the
optimal depth. Although Hilken et al (1995) observed no effect on growth rateof frogs after placing them in tanks with differing water depths (of 5, 10 or 20cms), water which is too shallow can increase stress, startle and escaperesponses in these wholly aquatic frogs. Insufficient water levels can also
cause ovary regression in females (Alexander & Bellerby, 1938). Furthermore,
a field study of natural behaviour (Elepfandt, 1996b) showed that in any given
water body, calling males favoured deeper areas where their calls propagate
better.
A range of recommendations for water depth can be found in the literature(see Figure 2). The rationale behind the recommendations is rarely provided:
Figure 2: Summary of recommendations for water depth for housing Xenopus
laevis frogs
SourceRecommendation

University of California
ARC guidelines (2000)
Council of Europe (2004)

5-10cms(approx. 2-4 inches)
Minimum depth depends on
the size (snout to ventlength) of the animal: < 6cms body length =water depth of 6cms
(2.4 inches) 6-9 cms body length =water depth of 8 cms(3.1 inches) 10-12 cms body length =water depth of 10 cms(3.9 inches) 13-15 cms body length =water depth of 12.5 cms(4.9 inches)
Allows ‘serenade behaviour’4-5 inches
where frogs stand on their hindlegs and protrude heads fromwater (and may even ‘chirp’,
particularly in the evenings).
At least 6 but no morethan 12 inches
20cms (approx. 8 inches)

University of Arizona
IACUC guidelines (2001)

Beck (1994)
Williams (1991)
LASA
Good Practice Guidelines(2001)
Van Zutphen et al (2001)
Wolfensohn and Lloyd(2003)The Berlin Workshop(1994)

Rationale (where provided)

At least 25 cms(approx. 10 inches)
30 cms(approx. 12 inches)
Up to 40 cms(approx. 16 inches)
Should not exceed 50cms(approx. 20 inches)

To facilitate lung breathing at low
energy costs.

18

Guidance on the housing and care of the African clawed frog Xenopus laevis)

A water depth of at least 8 inches is recommended for housing
adult Xenopus laevis frogs. This is based on current evidence andknowledge, and should allow them to perform most normal
behaviours.
ii)

VolumeWhen maintained under natural conditions, Xenopus do not live in directcontact with each other (Council of Europe, 2003). Without doubt, population
density is one of the most important factors affecting the growth of Xenopus
laevis. Frogs with more area available per animal grow significantly faster than
animals kept in a tank with a higher population density (Hilken et al, 1995).
Suggestions in the literature for volume of water to be provided per frog at
breeding age vary (see Figure 3), usually from around 2 litres (0.53 gallons) of
water per frog to 8 litres (2.11 gallons) of water per frog (Schultz and Dawson,2003). However, figures can be up to around 19 litres (5 gallons) per frog.
Figure 3: Summary of recommendations for water volume for housing Xenopus
laevis frogs
SourceRecommendation

University of California
ARC guidelines (2000)
Marine Biotech, Inc

One adult frog per 2 litres ofwater.
Tanks such as the XR3 and
XR4 are designed with arationale of around one adult
Xenopus laevis frog per 2.64
litres of water.
At least one gallon (US) (3.79
litres) per frog.
Maximum of four frogs in 5 to
10 litres of water (anapproximate minimum of 1.25
- 2.5 litres of water per frog).
15 animals may be kept in 70
litres of still water(approx. 4.7 litres per frog).

NASCO (2003)
University of Arizona
IACUC guidelines (2001)

LASA
Good Practice Guidelines(2001)

Sive et al (2000)

Wolfensohn & Lloyd(2003)
Xenopus Express (2003)

Four females or six males inapproximately 16 litres ofwater (2.7 to 4 litres per frog).
Approximately 6-10 litres ofwater per animal.
2-5 gallons (US) (approx. 7½
to 19 litres) per adult.

Rationale (where provided)

Frogs like lots of room to swim.

19

Guidance on the housing and care of the African clawed frog Xenopus laevis)

5(for Council of Europe recommended volumes see footnote at bottom of this page).

There is wide variation in the volume of water recommended per
frog. More discussion and research is needed to determine bestpractice, with due regard to the behavioural needs of the animals(see Section 4.3.1).
4.3.2 Temperature
Xenopus laevis frogs have been found to exist in the wild across a wide
geographical range and across a spread of environmental temperatures(Tinsley and Kobel, 1996). As with other amphibians, they are cold-blooded
animals whose body temperature is dependent upon that of the surrounding
environment, and as a consequence, they are very susceptible to healthproblems if the surrounding environmental conditions are unsuitable.
In their natural environment, cold-blooded animals will literally ‘select’ theirbody temperature by finding the appropriate thermal environment throughbasking, burrowing, hiding under rocks or logs, or entering water (Kreger,
2002). For Xenopus laevis, who, it is stated, typically prefer water around 21ºC
to 24ºC (Green, 2002), this behavioural modifier may be expressed by thedepth at which they rest in the water. Other changes in behaviour as a resultof exposure to unfavourable environmental temperature have also been
observed in this species in the wild. At water temperatures of 30ºC (a level
which can in fact be lethal to Xenopus (Green, 2002)), these frogs have been
observed to excavate pits 30-40cm deep in the soft bottom mud where thetemperature remained a more comfortable 20ºC (Tinsley and McCoid, 1996).
Their ability to adapt their bodily systems (the locomotor system for example)
to surrounding temperatures has also been noted (e.g. Wilson et al, 2000).
i)

Adverse effects associated with temperature

If water temperature is sub-optimal, frogs will not eat and their metabolism andimmune systems will be depressed (University of Arizona, 2001). The
Xenopus immune system operates best above 21ºC (Tinsley and Kobel, 1996)
and is depressed at low temperatures. Keeping these frogs in laboratories at
unsuitable temperatures, such as 15ºC or lower, may be the reason forpathogenic infection occuring (Tinsley and Kobel, 1996). It is certainly believed
5

Council of Europe (2003) recommended minimum space requirements are provided in adifferent format to that given by others and are as follows:
Body length (snout tovent) (cms)Min. water surface
area (cm2)

<6
6-9
10-12
13-15

160
300
600
920

Min. additional water
surface area per
additional animal (cm2)
40
75
150
230

Min. water depth
(cm)
6
8
10
12.5

20

Guidance on the housing and care of the African clawed frog Xenopus laevis)

that Xenopus are stressed by prolonged exposure to temperatures below 14ºC
and above 26ºC (Green, 2002).
At marked departures from the preferred range, Xenopus growth anddevelopment is delayed (The Berlin Workshop, 1994). Nevertheless, in a rare
study undertaken to investigate temperature preference in Xenopus, Hilken etal (1995) observed no differences in growth development between frogs kept
at 19, 22 or 24 º C over a five month period starting when the animals were 3.5
months old. However, this may have been because the temperature levels
used fell within a relatively narrow range (5º C), all of which may have been
suitable. It should also be borne in mind that growth rate is just one potential
indicator of health.
ii)

Optimal temperatures

In order to offer the best conditions for overall frog health and egg quality, theoptimal conditions should be provided not simply those that the frogs cantolerate. However, it is not clear what the optimal temperature range should
be. The recommendations in existing guidelines are shown in Figure 4.
Providing areas of slightly varying temperature which allow animals to seek
their preferred microenvironment is beneficial (Council of Europe 2003).
However, this is not usually possible in the type of housing usually provided in
the laboratory (Hayes et al 1998). Research is needed to determine both the
optimal conditions and how best to provide them.
It is also important to minimise significant fluctuations of either room or watertemperature. Although frogs may be tolerant to gradual increases ordecreases in temperature, they may be susceptible to thermal shock andsuffer mortality if they are abruptly exposed to variation in temperature of 2ºC
to 5ºC (Green, 2003). In addition, where frogs are to be moved between tankswith water of different temperature, gradual and staged acclimatisation inintermediary holding tanks is advised.
Figure 4: Summary of recommendations for water temperature for housing
Xenopus laevis frogs6
SourceRecommendation

Wolfensohn and Lloyd(2003)
NASCO (2003)

15 - 18ºC

Sive et al (2000)
Van Zutphen et al (2001)

Council of Europe (2004)

Optimal temperature is
16 - 18ºC
16 - 20ºC
18 - 20ºC

Rationale (where provided)

Note: Variations in the ambient
temperature in excess of 5ºC per
24h may harm the animals.

Optimal temperature is
18-20ºC

6

A survey of establishments using Xenopus mainly in the United States (Major & Wassersug,
1998) revealed temperatures are maintained at levels within the wide range of 15 to 25ºC (5977ºF).
21

Guidance on the housing and care of the African clawed frog Xenopus laevis)

University of California
ARC guidelines (2000)

15 – 27ºC (60 to 80 º F)
though believe the ideal is
19 - 22ºC (66 to 72 º F)

Xenopus Express (2003)

20 - 23ºC

Schultz and Dawson(2003)

18-24ºC

University of Arizona
IACUC guidelines (2001)
LASA
Good Practice Guidelines(2001)

Avoid temperatures over
24ºC
Ambient room temperatureof 20ºC

Sub-optimal temperature will affecteating, metabolism and theimmune system will be depressed.
Xenopus are sensitive totemperature changes within theroom as well as the water.
Ranges of 18-24ºC are considered
acceptable for adequate growth.
‘Laboratories have discovered’ thatwater temperature maintained at
20-22ºC optimises oocyte
production.
Warmer temperatures will affectbreeding.

Water temperature should not be maintained below 16ºC or above
24ºC, with strong consideration given to providing a relatively
controlled temperature at a point between 18 and 22ºC.
Allowing an individual animal some control over the immediate
microhabitat is desirable. Current animal housing practices
should be reviewed to determine if measures can be taken (suchas the inclusion of temperature gradients) within the constraintsof the laboratory environment to allow more scope for this.
Abrupt significant fluctuations of either room or watertemperature should be avoided.
4.3.3 Water qualityi)

pH level

The pH tolerance range for Xenopus described in the literature is variable. In
the Cape, South Africa, Xenopus laevis are noted to breed in both acidic andalkaline water (Tinsley & Kobel, 1996). Some authors state that Xenopus frogs
can tolerate a wide range of pH and that a suitable level might be between 5
and 9 (e.g. NASCO, 2003). However, a far narrower tolerance zone (e.g.
between 6.5 and 8.5) is given by others (University of Arizona, 2001) with awarning that deviation from this (where water is too acidic or too alkaline)
could even cause the sudden death of an entire colony (Wolfensohn & Lloyd,2003). Sive et al (2000) consider a pH of 6.5 to be optimal for these frogs,
while the commercial frog supplier Xenopus Express (2003) recommend
maintenance between pH 6.5 and 8.5.
A potential danger to frog health and welfare is that a higher pH increases the
toxicity of chemicals, such as ammonia, in the water. Even without ammonia
22

Guidance on the housing and care of the African clawed frog Xenopus laevis)present, it is believed a change of 1pH unit (e.g. from 6.5 to 7.5) can lead tothe loss of the frogs’ protective mucous, a higher susceptibility to pathogen
attack, and other stress-related conditions (Sive et al, 2000).
Clearly it is important to monitor the pH of the water in the tanks regularly,
using pH test tape or preferably, a more precise electronic meter.
ii)

General hardness

Analysis of samples taken from ponds containing wild Xenopus laevis in South
Africa often show the general hardness of the water to be moderate to very
hard (Godfrey & Sanders, 2004). It has also been shown that housing adultfemales in hard water (containing a high concentration of calcium andmagnesium ions) can greatly improve the firmness of their oocytes and early
embryos and their survival and normal development (Godfrey & Sanders,
2004). However, these frogs have been held in 'soft' water in someestablishments without apparent problem.
iii)

Other water quality parameters

It is important to have a full knowledge of the origin and properties of the waterused for maintaining frogs. Properties will vary widely depending on whether
water is obtained from municipal sources (e.g. tap water, which in effect istreated sewage), natural springs, lakes or rivers, or whether it isdistilled/desalinised.
Water should be completely de-chlorinated before use (Sive et al, 2000) as
chlorine attacks the protective mucous layer over the skin and can predispose
frogs to infections (Wolfensohn & Lloyd, 1998). This can be achieved byexposure to air for several days in standing tubs. If local water authorities add
chlorine agents to the water then this can be removed by running the water
through a carbon filter (Sive et al, 2000). Allowing the water to stand before
use and using filters should also help deal with any chlorine surges in thewater supply. The pipes used for transporting water into and around theaquatic system should not be galvanised or copper, since heavy metals canleach from such pipes and may be toxic (Wolfensohn & Lloyd, 2003).
The water quality standards in Figure 5 have been proposed and successfully
implemented for Xenopus laevis7 (Sanders, 2004b).

7

Note: this is just one recommendation for good practice, other sources suggest different
parameters - for example, the NASCO (2003) water supply has 350mg/L alkalinity, 340mg/L
hardness and 490µS conductivity
23

Guidance on the housing and care of the African clawed frog Xenopus laevis)

Figure 5: Water quality standards for Xenopus laevis

Alkalinity
Hardness
PH
Salinity
Conductivity
Un-ionised ammonia (NH3)
Nitrate (NO2)
Nitrate (NO3)
Chlorine
Dissolved Oxygen content
CO2

>
=
=
=
=
<
<
<
=
>
<

50 mg/litre CaCO3
75-150 mg/litre
6.5-8.5
0.4 mg/litre(ppt)
50 -2000 µS
0.02 mg/litre
1 mg/litre
50 mg/litre
0 mg/litre
80% saturation
5 mg/litre

Maintenance of a stable water pH between 6.5 and 8.5 is advised.
Water quality and pH level should be routinely monitored.
Contingency plans should be made in case of system breakdown
or other emergency.
When devising water quality assurance schemes for frog housing,a water expert knowledgeable of the local water treatment
procedures should be consulted (Dawson et al, 1992).
4.3.4 Cleaning
Frogs are naturally found in murky water, which provides a visual barrier topotential predators (Schultz & Dawson, 2003). However, there is a difference
between murky, and dirty. Frogs routinely shed skin particles, and releaseammonia and faeces into the surroundings. This along with floating decaying
food particles will foul the water and may have implications for frog healthwhere space and animal movement is limited, as in the laboratory tank.
Consideration must therefore be given to how best to maintain the quality of
the water whilst at the same time minimising disturbance to the animals.
Xenopus laevis are routinely housed either in tanks of standing water(periodically ‘dumped and refilled’ every day or few days), or in tanks where a
drip-through system continuously and slowly changes the water. In dripthrough systems the water coming in may be new, or to reduce overall wateruse, may be treated and cleaned re-circulating water. Static systems require
frequent cleaning of tanks but have the benefit of enabling disease outbreaks
to be more easily controlled. This can be harder in re-circulating systems(Sanders, 2004b). Recommendations for cleaning practices will be influenced
not only by the tank or system design in place but also by the feeding regime
and quality of water entering the system. There is no current universally
applicable regime, but there are many suggestions for suitable generalpractice.

24

Guidance on the housing and care of the African clawed frog Xenopus laevis)i)Standing water tanks

Some suggest changing the water in standing tanks at least three times aweek (Sive et al, 2000). A number of establishments change the water evenmore frequently. However, it has been discovered that frogs disturbed daily forwater changes have a slower growth rate and this might negate any
improvement gained by better water quality (Hilken et al, 1995). Indeed, theauthors observed that frogs grew better in a tank where water was replaced
only once a week.
If complete water changes are undertaken, then this should be done at aroundthe same time each day (Schultz & Dawson, 2003), preferably post-feeding(see Section 3.3.6) since this is when the water quality is at its worst(DeNardo, 1995).
Establishments using individual standing water tanks should
review their water changing protocols. Although mostestablishments change the water in tanks at least three times aweek, and maybe even everyday, the benefit of such frequent waterchanges in view of the increased disturbance and stress caused tothe animals has yet to be comprehensively evaluated.
ii)

Drip-through water systems

It has been proposed that drip-through systems are the optimal way to house
Xenopus laevis since levels of toxic waste are kept low and solid waste (insuspension) can be drained continuously (Sive et al, 2000). The downside ofthese systems is that they use a lot of water (if not re-circulating) and thequality of the input water must be monitored constantly which often means asignificant capital investment. In addition, these animals would naturally favour
still, stagnant areas of water or, at most, slight and slow moving waters, so astrong water inflow may cause them stress.
As frogs sense water movement through a highly developed lateral line
system, the position of in- and out- flowing taps in the tanks and the rate ofwater flow should be set so water turbulence or motion is minimised. Whenusing continuous drip-through systems (which are more cost-effective when
housing large numbers of frogs) a rate of 10-50ml per minute has been
recommended by Sive et al (2000). Consideration should also be given to
submerging the incoming water jet as this may lead to less water turbulence(and noise) than where the water filters down from a height onto the surface ofthe existing body of water.
If exposing frogs in tanks to constantly running water, the flow rateshould be the minimum required in order to reduce the potential
for causing the animals distress.

25

Guidance on the housing and care of the African clawed frog Xenopus laevis)

iii)

Minimising temperature variation in the water supply

Though frogs may be able to tolerate a wide range of variation in temperature,e.g. a 5ºF increase or 10ºF decrease (NASCO, 2003), abrupt changes greaterthan just a degree or two could sometimes induce shock or even death(University of Arizona, 2001). Therefore, whatever the system, incoming
replacement water should be consistent in temperature with that it is replacing(see Section 4.3.2).
iv)

Removing dissolved gases

Water used directly out of the tap is saturated with dissolved gases and willcause bubbles under the skin and in the toe webs of frogs known as 'gas
bubble disease' (University of Arizona, 2001; Xenopus Express, 2003) leading
to possible emboli, emphysema, and even death (Orwicz, 1985, in DeNardo,
1995). Therefore, incoming water should be left for 24 hours prior to use toequilibrate with the surrounding environment. This allows the dissolved gasesin the water to dissipate before the water is used.
Incoming water can also be passed through a de-gassing device (verticalpacked column) to speed up the process of equilibration of the water(Sanders, 2004b).
v)

Careful use of cleaning agentsThe use of bleaches and detergents in aquaria must be treated withconsiderable caution (LASA, 2001). Most amphibians are susceptible tointoxication by phenol and cresol-type disinfectants such as Lysol® and will
exhibit symptoms including convulsions, flaccid paralysis and death (Universityof Arizona, 2001).
LASA (2001) suggest that tanks can be sterilised using food grade
hypochlorite, (e.g. ‘Klorite’) at 200ml/70L (0.28% solution) but emphasise theimportance of rinsing them thoroughly several times before frogs are returned.
Other recommendations for use in tanks housing amphibians, are 10% bleach(University of Arizona, 2001), 3% bleach (DeNardo, 1995) or betadine scrub,
followed by thorough rinsing.
Cleaning strategies should be designed to minimise disturbanceand distress to the frogs. Disinfectants should be used withextreme caution.

26

Guidance on the housing and care of the African clawed frog Xenopus laevis)

4.4

Tank housing

4.4.1 Labelling
Tank housing should always be clearly labelled with the number, sex andspecies housed inside. If the animals contained are currently being used in anexperiment, the reference of that research should be clearly identifiable. Thisis so that all personnel involved in the research are fully aware of theexperimental procedures involved, the objectives of the work, the potential
adverse effects the animals may experience and the agreed humane
endpoints (if applicable).
4.4.2 Tank material
Tanks should be constructed from material that is smooth, impervious, easy toclean and without sharp edges. Plastics used must not add toxicants to thewater; acceptable plastics include those used for the storage of human food
(Schultz & Dawson, 2003). Tanks for Xenopus laevis have traditionally been
constructed from fibreglass, stainless steel (Schultz & Dawson, 2003), glass or
polycarbonate (Wolfensohn and Lloyd, 2003). Whether the materials that are
currently being used have any effect on growth or reproduction in Xenopus
species has not yet been established (Major & Wassersug, 1998).
4.4.3 Colour and transparency
Tanks with darkened or opaque sides better approximate pond conditions(Sive et al, 2000) and are also likely to be less stressful for the frogs. Indeed,
the literature suggests that both young and adult frogs show preference fordark background colouring (Goldin, 1992) and that a black backgroundcolouring may be better for the frogs’ wellbeing than a grey or white one(Hilken et al 1995). In addition, a dark floor may enhance these animals’ senseof security (Council of Europe, 2004) thereby improving their wellbeing. This is
not surprising given that light emanating from underneath the frogs where thetank has a transparent floor, is the exact opposite of conditions encountered in
the natural environment.
In white opaque tanks it is easier to see the build-up of grime, and this may
facilitate cleaning. However, white or completely transparent tanks are notconsidered best practice for housing these animals, particularly those held
over long periods.
An argument may be made for leaving some part (e.g. just the front panel) of
the tank transparent (Council of Europe, 2003) so that the wellbeing of thefrogs can be checked with minimal disturbance to the animals8.

8Note: where frogs are recovering from surgical procedures or treatment for disease forexample, there may be some justification in the short-term for using a holding tank with aclear bottom, to allow the non-invasive assessment of wound healing.
27

Guidance on the housing and care of the African clawed frog Xenopus laevis)

4.4.4 Lids
A well-fitting wire or nylon mesh cover can be used to prevent animals fromjumping out of the tank. It will also prevent objects accidentally falling into it.
Tanks should never be completely covered with a solid cover as this will affectair quality and may possibly lead to hypoxia in the frogs.
4.4.5 A specialised design
Currently, many frogs are kept in containers not originally designed for the
purpose. Containers of various volumes and dimensions are used, many ofwhich were originally designed for other species (e.g. fish aquariums, rodent‘shoebox’ cages filled with water) or those that were not originally designed for
housing animals at all (e.g. bath tubs). Strong consideration must be given to
the suitability of available containers to house these frogs. The development ofspecifically designed tanks by laboratory equipment manufacturers isincreasing, and this is welcome.
4.4.6 Dimensions
Tank dimensions must allow adequate space for a suitable volume of water(see Section 4.3.1) and for environmental enrichment (see Section 4.9).
A tank size of 65 x 45 x 45cms has been recommended by LASA (2001) for
holding four to five frogs in 5 to 10 litres of water. A tank of this depth should(depending upon the water depth) prevent Xenopus from jumping out. An
overhanging lip will further act to prevent escapes. Xenopus escaping during
the night from laboratory aquaria are often dehydrated, debilitated or dead by
the next morning (Tinsley and Kobel, 1996), so such incidents should be
prevented.
The shape of the tank is also important. Long, narrow designs should be
avoided as they restrict locomotor activity and social behaviour such as
feeding frenzies (Council of Europe, 2004).
Opaque containers or those made of darkened plastic are
preferable to completely transparent ones – in particular, thebottom of the container should not be permeable to light.
Containers must prevent escape and should allow space forsufficient volume and depth of water and for enrichment such as
refuges to be added.

28

Guidance on the housing and care of the African clawed frog Xenopus laevis)

4.5

Identification and marking techniques

Marking can have an effect on an animal and its wellbeing through the act ofmarking itself, through the wearing of the mark and/or through the procedures
required for observing the mark (Mellor et al 2004). The actual need forindividual or group identification must therefore first be established, as only
some research protocols may require this. If identification of animals isnecessary then it is of paramount importance that only the most humane
methods are used. These will be the ones likely to cause the least pain or
distress, and which minimise the risk of secondary health problems whilst also
being effective over the required period of use. When considering theacceptability of potential methods it is important to bear in mind that pain
perception in amphibians is likely to be analogous to that in mammals(CCAC, 2004; Green, 2003).
4.5.1 Criteria for an acceptable method
The method of identification employed must:






cause minimal suffering or impact on the animal both during the marking
process or anytime subsequently;
be long-lasting (although this is dependent upon duration of study/use);be reasonably quick and simple to apply to the animal;
be easy and quick for the human to read/identify;
be economically viable (particularly pertinent for laboratories housing large
numbers of animals).

4.5.2 Assessment of methods
There are numerous methods of identification that have been tried with
Xenopus laevis frogs over the years, though many of the more traditional
methods are now actively discouraged and should no longer be practised. Thisis usually because of the adverse effects on the animals, but also often
because of their relative ineffectiveness. Some of the merits and shortcomingsof the various methods described in the literature are considered in Figure 6.
There is currently no ‘ideal’ method favoured by all.
Figure 6: Methods of identification and some points to consider regarding theirsuitability

Key: 9X?

This method can be consideredThis method should not be used
The merits of this method are debatable – it might be considered
suitable in certain circumstances

Taking into account both animal welfare and scientific requirements, the‘pros’‘cons’and ‘other factors needing consideration’highlighted for each method.

are

29

Guidance on the housing and care of the African clawed frog Xenopus laevis)

MethodPoints to consider (with reference)

Record cards

 No impact on the animal.
 Inexpensive.
 Useful when just needing

firmly secured on
the front of tanks

to distinguish between groups ofanimals.
 Does not allow comprehensive lifetime history to be maintained foreach individual.
9 Non-invasive.
Photographs
 Inexpensive.
for recognisingindividuals by body  Already being used successfully (e.g. Mathers, 2004).
 Particularly useful for small numbers of animals.
markings
 The pattern of markings on the frog will remain consistent but
relative skin colour can alter as a result of stress (Wolfensohn &Lloyd, 2003), or fluctuations in the environmental lighting, humidity
or air temperature and this may make the pattern harder todistinguish.
 Could be time consuming where large numbers of animals are
kept in each tank.
 Development of electronic scanners (linked to a computer) for
detecting marking patterns could increase potential of this method.
 Markings fade once the animal dies.
 Patterns in very young or albino individuals are not as easy to see.
9

Microchiptransponders

 Somesuggest this to be one of the best methods (e.g.
Wolfensohn & Lloyd, 2003).
 Very large numbers of animals can be individually identified
are increasingly(Halliday, 1999).
being suggested
 Transponders have been found to be effective (Fineberg, 2003)
for use inpractical and reliable (Mrozek et al, 1995) and able to serve as theamphibians andmethod of identification for the duration of the frog’s life, normally
reptiles (Schaeffer
without adverse effect (Mrozek et al, 1995; Hoogstraten-Miller &1999).
Dunham, 1997).
Transponders
 Chips can be retrieved, re-sterilised and re-used when a frog isaround 12mm longeuthanased.
by 1.2-2mm
 Immediate skin tissue damage should only be minimal and if it
diameter are
does occur then it can be treated with tissue adhesive such as
implanted in the
VetBond® (anon, pers.comm).
lymphatic cavity(WSAVA 1999) or  Frogs normally have to be removed from the water to allow
scanning – but individual identification is usually only necessary
injected subwhen the frog is to be used in an experimental procedure or iscutaneously in the
being examined for illness and as such would probably need to bearea behind theremoved from the water anyway.
neck.
 Anecdotal evidence suggests that on rare occasions, a microchip
when placed subcutaneously in the neck, may move around thescapular region of the frog and even be expelled from the body by
the frog. More data is required to confirm the prevalence of thisand the potential for the chip to cause a blockage (i.e. in a lymph
heart) – such occurrences are apparently highly infrequent.
 If implantation of the transponder requires surgery (i.e. when it isplaced into the dorsal sac) it would be classified in the UK as aregulated procedure under the ASPA 1986.
 Due to their smaller size, microchip transponders used for
Xenopus laevis are not suitable for use in Xenopus tropicalis.
 Some people are concerned ulcers or inflammatory responses
may occur around the injection site.
 Most expensive of assessed methods (around £2-3 per?
transponder).

30Guidance on the housing and care of the African clawed frog Xenopus laevis)

Freezebranding

 Some

Applying acetic
acid to the skin This method is painful for the frogs - it should not be undertaken.

may deem this method as being more acceptable formarking frogs in field studies where alternative methods are fewer
in number.
- shaped copper Frogs regularly shed skin meaning branding may be relatively
wire is placed into
ineffective. In some cases, the mark may become illegible in a few
liquid nitrogen andmonths to a year (University of Arizona, 2001).
then placed on the Some believe this method to be difficult to justify due to thebelly of the frog
significant adverse effects (anon, pers.comm).
(Measey 2001)x
 Beads have been sewn onto the legs or back of anaesthetised
Sewingcoloured plastic frogs with no reported perceived health or welfare complications(e.g. Hoogstraten-Miller & Dunham, 1997).
beads onto the The suturing of coloured plastic or glass beads of appropriate sizemuscle mass of
into the toe webs of frogs with plastic suture or monofilament line
the leg or back or
is considered an acceptable method of marking by some e.g.
attaching studs orUniversity of Arizona (2001), Schaeffer (1999), Wolfensohn &tags to the web of
Lloyd (2003).
the feet The skin and feet web in particular is very delicate in frogs,
containing many blood vessels (Fineberg, 2003) which may bedamaged during this process.
 It could be hazardous to the animal if the thread or beads/tags getcaught by the hind claws of that or other animals as they movearound (Verhoeff-De-Fremery & Vervoordeldonk, 1982).
?
 It could disrupt the mucous membrane of the skin and increasex
the chance of opportunistic pathogenic infection.
 If this method is undertaken, appropriate anaesthesia should be
Tattooingused and the size of the numbering or lettering should be noof numbers orbigger than is necessary to enable clear interpretation, i.e. there isletters onto theno need to cover the whole of the belly.
skin Tattooing is often carried out with no anaesthetic used (i.e.
Fineberg, 2003). Because the frog does not struggle greatly whilst
laid on its back, people may deem the procedure not to be
aversive to the frogs. However, prey species often do not show
overt distress behaviours that might draw the attention of
predators to their plight (Green, 2003) and thus decrease thechances of survival. Furthermore, while on their backs, theseanimals may simply be exhibiting tonic immobility and as suchhumans might not recognise suffering.
 There is concern the use of vibrating needles and the resultant
disruption of the protective slime layer and dermis may predispose
the frog to diseases such as red leg syndrome (Hoogstraten-Miller& Dunham, 1997).
 Ink tattooing is not an ideal method due to its non-permanence
and the need often to re-tattoo (Hoogstraten-Miller & Dunham,
1997). As repeated re-tattooing over scar tissue should be
avoided, this method, if used, should only be considered where
identification of the animal will be required for 18 months or less.
 The presence of bruising, redness and swelling of the delicate
skin, observed for a period of days after tattooing (even when
done by a highly experienced professional), would suggest that
this method does have a negative impact on the animal.
x

to mark the animal

Leg ringingx If the bands are tight enough to stay on the slippery mucous

covered skin, then they are probably too tight. Blood circulation
around the foot could be restricted, causing the feet to swell andx
become necrotic within hours (University of Arizona, 2001).

31

Guidance on the housing and care of the African clawed frog Xenopus laevis)

Removal oftoes or fingers

 Traditionally a commonly used method.
 Causes animals pain and may cause distress.
 Should not be done (Council of Europe, 2004;

Wolfensohn &Lloyd, 2003).
 Causes tissue damage in frogs (Golay & Durrer, 1994).
 Could be detrimental to health – it appears to decrease frog
survival in the wild (Clarke, 1972; McCarthy & Parris, 2004).
 Frogs can re-grow removed digits (University of Arizona, 2001)x
meaning the process may have to be undertaken more than once.
 Proponents of this method (e.g. Van Zutphen et al, 2001) provide
Skin grafts
no information relating to the associated health or welfare- lighter coloured
problems or the longevity of the method – this makes it hard toabdominal wall
assess.
skin is removed
under anaesthesia  The procedure is time-consuming.
 Undertaking the procedure requires surgical expertise.
and implanted
 Use is limited to small number of individuals.
onto the darker
coloured back, orvice versa(usually byscissors or nailclippers)x

Careful consideration should be given to whether identification ofindividual animals is necessary, and if so, then to the method of
identification used, particularly where the methods are invasive
and likely to cause a degree of trauma.
Non-invasive methods of identification such as using group cards
or photographs of the dorsal markings of the frogs are themethods of choice.

4.6

Group housing

It is generally considered that Xenopus laevis frogs should be kept in a group,
and in most establishments, this is how they are housed.
Grouping is advisable as:

efficient group feeding in this species involves feeding frenzies that are notas prevalent when only very small numbers of animals are kept together(Council of Europe 2004);
grouping animals can help reduce fear responses.

Maintaining stability in these groups is advisable because:

Xenopus laevis naturally form hierachies within a territory (Elepfandt, inTinsley and Kobel, 1996). In many other species, if group structure isregularly altered (or if groups are too big) con-specific relationships are
more difficult to maintain and antagonistic encounters may be more
frequent leading to an increased potential for stress or injury.

When keeping animals together in the same cage or tank, there are manyfactors to consider including group size and stability and stocking density.
However, there is little evidence to suggest what the ideal group size should
32

Guidance on the housing and care of the African clawed frog Xenopus laevis)

be. Many laboratories keep groups of between 5 and 20 frogs although thesegroups are small compared to one commercial breeder whose smallest
aquarium contains 40 individuals and the largest, around 400.
Individuals must not be kept in overcrowded conditions as frenzied responses
to feeding can lead to traumatic injury of tank-mates (DeNardo, 1995). In thisrespect, it is probable that once above a number of animals conducive toencouraging feeding behaviours, area per frog is more important than actual
number of frogs in the tank. Hilken et al (1995) found that frogs with more area
available per animal grew significantly faster than animals kept in a tank with ahigher population density. Furthermore, it has long been known that too high a
population density (along with insufficient water levels) affects oocyte quality infemales by causing ovary regression (Alexander & Bellerby, 1938).
In the absence of comprehensive evidence it is difficult to gauge an optimumgroup size for African clawed frogs. The group should be small enough toallow the formation of a stable hierarchy yet large enough to promote feeding
frenzies. Animal carers commonly consider a minimum of 5 or 6 animals pertank to be advisable.
Group housing of these frogs in relatively stable groups isadvisable.
Overcrowding should be avoided.
It is important that animals kept together in the same tank are ofsimilar size.

4.7

Handling

There are many factors requiring consideration in relation to handling thesefrogs:


As potential prey species, amphibians do not like to be handled.
The skin is highly glandular (secreting a mucous layer to help protect
against abrasions) which can be easily damaged.
The lateral line sensory system is highly sensitive (Elepfandt, 1996a).

For these reasons repeated disturbance for capture and handling should be
minimised (Council of Europe, 2004), only being undertaken where absolutely
necessary (Wolfensohn and Lloyd 2003).
4.7.1 Capture
Xenopus laevis should be caught by catching the head between the first two
fingers as it crosses the palm of the hand, followed by the thumb gently
restraining the neck. In this way the forward straining movement of theescaping animal will only serve to push it further into the finger grip (Williams,
in Benyon & Cooper, 1991) and should prevent an animal jumping or falling33

Guidance on the housing and care of the African clawed frog Xenopus laevis)

and potentially injuring itself. Alternatively, a frog may be picked up by placing
one hand across its back with a forefinger between the animal’s hindlegs,
wrapping the rest of the hand around the animal’s middle. The other hand may
be used to cover the frog’s eyes. It is believed that this will ‘calm’ the animalwhilst also allowing physical restraint (Sive et al, 2000). Picking frogs up by thehind legs is not acceptable.
Before handling a live animal for the first time, it is helpful for animal carers topractice holding a model frog (Sive et al, 2000) or if appropriate, one that has
been recently euthanased (although frogs should not be killed specifically forthis purpose).
4.7.2 Use of hands
The skin of amphibians is more delicate and sensitive than that of any othervertebrate (Halliday, 1999). Traces of hand lotion, colognes or medicatedointments may harm and even have the potential to kill these frogs.
Additionally, some amphibians have been observed to die if handled bysomeone who smokes (Halliday, 1999). It is also believed that the thin
epidermis of the frog’s skin could be abraded by the ridges and callosities of
the restrainer’s hand (Wright & Whitaker, 2001). If personnel are going tohandle frogs then their hands should be thoroughly cleaned and moistenedwith water.
4.7.3 Use of gloves
Many believe moistened gloves should be worn by personnel changing wateror handling frogs to avoid the problems potentially arising when the barehands of personnel come into contact with frogs skin (Schaeffer, 1999;
University of Arizona, 2001; Wolfensohn and Lloyd 2003). If used, Sive et al(2000) state that gloves must not be powdered or textured since they may
abrade the animal’s skin, or cause skin irritation. However, there is also
concern that gloves reduce the sensitivity of the human hand and so a frog
might be harmed from over zealous handling (Xenopus Express, 2003).
Wearing plastic or latex gloves does afford additional protection to the handler.
It is important for personnel to remember not to touch their own mucous
membranes (e.g. rub their eyes) after handling frogs as secretions from theparotid glands rubbed into the conjunctival sac of the human eye can cause
irritation (University of Arizona, 2001)9.
4.7.4 Use of nets
Frogs in a tank will often struggle or try to evade being picked up. Therefore,
some believe using a net may be the best method for capture and handling(University of Arizona, 2001).
Using a net to initially scoop the frog out of the water avoids damaging the
mucous layer of the frog and is less traumatic than physical contact (handling)
9

These frogs should pose no additional health risks to humans.
34

Guidance on the housing and care of the African clawed frog Xenopus laevis)

with the animal (University of Arizona, 2001). Swift capture can be made infine nylon nets but care must also be exercised here to avoid trauma(Williams, in Benyon & Cooper, 1991). However, others believe nets might stillcause micro-lesions of the skin and therefore should not be used (e.g. The
Berlin Workshop, 1994; van Zutphen et al, 2001). Beck (1994) has evensuggested that the risk of the animals having fingers inadvertently trapped andeven amputated in netting should preclude use of this method. However, if softand fine mesh is used for the net then these potential problems should be
avoided. LASA (2001) state that such nets should be deep and strong.
4.7.5 Use of plastic containers
A small plastic bowl or container may be used to lift frogs from the tank alongwith the water immediately around it (Elepfandt, 2004). This method would
appear both time-efficient and likely to cause less stress or harm to the animal.
A cover should temporarily be placed over the container to prevent the frog
from escaping.
A drawback of this technique is that it may be less practical in tanks with alarge group size of animals, a deep water level and where trying to capture
particular individuals.
4.7.6 Restraint
Restraint of these frogs might be difficult due to the secretion of a slipperymucous covering. Animals can be gripped gently but firmly and a soft damp
clean cloth can be used to increase grip (LASA, 2001; Wolfensohn and Lloyd,
2003; IAT, 2003). It has been suggested that permanently aquatic species like
Xenopus may be held with a rough cloth (Halliday, 1999). However, in view ofthe sensitive nature of the skin, this has the potential to cause mild skinabrasion and is therefore discouraged.
4.7.7 Other considerations
Whatever the method of handling, when approaching the tank (or when simply
moving around in the vicinity of the tank) personnel should be aware of thepotential effects of their own behaviour on that of the animals. The classic‘escape’ response can be observed where frogs are startled and thosepreviously hanging at the waters’ surface, dive to the bottom to hide when
objects move rapidly over the tank. This response is not usually stimulated by
objects passing by the side of the tank (Elepfandt 1996a). Unnecessary
movement of hands or objects by personnel over the tank should therefore be
avoided.
Handling should be kept to a minimum and every precaution to
avoid stressing or injuring the animals should be taken.
Personnel should consider taking a precautionary approach andwear gloves (non-powdered) whilst removing frogs from the waterwith soft-mesh non-abrasive nets.
35

Guidance on the housing and care of the African clawed frog Xenopus laevis)

Unnecessary movements of objects or hands over the tank should
be avoided.

4.8

Food type and feeding regime

4.8.1 Natural behaviour in the wild
Xenopus laevis are carnivorous by nature (Phillips, 1979) and although they
will scavenge the carcasses of dead animals, they prefer live food (Green,2002). They have a voracious appetite and will attack almost anything thatpasses in front of them (Garvey, 2000). They take whatever organisms can beingested, with insects the most common prey in adult animals (Tinsley &Kobel, 1996). Field studies reinforce this finding - examination of the stomach
contents of Xenopus laevis have found the remains of fish, birds, and otheramphibians, but it is small insects, slugs, worms and other aquatic invertebrate
which predominate (Green, 2002).
4.8.2 Feeding requirements of African clawed frogs
It has been recorded that Xenopus can usually survive long periods (months)of starvation (Tinsley and Kobel, 1996) but such events carry a physiologicalcost for the frog which breaks down body tissue in the attempt to cope. Foodshortage strongly affects egg production and it has been cited as a major
factor causing the ovaries of Xenopus laevis to regress (Bellerby & Hogben,
1938). Therefore, to help ensure optimal frog health and that egg quality is
maintained, Xenopus laevis must be provided with sufficient quantity andquality of food.
The dietary requirements and feeding behaviours of Xenopus laevis are easierto provide for in the laboratory environment than many other amphibian
species, due to their willing acceptance of non-living food items.
4.8.3 What food should be given?
A number of commercially prepared diets for captive frogs are available. For
example, NASCO produce Frog Brittle®, which contains fish meal, meat meal,
soybean meal, corn meal, wheat flour, dried yeast, distillers solubles, whey,
wheat germ meal, salt, vitamin supplements-diacalcium phosphate10.

10

Vitamin content of Frog Brittle (NASCO 2003): Vitamin A - 14000 I.U. per kg,
Vitamin D - 5000 I.U. per kg, Vitamin E - 88 I.U. per kg, Vitamin B12 - 0.04 mg per kg,
Folic Acid - 1.5 mg per kg, Thiamine - 4.0 mg per kg, Riboflavin - 9.0 mg per kg,
Pantothenote Acid - 11.0 mg per kg, Niacin - 60.0 mg per kg, Choline - 1550 mg per
kg, Calcium - 2.0%, Phosphorus - 1.6%, Sodium chloride - 1.2%, Magnesium - 2%,
Linoleic Acid - 2.8%, Iron - 300 mg per kg, Copper - 10 mg per kg, Cobalt - 2.2 mg
per kg, Manganese - 9.0 mg per kg, Zinc - 100 mg per kg, Iodine - 48 mg per kg,
Pyridoxine - 8.8 mg per kg

36

Guidance on the housing and care of the African clawed frog Xenopus laevis)

Beef liver is another item commonly provided but when given on its own has
been implicated as a cause of vitamin A toxicity, metabolic bone disease andother amphibian diseases (University of Arizona, 2001; Wright & Whitaker,
2001). Meat (such as liver or heart) should therefore be mixed with vitamins(Mrozek et al, 1995).
Maggots and crickets are also provided by some establishments and arestated to appear to provide welcome stimulation and enrichment for the frogs.
4.8.4 How often should food be provided?
Establishments vary in the feeding regime they provide for their frogs. The
regime is influenced by the quantity and frequency personnel deem to beappropriate and also by the type of food provided (see Figure 7).
Figure 7: Summary of recommendations for food type and feeding regime for
Xenopus laevis

SourceWhat

How much

How often

Xenopus Express(2003)

A nutritional highquality balanced
diet such as
Xenopus Express’‘sinking frog food’.

As much as can be
consumed in 15 minutes.

Daily

University of California
ARC guidelines (2000)
LASA
Good Practice
Guidelines(2001)

As much as will be eaten
in one hour30-40g of dry diet for
Xenopus laevis
can be fed
females with an average
proprietary pellets, weight of 145g (mature
minced ox heart or males with an averagea meat and pellet
weight of 60g should be
mixture although
fed about half as much asmeat should only
females). Adjust if
be given as anecessary so that amountsupplement rather provided is consumed inthan a staple diet. about an hour.

Wolfensohn &(2003)

Lloyd Pelleted food, or
strips of beef heart(with tough tissue
removed) withvitaminsupplements.
NASCO (2003)
Frog Brittle
University of Arizona
Frog Brittle,
IACUC guidelines
tubiflex worms,(2001)
earthworms,
commercial
salmon pelleted
diet, beef liver(though not on its
own).
Schultz and Dawson(2003)

Daily or every
other day.
3 times a week.

Two or three strips of beef
heart per animal.
Pelleted food will normallybe consumed within 30
minutes.

Two to three times
per week.

1 gram/frog‘Good’ meals.

Twice a week.
2 to 3 times a
week.

Most facilities feed
their frogs between
2 and 5 times a
week.

37

Guidance on the housing and care of the African clawed frog Xenopus laevis)

Van Zutphen et al(2001)

Council of Europe(2004)

Slices of meat into
which a multivitamin andmineral mixture
should have been
intensively rubbed.
Captive animals
should be
maintained on
their natural foods
or on foodstuffs
approximating
those of theirnatural diets.
Captive aquatic
amphibians can
successfully bemaintained onpieces of fish fillet
or scrapings fromfrozen liver andheart.

Ad libitum (leave food intank for at least an hour
and a half).

3 times a week.

To satiation.

Daily feeding is notadvisable for adultanimals. Once tothree times weekly
is recommended.

4.8.5 Regurgitation of food
Uneaten particles of food may foul the water and so many establishments
remove such debris after feeding has taken place. Care should be taken toleave a sufficient time interval between feeding and cleaning as frogs may
regurgitate food if startled, disturbed, or handled shortly afterwards (Universityof California, 2000).
Some personnel (e.g. Schultz & Dawson, 2003) do not experience such aproblem and recommend waiting only two hours before changing the water(for static water systems). However, the scientific literature shows that foodregurgitation by frogs after disturbance can be a common occurrence and so
waiting a little longer (i.e. between 3 and 5 hours as suggested in University of
Arizona (2001)) before cleaning or introducing new water is probably best.
Some establishments (e.g. NASCO) feed their frogs at the end of the day so
the frogs consume and digest their food undisturbed during theevening/overnight. This seems to more closely reflect the natural behaviour of
this species and is recommended.
Frogs and their surrounding water should remain undisturbed for a
3-5 hour period after feeding.
Feeding of frogs should preferably take place towards the end of
the day – in this way, frogs can be observed to be eating
adequately and then be left undisturbed to digest their meal.

38

Guidance on the housing and care of the African clawed frog Xenopus laevis)

4.9

Environmental enrichment

4.9.1 What is it?
Environmental enrichment is a means of enhancing the quality of captive
animal care by identifying and providing the environmental stimuli necessary
for optimal psychological and physiological wellbeing (Shepherdson, 1998).
4.9.2 Why is it important?
The provision of space in terms of suitable cage or tank size alone, is notenough to ensure animal wellbeing, and must not be considered in isolation.
What is provided within the housing by means of environment complexity isalso critically important. Allowing animals the opportunity to exhibit a range of
species typical behaviours can reduce the negative effects captivity may haveon health and welfare. This is also important for the science - ‘happy animals
make good science’ (Poole, 1997). Animals whose wellbeing is compromised(e.g. by being placed in unsuitable social groupings or an inadequate
environment) are often physiologically and immunologically abnormal and thus
experiments using them may reach unreliable conclusions.
Despite the wide acceptance and promotion of environmental enrichment, itremains an approach largely applied to mammals, with relatively little having
been developed for other groups such as reptiles and amphibians (Hayes etal, 1998). In terms of complexity of both environment provided and behaviours
shown, the Xenopus laevis frog can seemingly be catered for fairly simply.
This belief is based on the information currently available regarding the natural
behaviour of Xenopus laevis and their perceived needs. There may be a bias
though, as humans are able to better empathise with the more familiar
mammals than with amphibians, and may not be aware of, or may
underestimate, the actual complexity of amphibian requirements.
Importantly, it has been shown that these frogs prefer an enrichedenvironment to a barren tank, and that previously evaluated enrichment
can be provided without any detrimental effect on egg production or
quality (Brown & Nixon, 2004).
4.9.3 Hides and refuges
Frogs are a prey species, therefore it is highly important to provide them with aform of shelter to retreat and hide in. Despite this, a study of US
establishments by Major & Wassersug (1998) found 54% were providing noform of refuge for adult Xenopus laevis. There are also many frogs in UK
establishments who are not currently provided with a hiding place. One reason
is that there may be concerns about the possible release of toxicants into thewater or that frogs may injure themselves when these items are in the tanks.
However, the plastic pipes used commercially to safely deliver water to point ofuse for human drinking can be easily obtained and adapted, and where
provided, appear to be frequently utilised by the frogs (Brown & Nixon, 2004).
39

Guidance on the housing and care of the African clawed frog Xenopus laevis)

The piping is extremely tough, durable and long lasting and has a very smoothsurface, so the frogs will not harm or scrape themselves on it (one of theprerequisites of a good enrichment device). Ceramic tiles or terra cotta pots
are also stated to provide suitable refuges and possible to decrease stress inthese animals (Schultz & Dawson, 2003). These items should only beconsidered once their potential for leaching agents toxic to amphibians into thewater has been properly evaluated.
Containers used for storing human food can also be customised and used as
improvised ‘caves’ for the animals, as these too will not affect the water quality(Schultz and Dawson, 2003).

Photo credit:
M.Brown(MRC)

Providing cover from above and additional hiding for frogs can also be
achieved by placing floating objects in the water (Brown & Nixon, 2004).
These vary from commercially available plastic 'lily pads' to simply cutting up
black bin liners into various floating shapes. Objects placed on the surface of
the water however, must still allow frogs to easily access the surface of thewater to breathe.
Frogs must be provided with refuges to hide in or under (Kreger,
2002; CCAC, 1984; Schaeffer 1999).
4.9.4 Additional environmental complexity
The addition to the tank of a few medium-to-large stones or rocks will break up
the physical monotony of a plain tank (Beck, 1994). If personnel haveconcerns that sterilisation of such items will not fully prevent leaching ofminerals or kill any bacteria present, then perhaps plastic moulds of stones orrocks can be created from those materials previously assessed and classified
as safe. If placed an inch or so away from the walls of the tank, the frogs cansqueeze into the spaces between the objects and the tank floor (Kaplan,
1993). Enrichment structures should have smooth surfaces and roundededges to reduce the risk of injury to the animals (Council of Europe, 2004).

40

Guidance on the housing and care of the African clawed frog Xenopus laevis)

A tank floor substrate of small stones should not be used as these can be
accidentally ingested (Beck, 1994) and may also be impractical for regularcleaning. Furthermore, it is clear from ‘choice tests’ that frogs do not prefergravel flooring (Brown & Nixon, 2004).
No rest area at the water’s surface is required in the tank as Xenopus frogs
are totally aquatic (University of California, 2000).
Further research is required into the relative preferences of
Xenopus laevis frogs for different objects in the surrounding
environment

4.10 Assessment of health and disease prevention
An animal’s welfare can be compromised by poor health. This section will
therefore deal with the identification of discomfort or clinical signs of disease
and the subsequent treatment of common diseases in Xenopus laevis frogs.
Before frogs are acquired a veterinarian with a knowledge of Xenopus
health and welfare should be consulted to agree issues such as healthstatus of the incoming animals and those already at the establishment,
how animals will be monitored, and the potential use of preventive
medicine and treatment strategies. A vet should again be consulted
before any treatments are given to the animals and animal carers should
be made aware of any restrictions on the use of medicines.
Amphibians are prone to many diseases that are often difficult to treat andcontrol (LASA, 2001). It is therefore far easier to prevent, than treat disease inthese frogs (DeNardo, 1995). Xenopus may carry a range of pathogens
without the development of disease, until there is physiological upset causedby additional environmental stressors (Tinsley, 1996). This can occur as aresult of stress through overcrowding, improper handling, over-use, or poor
water quality (Xenopus Express, 2003). Providing the most suitable housing
conditions and care is vital for maintaining a healthy frog population (Sive et al,2000).
4.10.1 Diagnosis of ill healthDiagnosis of disease and assessment of wellbeing can be difficult as Xenopus
have little ability to express their state of welfare (The Berlin Workshop, 1994).
Xenopus laevis is a potential ‘prey’ species and therefore may well have
evolved adaptively to hide overt signs of distress, injury or susceptibility. Thismeans that disease may become established, and pathogenesis may develop
to an extreme degree before the frog exhibits obvious ill-effects (Tinsley 1996).
It is also harder for human carers to be able to recognise and assess signs ofsuffering the further away from humans (and mammals in general) the animals
are on the phylogenetic scale. This is particularly so in those species which
41

Guidance on the housing and care of the African clawed frog Xenopus laevis)

are not very animated or vocal during distress or sickness. Lack of acomprehensive understanding of amphibian behaviour, physiology andmedicine, or inability to recognise health problems, may mean users obtainnew animals rather than attempting to investigate or treat medical problems inexisting animals (Crawshaw, 1991). Significant reductions in the numbers ofanimals used can be achieved when animals are kept healthy and when earlysigns of disease are recognised and appropriate veterinary care is provided.
Frogs should be observed daily for indicators of poor health.
4.10.2 What does a healthy frog look like?
It is very important to know how a healthy individual animal looks and behavesin order to be able to make a comparative judgement. Such animals shouldbe:

placid, with moderately slimy skin and a nice pear shape (Sive et al, 2000)

4.10.3

Some key signs of ill health in Xenopus laevis

Key signs to look out for include:










dull skin with subcutaneous haemorrhaging (particularly petechial or‘pinpoint’ haemorrhages on the ventral surfaces of the legs)
failure to feed properly or weight loss
erosion or tremors at its extremities (e.g. legs and feet)
open cuts, lesions or abrasions of the skin or ulcerations
excessive coelomic cavity distension / swelling ('bloating')changes in activity level e.g. lethargy
postural changes
skin discoloration or flaking
diminished avoidance response and righting reflexes

Jumpy frogs, frogs with dry or excessively slimy skin, bloated frogs, and frogs
that look grey and thin or reddish are not healthy and should not be used inexperimental procedures as this would lead to further deterioration of theanimals’ condition. Additionally, eggs collected at this time, for example, would
be generally unsuitable for experimental purposes (Sive et al, 2000).

42

Guidance on the housing and care of the African clawed frog Xenopus laevis)

4.10.4

Common diseases

Sive et al (2000) and Wright & Whitaker (2001) observe that the two mostcommon diseases in Xenopus are bacterial septicemia and nematodeinfection. Symptoms and some suggestions in the literature for treatment ofthese conditions are detailed in the following table:
Red leg syndrome (bacterial septicemia)
Background
•
•
•
•
•
•

This is one of the most common diseases for frogs (and amphibians in general) in captivity
It is not a single disease entity but a septicaemia caused by a number of gram-negative orgram-positive bacteria - the most commonly associated bacteria being Aeromonas
hydrophila (Mahony, 1994).
The bacteria forms a normal part of the fauna encountered by Xenopus in theirenvironment and only under immunosuppressive conditions does it become pathogenic(Kelly, n.d.).
Unsanitary conditions, prolonged exposure to cold conditions, overcrowding and otherfactors that lead to stress can increase the incidence of red leg syndrome (Xenopus Zoo,
2003).
Red leg syndrome can spread through a whole colony and is only treatable if caught early(Xenopus Express, 2003).
Sive et al (2000) recommends that infected individuals must be isolated immediately.

Clinical signs••

Include cutaneous haemorrage (especially in the legs and feet), subcutaneous oedema,
and trembling (initially in the limbs). Frogs may also appear listless, bloated and may lose
their appetite (Xenopus Zoo, 2003).
In some instances death can occur without any visible evidence of illness (Mahony, 1994).

Proposals for prevention•

University of Arizona guidelines (2001) suggest placing frogs in a 0.6% calcium
hypochlorite solution (or a 0.06% sodium chloride solution) when first acquired will help
reduce the growth of the gram-negative bacteria Aeromonas hydrophilia and thus theoccurrence of red leg syndrome.
• Care should be taken not to damage the skin of these animals as this might lead to amore severe systematic infection.
Recommendations for treatment (under appropriate veterinary instruction)• Xenopus laevis frog colony care (2003) suggest that the salt solution of the tank water canbe increased to 100mM, and 100microgrammes per ml of oxytetracycline should be added
to the water for a week. They further state that antibiotics may also be used e.g.
tetracycline oral: 1mg/5g body weight for 5 days.
Note: Antibiotics should only be used after evaluation of bacterial culture and sensitivity.
Appropriate drug selection, dosage and treatment length will minimise the development ofantibiotic resistance by bacteria.
• Alternatively, for milder cases, LASA (2001) recommend frogs are placed in a bath of 1g/L
potassium permanganate. The infected animal should be dipped for 30sec on alternate
days, making sure that the water in the treatment tank is the same temperature as thestock tank.
• LASA (2001) also observe that Malachite Green can be effectively used as a 15 second
dip at 0.2g/L on 3 successive days 11.
• A salt bath is also a good treatment for many of the problems that are found inamphibians. Treatment may either be a 10- minute dip in a solution of 25g salt/L or a bathfor up to 3-5 days in a solution of 5-10g salt/L (LASA, 2001).

11

Although Malachite Green has been recommended as an effective treatment for bacterial
infection in Xenopus laevis frogs, its use for treating similar conditions in commercially
produced fish has been banned due to the substance's carcinogenic properties. Laboratory
43

Guidance on the housing and care of the African clawed frog Xenopus laevis)

Nematode infection (e.g. Pseudocapillaroides xenopodis)
Background••••In many cases, the nematode (roundworm) parasite involved is non-pathogenic though its
prevalence can be life threatening. The infection is highly contagious.
An opportunistic infection may have arisen as a result of damage to the protective mucousof the skin and the infection can be maintained and increased by the ingestion of sloughed
epidermis containing the parasite’s eggs (Cunningham et al, 1996).
This pathogen has a direct life cycle, therefore larval nematodes can penetrate into intact
skin (Wright & Whitaker, 2001).
In frogs with low levels of infection, the first clinical signs may not appear until 12 to 18
months after the initial infection (Wade, 1981, in Cunningham et al, 1996).

Clinical signs••

Include grey, rough, flaky skin usually starting on the thighs, excessive shedding of skinand rapid weight loss (Xenopus Express, 2003).
Thread-like, generally whitish bodies around injection sites (Sive et al, 2000).

Proposals for prevention•

Continuous drip-through water systems (whether re-circulating or not) have the advantage(over standing water tanks) of removing much of the sloughed skin and infective larvae
before it can be ingested by the frogs. As sloughed skin may contain pathogens, reduced
ingestion of skin could lead to reduced potential for infection.
Recommendations for treatment (under appropriate veterinary instruction)• Treatment is most effective if administered immediately symptoms are recognised.
• Xenopus laevis frog colony care (2003) recommend the use of ivermectin at a dose of 0.2
micrograms per gram of body weight.
• Sive et al (2000) states that MarOxy solution can be used to good effect.
• Thiabendazole (at 0.1g/L water) has also been suggested as a possible treatment by
Xenopus laevis frog colony care (2003) (note: this substance has been observed to behighly irritating to the frog’s skin when administered at an improper dose).

4.10.5

Injury to skinIn the (hopefully) rare event of injury, either caused by other frogs or during
handling or procedures, healing (of skin abrasions for instance) can be aided
by bathing the individual in a mild saline solution (NASCO, 2003).
4.10.6

Responses to acute noxious stimuli

The behaviour of the frog may indicate that the animal is finding a procedure
noxious or painful. Green (2003) observes that behavioural responses of
Xenopus laevis frogs to aversive stimuli include:

startle reactions (sudden movements);
activity to physically avoid the stimuli (including 'escape' behaviours);
wiping or rubbing of the irritated area.

4.10.7

Disturbance caused by noise

The potential of laboratory equipment and human activity to disturb or causestress to these animals must be considered. The hearing range of these frogs
is 200-3900 Hz, with optima at 600 Hz and 1400-1800 Hz (Elepfandt 1996(b))
personnel may therefore wish to reconsider the acceptability of this substance, and if still
used, then give special regard to how the water treated with this substance is disposed of.
44

Guidance on the housing and care of the African clawed frog Xenopus laevis)

and they are sensitive to surrounding vibrations (through the lateral line
system). The range of hearing for healthy young humans is 40 to 20,000 hertz(Blakemore & Jennett 2001). Although hearing ability does decrease with age,personnel should be sensitive to most of the range of environmental
noises/vibrations that these frogs can perceive. They should therefore be in aposition to act to minimise them, for example, by limiting the disturbancecaused by air conditioning systems, industrial vacuum cleaners or radios.

A good understanding of frog health, including diseases,
symptoms and treatments, is necessary to minimise suffering ordeath.
Frogs should be regularly monitored for signs of ill health.
Personnel should be well informed regarding any pain or distressfrogs may experience as a result of experimental procedures. They
should be aware of the responses frogs are likely to show, what
the humane endpoints of the project are, and what to do when they
are reached.
Many diseases that Xenopus are susceptible to are caused or
exacerbated by stress. The importance of providing the most
optimal environmental and care conditions for the frogs istherefore paramount.
As this section has only dealt with the small number of the mostcommon diseases and infections found in Xenopus frogs, those with amore specific interest or expertise may wish to consult the following
resources for more detailed information:•

The Biology of Xenopus - Tinsley, R.C. & Kobel, H.R. (1996)
Oxford University Press, Oxford (UK)In particular:
Pages 233-261 - Parasites of Xenopus•

Laboratory Animal Medicine (2002) (Second edition)
American College of Laboratory Animal Medicine Series
In particular:
Chapter 17 - Biology and Diseases of Amphibians•

Amphibian Medicine and Captive Husbandry - Wright, K. & Whitaker,
B.R. (2001)
Krieger Publishing Company, Florida (USA)45

Guidance on the housing and care of the African clawed frog Xenopus laevis)

5

Scientific procedures

There are a number of procedures commonly carried out on Xenopus laevis
frogs. These include egg harvesting, oocyte collection and the induction ofanaesthesia. All of these have the potential to cause pain, suffering or distressand the opportunities for refinement therefore need to be explored. Thissection therefore sets out background information on the purpose of somecommon procedures, together with recommendations for refinements that will
help minimise and ideally avoid suffering.
In this context, it is important to note that amphibian behaviour in relation topain perception is difficult to recognise and largely undefined. For this reason,
specific clinical signs or objective means of pain assessment in Xenopus
laevis frogs have not yet been determined (Green, 2003). However, thecurrent understanding of pain in non-mammalian vertebrates, based on recent
anatomic, physiological, and biochemical studies, suggests pain perception in
amphibians is likely to be analogous to that in mammals (CCAC, 2004; Green,2003). In particular, amphibians have nociceptive mechanisms analogous tothose found in mammals, so it is justifiable to give amphibians the benefit ofthe doubt and assume that they are aware of and can suffer as a result of
pain. Invasive, potentially painful procedures (such as surgical collection ofoocytes) should therefore be subject to appropriate ethical review, andaccompanied by both anaesthesia and post-operative care including
appropriate analgesia (Council of Europe, 2003; CCAC, 2004).

5.1

Egg harvesting

Researchers, and particularly those in the fields of developmental biology,
embryology or genetics, generate a high demand for a constant supply of
Xenopus eggs. There are a number of techniques that may commonly be
associated with the procurement of these eggs. The main ones are:•••natural mating;induction of ovulation;the ‘squeezing’ of females.

Nowadays, only a small proportion of the fertilised eggs used in experimental
studies are obtained through natural mating using a live male.
5.1.1 Natural mating
Natural mating (i.e. placing a female with a live male frog to fertilise the eggs)
is a way of obtaining many different stages of embryos at once, with minimal
stress to the frogs (Sive et al, 2000).
When a female is receptive, she shows little preference for individuals andinstead will be attracted towards any advertising male (Tobias et al, 1998).
This is because under natural conditions competition between males for46

Guidance on the housing and care of the African clawed frog Xenopus laevis)

access to the relatively scarce receptive females results in the establishmentof a hierarchy where subordinates rarely advertise vocally (Tobias et al, 2004).

Amplexus occurs where the male and female frogs clasp together. This canlast for 12 hours or longer (Sive et al, 2000) with the male releasing sperm as
the female lays her eggs (which are laid singly).
Oogenesis (the formation, development and maturation of eggs) is acontinuous process under favourable environmental conditions and, given
suitable temperatures, ovary production is determined primarily by food supply(Tinsley and Kobel, 1996). This means that wild individuals may have welldeveloped ovaries at almost any time of the year if there is an abundance ofprey.
5.1.2 Induction of ovulation and mating behaviour
Leaving reproduction to occur naturally in the laboratory affects both when,and how many, eggs are produced. However, the hormone human chorionicgonadotrophin (hCG) can be used to overcome this, enabling laboratories tohave access to a near constant supply of eggs all year.
Female Xenopus laevis frogs can be induced to ovulate by injecting them withhCG (usually at a level of 500 I.U). Ovulation generally occurs within 36 hours
following injection (NASCO, 2003).
Males too can be stimulated to perform mating behaviour by injecting 50 unitsof hCG into the dorsal sac, a few days before being placed with a female.
Readiness for mating is indicated in the males by the production ofadvertisement calls (in the form of a rapid series of 'clicks') and theappearance of a dark pigment on the front legs.
5.1.3 Squeezing/milking the eggs from the female frog
Applying gentle pressure with the hand to the ventral and lateral sides of thebelly mimics male behaviour during amplexus and stimulates hormonallyprimed females to expel eggs. Egg-laying should begin within a minute of
beginning to massage the belly. The frog may either not move, or she may
push the eggs out in a couple of vigorous bursts (Sive et al, 2000).
The back of the frog should not be rubbed. Some eggs may be obtained in thisway but as there is little subcutaneous fat on its back, the animal is very likely
to bruise and may succumb to a secondary bacterial infection as a result of
disturbance to the epidermis (Sive et al, 2000). After induction of ovulation, thecloaca becomes red and swollen and is probably rather sensitive, so
personnel should avoid touching it during subsequent handling.
‘Squeezing’ or ‘milking’ of females speeds up the egg laying process, which
normally takes several hours. Obtaining a batch of eggs in this way alsomeans that they can all be fertilised at approximately the same time by mixing
47

Guidance on the housing and care of the African clawed frog Xenopus laevis)

them with the ground up testes of a previously euthanased male frog.
Simultaneous fertilisation is often required when studying egg development.
However, the above technique has negative impacts on the frogs because it
requires greater and more prolonged handling of the females, as well as
euthanasing the male frogs to obtain their testes (although the ground testicles
of one male can serve multiple females). Furthermore, it has been stated thatthe stress of the induction of ovulation and subsequent egg collection is
the most common cause of disease outbreaks in frogs (Sive et al, 2000).
To minimise the risk of disease, these authors recommend that females should
be kept in a bath of 20mM NaCl, supplemented with 5µg/ml gentamycin during
egg laying and for 12 hours afterwards. It is then imperative to keep frogs inisolation in clean water for 12-24 hours after egg collection and to monitor
them for signs of illness.
There is less potential for frogs to suffer when eggs are obtained
as a result of natural ovulation, egg laying and mating. The
requirement for ‘inducing’ these behaviours must be justified on
scientific grounds.
It is imperative that only those individuals experienced and trainedin the ‘squeezing’ procedure attempt this process. Harm andbruising can be caused to the frog through incorrect or too heavily
applied pressure. This is in addition to any stress caused throughpossible unsuitable handling techniques.
5.1.4 Egg quality
Obtaining good quality eggs from the animals is important, not only for helping
realise research objectives, but also to keep the number of animals used to aminimum.
The number of eggs laid is less important than the quality of eggs; forexample, a hundred good-quality eggs is far preferable to thousands of poor
quality eggs (Sive et al, 2000). Since the majority of Xenopus eggs produced
at research establishments are for experimentation not reproduction, ‘quality’is stated to refer to the ability of the eggs to remain viable after experimental
manipulation, such as centrifugation and microinjection (DeNardo, 1995). For
most of the academic areas in which these eggs are utilised (e.g.
developmental biology, anatomy, genetics studies) it is particularly importantthat a high percentage (i.e. > 80%) of the eggs are fertilised successfully, thatthe eggs undergo a clean and even first cleavage, and that they remain
normally developed at gastrulation.
DeNardo (1995) observes that 'it is currently unknown what factors influence
the quality of eggs; however, numerous husbandry parameters are often
manipulated in an attempt to improve egg quality. Such factors include diet,lighting, water salinity, water flow, frequency of cleaning, tank size, type ofhormonal stimulation, frequency of egg collection, and age of females’.
48

Guidance on the housing and care of the African clawed frog Xenopus laevis)

Some establishments obtain better quality eggs than others. Since egg quality
can have a direct effect on the standard of scientific research produced, it isimportant that the sharing and publication of information regarding factors
affecting egg quality is improved.
5.1.5 Frequency of egg collection
Many laboratories induce or naturally mate their females three or four times ayear as might happen in the wild, though others induce their females to
produce eggs more frequently. Constant breeding and egg production places
physiological pressure on females mated regularly (e.g. every four to six
weeks) and so this might affect the length of time females may be considered
to be productive.
Rest periods of two to three (Sive et al, 2000) or four months (Xenopus frogcolony care, 2003) are suggested as being required between ovulations.
Possibly, frogs used less intensively (e.g. only every three months) may
continue to provide eggs of good quality for a far longer time. Dawson et al(1993), however, note that the period between laying must not be too great, as
frogs who are not bred from for four to six months, tend to deposit a higher
percentage of necrotic eggs and the general embryo quality tends to bepoorer.
5.1.6 Age of females
It is possible for animals to reach sexual maturity within one year of hatching(Measey, 2004). However, initial batches of eggs from such young animals
may be of poor quality and therefore unusable (anon., pers.comm). For
reproduction purposes, animals aged between 2 and 5 years are commonlyused (Van Zutphen, 2001; LASA, 2001).
It has been suggested that frogs may only be useful for egg production
purposes for one or two years (e.g. Dawson et al, 1993) though sometimes
longer. Some ‘older’ females may have atretic (degenerating) eggs and cannot
be used for breeding (Kelly n.d.). Nevertheless, a number of laboratories
successfully keep and use their female Xenopus over a period of many years(e.g. 6 - 8 years, anon, pers.comm). Xenopus frog colony care (2003) statesthat frogs can be induced to lay repeatedly for several years providing they arehealthy and have suitable rest periods.
It is better to use a larger number of animals to produce a given
number and quality of eggs than to use fewer animals more
intensively. Minimising levels of individual animal suffering is more
important than simply reducing the numbers of animals used.
Good environmental record keeping is essential for Xenopus
colonies, since there are many factors that influence egg quality
and their mechanisms and interactions are poorly understood.

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Guidance on the housing and care of the African clawed frog Xenopus laevis)

It is unlikely good quality eggs will be obtained from female
Xenopus frogs before they are at least 18 months to 24 months ofage.
As long as the animal remains healthy and egg quality is high,frogs can continue to be used. There is no upper age limit.
A minimum interval of 8 – 10 weeks12 is recommended between
episodes of induced egg laying in these frogs.

5.2

Oocyte collection

One of the major uses of Xenopus laevis frogs in biomedical research is toobtain oocytes (immature eggs not yet capable of being fertilised) for researchwithin molecular biology, biophysics, ontogenesis (Elsner et al, 2000) and inmRNA expression applications.
5.2.1 Surgery to obtain oocytes
The process of oocyte collection is more invasive than the process of egg
collection, as it requires surgical removal of ovarian tissue under anaesthesia
and the collection of developing stage I to VI oocytes from within the body.
Although all stages of eggs are present at any given time, investigators
typically require healthy looking mature stage VI oocytes, which generally
account for around 30% of a clutch (Green, 2002). Oocyte removal for ascientific purpose by laparotomy surgery (undertaken following injection 48
hours prior with low level hCG) with recovery can only be done in the UK
under Home Office licence. As a fully mature female Xenopus can contain as
many as 30,000 oocytes (LASA, 2001) which researchers may want to takeover a period of time, animals may be subjected to repeated episodes ofsurgery during their lifetime (Green, 2003).
There is a consensus that multiple episodes of surgery with survival on
animals should be discouraged, but exceptions are often granted for oocyte
removal from Xenopus (e.g. University of Arizona, 2001). The rationale for thisis that surgery is usually rapid (normally around 10 minutes or less), theoocytes are located in the immediate area of the incision and thus the othervisceral organs should be undisturbed, and because the animals are observed
to apparently quickly return to normal feeding and activity. However, in theabsence of effective behavioural indicators of pain in Xenopus, it is important
not to assume that there is no surgical trauma. When deciding whether toundertake multiple surgeries on a single animal or subject a larger number ofanimals to fewer occasions of invasive surgery, the total number of animals
used must be considered relative to the pain or distress experienced by an
12This recommendation (and also the interval recommended between episodes of surgery foroocyte collection - see next page) is based on the minimum periods suggested in the relevant
literature and on discussions with personnel using and caring for frogs on how the impact ofthese procedures can be reduced. They are designed to be the minimum advisable in order toallow the individual animal sufficient time for recovery. The period should be extended if
possible.
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Guidance on the housing and care of the African clawed frog Xenopus laevis)

individual animal (NIH, 2001). The welfare, experiences and potential
suffering of each individual frog must be considered.
The number of times a frog is subjected to surgery and recovery for oocyte
collection varies among studies and between research establishments aroundthe world. Frogs sometimes have oocytes collected up to six times (Elsner etal, 2000; University of California, 2003; University of Arizona, 200113, NIH,
2001). If repeat surgery is undertaken, the use of individual frogs is generally
rotated so that the interval between episodes of surgery on each animal is
maximised (NIH, 2001).
In the UK, the wellbeing of frogs is better protected through greater restrictions
on the frequency of surgery. Surgery is usually restricted to two occasions perfrog, with the second being undertaken under terminal anaesthesia. Specificscientific justification must be presented for allowing a frog to undergo andrecover from laparotomy surgery under general anaesthesia on more than one
occasion.
Many establishments seek to avoid causing suffering to the animals throughsurgical trauma, and undertake oocyte collection only once per animal under
terminal anaesthesia.
If oocytes are to be taken from a frog on more than one occasion, it
is recommended that the second collection takes place under
terminal anaesthesia.
Where specific scientific justification has been presented forcollecting oocytes (with recovery from general anaesthesia) fromthe same frog on more than two occasions, a minimum interval of
12 weeks is recommended between episodes of surgery.
i)

Post-operative care

The University of Arizona (2001) suggests that post-operative care after
surgery should, as a minimum, include singly housing animals and monitoringfor 24-48 hours. During this time the frogs should be closely and regularly
observed, but if possible, be left as undisturbed as possible. After this time, it
is stated most Xenopus swim well enough to be returned to regular housing(Green, 2003). Skin sutures and wound clips, if non-absorbable, must be
removed two to three weeks after surgery (Green, 2003).
As much serious consideration must be applied to surgery on
Xenopus as to any other species – the justification and necessity
for surgical procedures should be critically evaluated and theprovision of good post-operative care is essential.
13

The University of Arizona (2001) sets a limit of six surgeries per animal (three on alternatingsides with the sixth surgery being a terminal procedure) and stipulates that animals should begiven no less than one month to recover between surgeries.
51

Guidance on the housing and care of the African clawed frog Xenopus laevis)

5.2.2 Oocyte quality
Since procedures that can cause stress and discomfort are required to obtainoocytes, it is essential to maximise oocyte quality so that the frogs do not
suffer needlessly. Some laboratories undergo periods where oocytes
produced are consistently of poor quality and the reason is often hard todetermine.
Poor quality oocytes may be characterised by any of the following (Waterman
et al, 2001):






discoloured or speckled yolks;
bursting oocytes (white, bloated eggs);
stringy coats and attachment to other oocytes;
small size;
dirty jelly coats with debris adhering to the surface.

Possible causes can be parasitic disease in the colony, water quality14, watertemperature or contamination with bacteria such as Pseudomonas fluorescens
via contact of the ovary with the frogs' skin during surgical removal.
Elsner et al (2000) emphasise the importance of ensuring pre-operative skinantisepsis, which reduces the development of marbled spots and prematurecollapse of electric resting potential in oocytes. Pre-operative skin antisepsis
should be established using very dilute povidone-iodine solution, sterile drape
and instruments, and careful consideration of the antibiotic supplement in the
Barth medium.

5.3

Blood collection

Blood collection in amphibians is often difficult because of their low bodyweight and poor accessibility of vessels (University of Arizona, 2001). The
clipping of the toe web of frogs with a sharp fingernail clipper to collect a few
drops to 0.5ml of blood is considered an acceptable method by someestablishments (e.g. University of Arizona, 2001). However, it is unclear how
commonly this procedure takes place, or to what extent it causes the frog painand distress. As a precaution, local anaesthetic should be used for thisprocedure (Wolfensohn & Lloyd, 2003).
Cardiac puncture under general anaesthesia is usually employed where agreater volume of blood needs to be obtained. This should only be used as aterminal method. Death after exsanguination should be ensured by theadministration of an overdose of an anaesthetic or other physical method
before the animal recovers.

14

see specifically Godfrey & Sanders (2004) study on the effect of general water hardness onoocyte and embryo firmness and successful development
52

Guidance on the housing and care of the African clawed frog Xenopus laevis)

5.4

Injections

Injections are normally given to frogs via the dorsal lymph sac because
substances administered via this route should be rapidly absorbed into theblood (University of Arizona, 2001). This is also the common site for injectionof hormones to stimulate breeding in Xenopus.
Intramuscular injections are generally administered into the thigh muscles,
while intraperitoneal injections may be given while the frog is placed on its
back in the hand with the head directed downward (Wolfensohn & Lloyd,2003). Inserting the needle into the groin area lessens the likelihood ofpuncturing any internal organs (University of Arizona, 2001).
Further information on refinement of the administration of substances,including by injection, can be found in Morton et al (2001).

5.5

Analgesia and anaesthesia

5.5.1 Analgesia
Little research has been undertaken into the efficacy of analgesic agents inamphibians. Extrapolating doses of analgesic agents from studies using otherspecies can be dangerous and there are concerns over potential adverse side
effects that could have a negative impact on the animals’ health or welfare orthat could affect oocyte or egg production and quality (Green, 2001; Wright &Whitaker, 2001).
There are few recommendations in the literature for appropriate analgesia,
and where suggestions are made, little detail is provided on how successfully
the agents have been used and how their efficacy and potential side-effects
have been measured. For example, it has been suggested that xylazine is apotent and appropriate analgesic in ‘frogs’, being very effective for up to 24
hours duration when delivered at a dose of 10 mg/kg (University of Minnesota,2003). However, there is no assurance given that it is suitable for all species.
Elsewhere, CCAC (2004) guidelines suggest that 2% lidocaine applied to thesurgical site might be an effective analgesic for some procedures. Given thelack of additional guidance provided, the potential use of either of thesesubstances for providing pain relief in Xenopus laevis frogs requires further
analysis.
There is no evidence that whole body cooling of frogs reduces pain or isclinically efficacious (AVMA, 2001). Hypothermia should not be used as amethod of analgesia.
It is essential to recognise that amphibians need, and should have,appropriate perioperative pain relief.
Relevant animal care and use committees should regularly review
and discuss the possibility for trials of potentially useful agents.
Trials should take note of changes in behaviour and egg quality inresponse to the use of different analgesic agents.
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Guidance on the housing and care of the African clawed frog Xenopus laevis)

More research is necessary to determine appropriate analgesic
agents and doses for Xenopus frogs, and to disseminate relevant
information on suitable protocols.
5.5.2 Anaesthesia
Amphibian anatomy and physiology gives rise to some special considerations
in relation to anaesthetising Xenopus frogs. Some key points are set out
below.
Considerations



Xenopus laevis frogs respire through both the lungs and the skin. They
usually rely on lung respiration for the majority of normal gaseous
exchange, but this can be affected during periods of anaesthesia. It istherefore essential to ensure that cutaneous respiration (i.e. through theskin) is not also interrupted. The animal should be kept moist whilst underanaesthesia (Johnson, 1991) by placing wet tissue in contact with the skin(Council of Europe, 2004).



Fasting of frogs prior to anaesthesia is recommended as frogs who havebeen disturbed may regurgitate food, which fouls the water and increases
the chance of inhalation of foreign material while the animal is being
anaesthetised (Smith and Stump, 2000).

•

Since amphibians are ectotherms, the environmental temperature during
anaesthesia will affect the frog’s metabolism. This, in turn, influences therate of absorption and excretion of the anaesthetic agent and its
subsequent effectiveness.

Successful anaesthesia in Xenopus laevis is judged by loss of righting
reflexes and respiratory effort with subsequent slowing and halting of gular(throat) movements (LASA, 2001; University of Arizona, 2001).



The length of time required for recovery from anaesthesia depends on theanaesthetic agent, frog’s life stage, environmental temperature and depthof anaesthesia. Frogs should be propped on wet tissue or foam rubber toprevent drowning during recovery (LASA 2001). They should not be
returned to their tank until completely recovered.

Comments regarding suggested anaesthetic agents
MS-222® 15(tricaine methane sulphonate) is the most common and convenientanaesthetic used for Xenopus laevis 16 (Halliday, 1999; CCAC, 2004). It can
15

Argent Laboratory Redmond, WA
There are some concerns that MS 222 affects Xenopus oocytes by inducing maturation,
however, according to the current literature these concerns have not been properly evaluatedor verified and so MS 222 use should not be avoided (Martin 1995).

16

54Guidance on the housing and care of the African clawed frog Xenopus laevis)

be administered by injection (50-150mg/kg) or immersion (300-500 mg/L
water) (Wolfensohn & Lloyd (2003). In the latter case, induction should takearound 5 minutes, though others suggest it could up to 20-30 minutes (LASA2001). MS-222 is acidic when dissolved in water, and so it should be bufferedwith 50ml of 0.5 M NaHCO3/L (sodium bicarbonate) to a pH of between 7 and
8 (O’Rourke 2002).
A frog should recover from MS-222 within 15 to 30 minutes after being rinsed
in clean water (Wolfensohn & Lloyd 2003), though some individuals may takelonger.
Most establishments are content with the efficacy of immersion in MS-222.
However, the induction of anaesthesia in frogs may sometimes take up to half
an hour at recommended concentrations (Smith & Stump 2000, LASA 2001),
so attempts have been made to develop alternative methods.
Isoflurane - the topical application of liquid isoflurane has been suggested bySmith & Stump (2000) as a viable alternative method. A single application of
isoflurane (dose range 0.03 - 0.06 mL/g) applied to an absorptive pad withimpermeable backing so that the pad is saturated, then the saturated side isplaced in contact with the skin. This provides a working anaesthetic time
averaging 6 minutes that Smith and Stump deem sufficient for shortprocedures such as obtaining oocytes.
Bubbling isoflurane into the water has also been attempted, but the frogs
showed repeated and vigorous escape behaviours (interpreted as a sign ofdistress) and did not reach a suitable depth of anaesthesia (Smith & Stump
2000). Administering isoflurane in this way should therefore not be attemptedon welfare grounds. Intracoelomic or subcutaneous injection of isoflurane canbe fatal to Xenopus or can lead to prolonged recovery times of up to 15 hours.
Given these results, these methods too should not be utilised, whilst thehumaneness of applying isoflurane via an absorptive pad should be further
scrutinised.
Ketamine or Telazol® (tiletamine/zolazepam) should be restricted to minor
procedures (e.g. radiography), as animals sedated with these drugs may
remain sensitive to pain, even at high doses (Wolfensohn & Lloyd 2003).
University of Arizona (2001) suggested that the surgical use of these drugs
should therefore be limited to pre-anaesthetic use only.
Hypothermia is not supported by available published articles as a clinicallyefficacious method of anaesthesia in this species (Martin 1995). As there areother, more appropriate protocols, hypothermia should never be used for thispurpose.

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Guidance on the housing and care of the African clawed frog Xenopus laevis)

At the time of writing, MS-222 appears to be the anaesthetic agentof choice in most circumstances.
Current in-house protocols for Xenopus anaesthesia or analgesia
may not be the most appropriate or effective. It is very important toensure that the literature is regularly reviewed so that protocols
can be updated to take account of new and better agents, dosing
regimes or routes.

5.6

Euthanasia

5.6.1 The principle
The word euthanasia means “a gentle or good death” and should be regarded
as an act of humane killing with the minimum of pain, fear and distress(European Commission 1996). Killing an animal has the potential to cause
substantial pain and distress if it is done incompetently or by using an
unsuitable method. Whoever carries out euthanasia should therefore beappropriately trained, competent and willing to use the approved method
deemed to cause the minimum stress or pain to the animal.
When deciding upon the method of euthanasia, consideration should be given
to the following points:












There must be minimal psychological stress on the animal (and any others
who may be present – it may be necessary to remove the animal to beeuthanased, although this will need to be balanced against any stresscaused by such movement)
Loss of consciousness must ensue as rapidly as possible
Death, following loss of consciousness, must be as rapid as possible
Death must occur without causing pain
The method must be reliable and the animal must not regain
consciousness
There must be minimal psychological stress to personnel and any
observers
It must be safe for personnel carrying out the procedure
It must be compatible with the requirements of the experiment
It must be compatible with any requirement to carry out further studies on
the tissues
It should be simple to carry out, with little room for error(Note: there may be conflicts between some of these points in practice - themost appropriate method should be determined on a case by case basis,
giving animal welfare top priority).

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Guidance on the housing and care of the African clawed frog Xenopus laevis)

5.6.2 Methods for euthanasia of Xenopus laevis frogs
Acceptable methods for the euthanasia of amphibians have been listed (e.g.
Home Office 1997) as either:• overdose of an anaesthetic using a route and an anaesthetic agent
appropriate for the size and species of animal;• concussion of the brain by striking the cranium - in amphibians up to 1kg with destruction of the brain before the return of consciousness
Figure 8: Key points from the literature relating to methods that have been
recommended for the euthanasia of Xenopus laevis frogs
Injectable agents• Injection of tricaine methane sulfonate (MS 222) either intravenously or intraperitoneally.
Where MS 222 is used as an injectable agent, 200-300mg/kg of a 1% buffered solution has
been recommended (e.g. Stanford University 2003).
• Sodium pentobarbital (60-100 mg/kg) may be injected into the dorsal lymph sacs orintracoelomically (Sanders 2004(b), CCAC 2004).
• Pentobarbitone must not be given intramuscularly as it is very irritant to the tissue and thiswill cause the animal pain (Wolfensohn and Lloyd 2003).

Immersion methods• Potential stress for an animal (i.e. through handling and injection) can be reduced if drugs
are administered by dissolving them in the water in which the amphibians are placed(European Commission 1997) NB. some agents may be irritant, e.g. Isoflurane.
• The most common and preferred method for euthanasia in Xenopus laevis frogs is
immersion in an overdose of MS 222. This method appears to be one of the least stressful
to the animals involved (Wright & Whitaker 2001). A concentration of >3g/litre (Schultz2003) or 0.2% solution should be used for at least three hours (Halliday 2003, LASA 2001).
The agent is absorbed into the animal through the skin. To prevent the frog suffering pain
or irritation from this agent (which is low in pH), MS 222 should be buffered to near pH 7with sodium bicarbonate. As the immersion time needed to assure death can range from20 minutes to three hours, many researchers may use MS222 as an anaesthetic followed
by a physical method of euthanasia (Stanford University 2003).
• Benzocaine hydrochloride is water-soluble and has been used directly for euthanasia – abath with concentration >250mg/L (for at least 10 mins) has been suggested (AVMA 2001).
• Death after using a chemical method should be confirmed by a physical method (European
Commission 1996, DeNardo 1995).

Concussion methods• Concussion can be an effective and humane way of stunning amphibians if carried out by aperson who is well trained in the method (European Commission 1997)• In the UK, the Home Office (1997) authorises ‘concussion of the brain by striking the
cranium’, with the destruction of the brain before the return of consciousness, but advises
caution when using this method. The ‘Code of Practice for the Humane Killing of Animals
under Schedule 1 to ASPA 1986' (Home Office 1987) states that the 'brain of amphibians,
reptiles and fishes is extremely tolerant to hypoxia. It cannot be assumed therefore that theeffects of concussion will be irreversible, or that even subsequent decapitation would
necessarily destroy brain function in the time to avoid the return of sentience. In the case ofamphibians, reptiles and fishes, if the brain is not destroyed by the initial blow, there must
be no delay in destroying the brain by a penetrating probe or by a blow sufficient to cause asevere brain contusion with fracture of the cranial bones’.

57Guidance on the housing and care of the African clawed frog Xenopus laevis)

5.6.3 Unacceptable methods
Decapitation or double-pithing if used in isolation is no longer approved bymany ethical committees (e.g. University of Arizona) on welfare grounds.
Decapitation, by itself, does not produce immediate unconsciousness within
the severed heads of amphibians (Stewart, in UFAW 1989) and should
therefore only be used if the amphibian has been made unconscious first byother methods (European Commission 1997). To ensure brain death,
subsequent double-pithing (insertion of a sharp needle through the base of thebrain and the spinal cord) while the animal is still unconscious is advised.
Hypothermia should never be used either for the purposes of anaesthesia oreuthanasia in amphibians (Schaeffer 1999, AVMA 2001). This is because
cooling the body does not reduce the animal’s ability to feel pain (ANZCCART
2001, AVMA 2001). Cooling followed by freezing of frogs for euthanasia could
also cause suffering due to ice crystal formation, both on the skin and within
the body (European Commission 1997).
Formaldehyde solution - amphibians, even in larval form, should not be killed
either by placing them directly in formaldehyde solution (University of Arizona2001).
Carbon dioxide - may cause irritation to the skin and induction takes too long
Ether - this is irritant to the mucous membranes, as well as being highly
inflammable and potentially dangerous to humans
Chloroform - this is hepatoxic and carcinogenic and is dangerous to humans
Hyperthermia - this is slow and painful, so is inhumane
Exsanguination - may not render amphibians immediately unconscious,
which causes suffering and distress
Strangulation - impractical, is slow and is inhumane
Volatile inhalational anaesthetics - these are slow to act and may be
irritating to the skin (European Commission 1997)
Ketaminehydrochloride,
chlorbutonol,
methylpentynol,
2phenoxyethanol, tertiary amyl alcohol, tribromoethanol, urethane areother agents not considered acceptable (European Commission 1997)
Chloral hydrate - immersion in a container holding 2-3mm of a 3% solution ofchloral hydrate has been recommended by ANZCCART (2001). In contrast,
the European Commission (1997) suggests this agent should not be used byitself on amphibians as it lacks analgesic effects, is slow to take effect, large
volumes are required and it can cause distressing animal movements.
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Guidance on the housing and care of the African clawed frog Xenopus laevis)

Where there are reservations regarding a method (whether due to thewelfare of the frogs or the health and safety of personnel carrying out theprocedure) alternative methods should be sought.

Euthanasia - a “good” death - is as important for amphibians as it
is for mammals.
It is essential to recognise that (i) brain activity can persist forsignificant periods of time in amphibians, even though they may
show no behavioural responses, and (ii) the animals may besuffering if they are held in aversive chemical agents or haveundergone physical damage.
Wherever possible, non-aversive chemical means of euthanasia
should be chosen over physical methods. This is because there isless perceived scope for error, it will generally cause less sufferingto the animal and less distress to the personnel carrying it out.
Immersion in buffered MS 222 solution (preferably followed bypithing to ensure death) is a straightforward method that shouldcause minimal stress to both the frog and the person performing
the procedure. There should be exceptional justification given forusing any other method.

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6

Training of animal care staff and users

The importance of personnel having a good understanding of the animals withwhom they interact, cannot be underestimated. Persons not suitably trained
are likely to harm animals when performing procedures such as capture,marking or squeezing for harvesting eggs. Inappropriate care or behaviour will
have a direct impact on scientific validity, the achievable benefits of the work
will be reduced and the potential harms increased (Orlans 2001).
The general principles of training in good laboratory animal care and use apply
to amphibians but additional species specific training is essential. Personnel
should have a detailed species-specific knowledge of the natural history,
behaviour and requirements of the animals in their care. They should be up todate with the latest thinking and publications on best practice with regard togood housing and care and, where appropriate, with advances in therefinement of scientific procedures. A sound understanding of the importanceand practical aspects of the prevention, recognition and alleviation of ill health,pain and stress is also essential.
Currently, many training courses for laboratory personnel cover amphibians ingeneral. Although many laboratories may also provide some in-house training,
it could be considered beneficial that those working with animals should
undertake training specifically tailored to the species in their care and not
simply amalgamated into general groups of animals e.g. courses on‘carnivores’ or ‘amphibians’. As the use of Xenopus species is wide ranging
and expanding, there is scope for such species- specific training to be useful.
Training for those persons caring for and using animals should bespecies-specific. More focused courses should be available
addressing the behaviour and requirements of Xenopus laevis.

60Guidance on the housing and care of the African clawed frog Xenopus laevis)

7

Conclusions

There is a widely held belief that Xenopus laevis frogs are hardy (Wolfensohn& Lloyd, 2003) and robust (The Wellcome Trust, 2003), capable of tolerating a
range of environmental conditions and experimental procedures. There hashistorically been little investigation into the capacity of these animals to suffer
and failure to appreciate that they are at times housed in sub-optimal
environments.
As knowledge relating to Xenopus laevis increases so does the understandingof the physical and social complexity of these animals. The attitudes ofcontemporary animal technicians and those using the animals in research areshifting away from the traditional view that Xenopus laevis ‘have no special
housing requirements other than a tank of water’ (i.e. Davys, 1986). There is a
growing acceptance of the necessary requirement to first investigate, evaluate,
and then implement the most suitable conditions relating even to the finest
specifications and features of environmental design including tank material,tank size, degree of wall and floor transparency, lighting regime and spectrum,
provision and type of hiding refuges, and group composition. Failure to meet
species-specific needs can and will result in stress-induced disease, low
growth rate and poor feed efficiency (AWIC, 2001). In Xenopus frogs thismight subsequently lead to sub-optimal health and a decrease in the quantityand quality of eggs produced, thereby increasing animal usage and reducing
welfare. With the expansion in our understanding of this species and of theirrequirements for good health and welfare, has come a broad desire to try tobetter define and then provide the most appropriate housing and careconditions.
The knowledge and experience of animal technicians, veterinarians andresearchers using Xenopus laevis within laboratories is extensive, but thisvaluable resource is not currently always widely published, transferred orcommunicated in a co-ordinated way, either within the UK or further afield.
There have been some useful attempts to pool knowledge, for example with
the British Xenopus Group meetings and LASA workshops in the UK.
However, these formal opportunities for information exchange are irregularand there are many people ‘outside the loop’, using and caring for these frogs,
who need and want more information regarding perceived best practices.
Many key factors have been highlighted in this report, which need to beconsidered by those using and caring for these frogs. Existing guidelines andrecommendations have been summarised, with perceived best practicehighlighted where possible. Ultimately, further research, discussion andcontinued study must be encouraged in other areas where science based
consensus is currently lacking so that there can be a properly evaluated basis
for future recommendations. This report has argued, that from theperspectives of ethics, science and animal welfare, effort must be focused
towards discovering and then implementing the optimal protocols for housing
and care, not simply those that these animals can tolerate.

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8

Summary of recommendations

 Supply and transport••

Purpose-bred animals should be used in preference to animals taken fromthe wild - a researcher must be able to provide specific justification for theneed to use wild-caught individuals.
It is preferable for frogs to be bred at the establishment where they will be
used - this will avoid any potential risks to health or welfare associated with
their transport.
Transport containers must have an adequate air supply.
During transport frogs must be kept within acceptable temperature limits
and measures should be taken to prevent desiccation.
Transportation of frogs should be postponed if extreme weather conditions(e.g. extreme cold or heat) could threaten the health or welfare of theanimals.
Journey times should be the minimum practicable.
Adverse weather, mechanical problems or industrial action can cause
flight delays or cancellations and it is important to check and plan for such
eventualities.
Frogs being transported together in the same box should be at a similarstage of development, size and weight, and should not be overcrowded.
An appropriately trained and competent person must check the health andwelfare of frogs on arrival. Animals should then be closely monitored and,to avoid introducing disease, should be quarantined away from any
existing resident colonies.
To help prevent the potential spread of disease or bacteria, when animals
are transferred from shipping containers to the new holding tanks, thesphagnum moss and foam cubes with which they were transported should
not be transferred with them.
A minimum quarantine period of 30 days is advised for all new animals
entering an establishment with strong consideration given to extending
this period to 90 days for those animals obtained directly from the wild.
Routine husbandry procedures for frogs in quarantine should beundertaken after the resident colony has been attended to and must be
carried out with separate dedicated equipment.
Frogs should be observed daily for signs of ill health.



Housing and care••

Continuous 24-hour lighting must not be used.
A lighting regime of between 12 and 14 hours light and 12 and 10 hours
dark is recommended.
Where artificial lighting is used, a gradual brightening/dimming period ofaround 20 - 30 minutes in the morning/evening should be incorporated.
Any artificial lighting used should simulate outdoor sunlight levels, withconsideration given to including the ultraviolet (UV) part of the spectrum.

•

•
•
•

•
•

•
•

•

•

•

•
•

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Guidance on the housing and care of the African clawed frog Xenopus laevis)

•
•

•

•
•
•
•

•

•

•
•

•

•

•

•
•
•
•
•

Tanks should not be openly exposed to direct sunlight or very bright
artificial light and frogs should have access to darkened refuges.
A water depth of at least 8 inches is recommended for housing adult
Xenopus laevis frogs. This is based on current evidence and knowledge,
and should allow them to perform most normal behaviours.
Water temperature should not be maintained below 16ºC or above 24ºC,
with strong consideration given to providing a relatively controlledtemperature at a point between 18 and 22ºC.
Abrupt significant fluctuations of either room or water temperature should
be avoided.
Maintenance of a stable water pH between 6.5 and 8.5 is advised.
Water quality and pH level should be routinely monitored. Contingency
plans should be made in case of system breakdown or other emergency.
When devising water quality assurance schemes for frog housing, a water
expert knowledgeable of the local water treatment procedures should be
consulted.
Establishments using individual standing water tanks should review theircurrent water changing protocols. Although most establishments change
the water in these tanks at least three times a week, and maybe eveneveryday, the benefit of such frequent water changes in view of theincreased disturbance and stress caused to the animals has yet to becomprehensively evaluated.
If exposing frogs in tanks to constantly running water, the flow rate shouldbe the minimum required, in order to reduce the potential for causing theanimals distress.
Cleaning strategies should be designed to minimise disturbance anddistress to the frogs. Disinfectants should be used with extreme caution.
Opaque containers or those made of darkened plastic are preferable tocompletely transparent ones – in particular, the bottom of the container
should not be permeable to light.
Containers must prevent escape and should allow space for sufficientvolume and depth of water and for enrichment such as refuges to be
added.
Careful consideration should be given to whether identification ofindividuals is necessary, and is so, then to the method of identification
used, particularly where the methods are invasive and likely to cause a
degree of trauma.
Non-invasive methods of identification such as using group cards orphotographs of the dorsal markings of the frogs are the methods ofchoice.
Group housing of these frogs in relatively stable groups is advisable.
Overcrowding should be avoided.
It is important that animals kept together in the same tank are of similar
size.
Handling should be kept to a minimum and every precaution to avoid
stressing or injuring the animals should be taken.
Personnel should consider taking a precautionary approach and wear
gloves (non-powdered) whilst removing frogs from the water with softmesh non-abrasive nets.

63

Guidance on the housing and care of the African clawed frog Xenopus laevis)

•
•
•

•
•
•
•

Unnecessary movements of objects or hands over the tank should be
avoided.
Frogs and their surrounding water should remain undisturbed for a 3-5
hour period after feeding.
Feeding of frogs should preferably take place towards the end of the day in this way, frogs can be observed to be eating adequately and then be left
undisturbed to digest their meal.
Frogs must be provided with refuges to hide in or under.
A good understanding of frog health, including diseases, symptoms andtreatments, is necessary to minimise suffering or death.
Frogs should be regularly monitored for signs of ill health.
Personnel should be well informed regarding any pain or distress frogs
may experience as a result of experimental procedures. They should beaware of the responses frogs are likely to show, what the humane
endpoints of the project are, and what to do when they are reached.

Scientific procedures•

There is less potential for frogs to suffer when eggs are obtained as aresult of natural ovulation, egg laying and mating. The requirement for‘inducing’ these behaviours must be justified on scientific grounds.
It is imperative that only those individuals experienced and trained in the‘squeezing’ procedure attempt this process. Harm and bruising can becaused to the frog through incorrect or too heavily applied pressure. This
is in addition to any stress caused through possible unsuitable handlingtechniques.
It is better to use a larger number of animals to produce a given number
and quality of eggs than to use fewer animals more intensively. Minimisinglevels of individual animal suffering is more important than simply reducing
the numbers of animals used.
Good environmental record keeping is essential for Xenopus colonies,
since there are many factors that influence egg quality and theirmechanisms and interactions are far from fully understood.
It is unlikely good quality eggs will be obtained from female Xenopus
laevis frogs before they are at least 18 to 24 months of age.
As long as the animal remains healthy and egg quality is high, frogs cancontinue to be used. There is no upper age limit.
A minimum interval of 8 – 10 weeks is recommended between episodes of
induced egg laying in these frogs.
If oocytes are to be taken from a frog on more than one occasion, it isrecommended that the second collection takes place under terminal
anaesthesia.
Where specific scientific justification has been presented for collecting
oocytes (with recovery from general anaesthesia) from the same frog onmore than two occasions, a minimum interval of 12 weeks is
recommended between episodes of surgery.
As much serious consideration must be applied to surgery on Xenopus as
to any other species – the justification and necessity for surgicalprocedures should be critically evaluated and good post-operative care isessential.

•

•

•

•
•
•
•

•

•

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Guidance on the housing and care of the African clawed frog Xenopus laevis)

•
•

•
•

•
•

•

•

It is essential to recognise that amphibians need, and should have,appropriate perioperative pain relief.
Relevant animal care and use committees should regularly review anddiscuss the possibility for trials of potentially useful analgesic agents.
Trials should take note of changes in behaviour and egg quality inresponse to the use of different agents.
At the time of writing, MS-222 appears to be the anaesthetic agent ofchoice in most circumstances.
Current in-house protocols for Xenopus anaesthesia or analgesia may not
be the most appropriate of effective. It is very important to ensure that theliterature is regularly reviewed so that protocols can be updated to takeaccount of new and better agents, dosing regimes or routes.
Euthanasia – a ‘good’ death, is as important for amphibians as it is for
mammals.
It is essential to recognise that (i) brain activity can persist for significant
periods of time in amphibians, even though they may show no behavioural
responses, and (ii) the animals may be suffering if they are held inaversive chemical agents or have undergone physical damage.
Wherever possible, non-aversive chemical means of euthanasia should bechosen over physical methods. This is because there is less perceived
scope for error, it will generally cause less suffering to the animal and lessdistress to the personnel carrying it out.
Immersion in buffered MS-222 solution (preferably followed by pithing toensure death) is a straightforward method that should cause minimal
stress to both frog and the person performing the procedure. There should
be exceptional justification given for using any other method.

Training of animal care staff and users•

Training for those persons caring for and using animals should bespecies-specific. More focused courses should be available addressing
the behaviour and requirements of Xenopus laevis.

65Guidance on the housing and care of the African clawed frog Xenopus laevis)

9

Future areas for focus

The report has highlighted some of the main areas where further information
to determine best practice, or decisions based on what is already known, arerequired. The most important issues are listed below:


Better information on Xenopus laevis use. Government statistics
detailing the use of animals in scientific procedures each year should list
figures for amphibians at species level so a clearer understanding of thescale of use can be obtained.



Health considerations regarding the supply of animals.
Comprehensive data on the comparative health of wild-caught versus
purpose-bred Xenopus laevis frogs throughout the duration of their use in alaboratory setting should be recorded and critically evaluated. Currently,
many of the comparisons made regarding egg production and quality, andhealth, are anecdotal.

Volume of water to be provided. There is wide variation in the volume ofwater recommended per frog. More discussion and research is needed todetermine best practice, with due regard to the behavioural needs of theanimals.

Optimal temperature. More research and discussion is required todetermine the optimal temperature for keeping Xenopus laevis.

Control over microhabitat. Allowing an individual animal some control
over the immediate microhabitat is desirable. Current animal housing
design should be reviewed to determine if measures can be taken (such as
the inclusion of temperature gradients) within the constraints of thelaboratory environment to allow more scope for this.



Nocturnal behaviour. Establishments should review current lighting,feeding and tank cleaning protocols to determine whether there arealternative regimes which may better cater for the nocturnal nature of theseanimals.



Environmental complexity. Further discussion and research is required
into the relative preferences of Xenopus frogs for different objects in thesurrounding environment.

Pain relief. Greater efforts must be made to determine appropriate (safeand effective) analgesic agents and doses for Xenopus frogs and todisseminate information on suitable protocols.

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10

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