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GHENT UNIVERSITY

FACULTY OF VETERINARY MEDICINE

Academic year 2016 – 2017

NUTRITION OF CAPTIVE AMPHIBIANS

by

Justin KEULEN

Promoters: Dr. Arturo Munoz Literature Review

Professor Geert Janssens as part of the Master’s Dissertation

© 2017 Justin Keulen

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This thesis contains confidential information proprietary to the Universiteit Gent or third parties. It is strictly forbidden to publish, cite or make public in any way this thesis or any part thereof without the express written permission by the Universiteit Gent. Under no circumstance this thesis may be communicated to or put at the disposal of third parties. Photocopying or duplicating it in any other way is strictly prohibited. Disregarding the confidential nature of this thesis may cause irremediable damage to the Universiteit Gent.

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GHENT UNIVERSITY

FACULTY OF VETERINARY MEDICINE

Academic year 2016 – 2017

NUTRITION OF CAPTIVE AMPHIBIANS

by

Justin KEULEN

Promoters: Dr. Arturo Munoz Literature Review

Professor Geert Janssens as part of the Master’s Dissertation

© 2017 Justin Keulen

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TABLE OF CONTENT SUMMARY ........................................................................................................................................................ 1

SAMENVATTING .............................................................................................................................................. 2

INTRODUCTION ............................................................................................................................................... 3

LITERARY REVIEW .......................................................................................................................................... 4 1 Nutrient content of feed ........................................................................................................................... 4

1.1 Vitamins ........................................................................................................................................... 4 1.1.1 Vitamin A ................................................................................................................................... 4 1.1.2 Vitamin B ................................................................................................................................... 5 1.1.3 Vitamin D ................................................................................................................................... 6 1.1.4 Other vitamins ............................................................................................................................ 6

1.2 Minerals ........................................................................................................................................... 7 1.2.1 Calcium ...................................................................................................................................... 7 1.2.2 Phosphorus ................................................................................................................................ 7 1.2.3 Other trace elements ................................................................................................................. 7

1.3 Pigments .......................................................................................................................................... 8 1.4 Protein .............................................................................................................................................. 9 1.5 Fat .................................................................................................................................................... 9 1.6 Carbohydrates ................................................................................................................................. 9 1.7 Fiber ............................................................................................................................................... 10 1.8 Probiotics ....................................................................................................................................... 10

2 Effects of nutrition on health and reproduction ..................................................................................... 10 2.1 Health ............................................................................................................................................. 10

2.1.1 Metabolism .............................................................................................................................. 10 2.1.2 Integument ............................................................................................................................... 11 2.1.3 Immune system ....................................................................................................................... 11 2.1.4 Pathologies .............................................................................................................................. 11

2.2 Reproduction .................................................................................................................................. 14 3 Means of feeding and supplementing ................................................................................................... 16

3.1 Feeding .......................................................................................................................................... 16 3.1.1 Invertebrate prey ...................................................................................................................... 16 3.1.2 Vertebrate prey ........................................................................................................................ 17

3.2 Supplementing ............................................................................................................................... 18 3.2.1 Dusting ..................................................................................................................................... 18 3.2.2 Gut-loading .............................................................................................................................. 18

4 Other husbandry related factors ........................................................................................................... 18 4.1 Water ............................................................................................................................................. 18 4.2 Enrichment ..................................................................................................................................... 18 4.3 Ultraviolet (UV) ............................................................................................................................... 19 4.4 Other lightning factors .................................................................................................................... 19 4.5 Temperature .................................................................................................................................. 19 4.6 Disease .......................................................................................................................................... 19 4.7 Toxicities ........................................................................................................................................ 20

DISCUSSION .................................................................................................................................................. 21

REFERENCES ................................................................................................................................................ 24

APPENDIX ...................................................................................................................................................... 30

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SUMMARY A lot of species in the diverse amphibian class are threatened by different causes. Habitat loss, pollution and disease are some of the processes that cause amphibian declines. In order to reinsure the survival of these endangered amphibian species, captive breeding colonies are maintained. The husbandry of amphibians has however some difficulties. Next to the environmental factors, nutrition is also important in keeping captive amphibians healthy. The content of the diet can have an important impact on amphibian health. Deficiencies and excesses of certain nutrients can cause different disorders that impair amphibian welfare. Vitamin A, vitamin D3 and calcium contents are for example important to prevent lingual squamous metaplasia and metabolic bone disease; two of the most occurring pathologies in captive amphibians. There are however other nutrients important to provide optimal nutrition. Protein is for example important to provide energy and facilitate reproduction. Reproduction is a process that is influential through nutrition. The fecundity should be as high as possible to sustain captive breeding programs by producing offspring. There is however a paucity of information regarding amphibian reproduction; more studies should be executed to determine the effects of certain nutrients. The circumstances of feeding determine more or less the ingestion of food. Time of feeding, size of prey and the environment can play a role in the feeding process. In captivity one should mimic the situation to the wild to provide the best feeding conditions. Overall amphibian nutrition is a difficult subject; amphibians are vulnerable and requirements are mostly unknown; the ideal diet is hard to compose. There is however some information that might help to form a nutrition that keeps the amphibians healthy.

Keywords: Amphibian – Captivity – Nutrition – Health – Reproduction

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SAMENVATTING De amfibie klasse is een zeer gevarieerde groep van dieren. Over de gehele wereld kunnen we verschillende soorten terugvinden; ze leven in uiteenlopende milieus. Veel soorten worden echter met uitsterven bedreigt. Ziekte, verlies van leefgebied, vervuiling en klimaatverandering zijn mogelijke oorzaken van de ‘amphibian declines’. Dit maakt het noodzakelijk om een oplossing te vinden om deze soorten te preserveren. Een mogelijke oplossing voor dit probleem is het inrichten van kweekprogramma’s in gevangenschap om de bedreigde soorten te beschermen. Deze kweekprogramma’s kennen vooralsnog enkele problemen die het moeilijk maken om de diersoorten te kweken. Amfibieën zijn kwetsbare dieren die zeer gevoelig zijn voor het milieu waar ze in leven. Voeding is een belangrijk aspect waarmee rekening gehouden dient te worden in het management van deze dieren. Een overzicht van de beschikbare informatie wordt hier gegeven, er zijn echter nog vele hiaten in de literatuur. Verder onderzoek is nodig om de fysiologie van de gehele klasse, dan wel specifieke amfibie-soorten in kaart te brengen. Tot nu toe zijn de meeste fokprogramma’s in gevangenschap echter redelijk succesvol, er is dus informatie beschikbaar om een gezonde amfibie populatie te houden in gevangenschap. Zoals bij andere diersoorten gelden voor amfibieën enkele algemene regels, maar er zijn ook specifiekere zaken die voor deze dieren van belang zijn. Een eerste aspect is de samenstelling van het voeder, daarna zal uitgeweid worden over de effecten van deze nutriënten op de gezondheid en voortplanting. Tot slot worden er nog enkele zaken besproken die van belang zijn over het toedienen van voedsel, het soort voedsel en andere management factoren die verbonden zijn met de voeding. Enkele nutriënten die een rol spelen in het metabolisme van amfibieën worden in het kort aangehaald. Enerzijds kan een tekort aan nutriënten leiden tot aandoeningen bij de dieren, daarnaast kunnen excessieve hoeveelheden ook ongewenste effecten hebben. Allereerst zijn vitaminen van belang in het onderhoud van de dieren. Vooral vitamine A dient in voldoende hoge concentraties aanwezig te zijn in de voeding. Hypovitaminose A kan leiden tot squameuze metaplasie met symptomen (vermindering van voedselinname) tot gevolg. Ook teveel vitamine A in de voeding kan leiden tot gezondheidsproblemen. Vitamine D3 is eveneens een vitamine dat een rol speelt in het metabolisme van amfibieën. Het bot metabolisme kan aangetast zijn wanneer er tekorten aan vitamine D3 optreden. Deze tekorten kunnen leiden tot de ontwikkeling van ‘metabolic bone disease’. Dit is een aandoening waarbij er decalcificatie van het bot optreedt. Eveneens kan een overschot aan vitamine D3 leiden tot afwijkende verschijnselen. Mineralen zijn ook een belangrijke factor in de voedselsamenstelling. Vooral calcium dient voldoende aanwezig te zijn in de voeding. Deze speelt samen met vitamine D3 en andere factoren (o.a. ultraviolet straling) een rol in het voorkomen van ‘metabolic bone disease’. Fosfor is een ander element dat van belang is in het metabolisme van het bot. De calcium:fosfor ratio in de voeding dient op een bepaald niveau gehouden te worden om afwijkingen aan de beenderen te voorkomen. Naast vitaminen en mineralen zijn eveneens eiwitten en vetten nutriënten die nodig zijn. Met name de energievoorziening is van belang, maar proteïnen zijn daarnaast ook belangrijk voor anabole processen. Andere nutriënten die betrokken kunnen zijn worden slechts kort aangehaald (koolhydraten, vezels, …). Voeding is een belangrijke aspect in het verzorgen van amfibieën in gevangenschap. Om gezonde dieren te onderhouden en daarnaast voortplanting te faciliteren dient een adequaat dieet samengesteld te worden. Het metabolisme, de immuniteit en reproductie zijn processen die beïnvloedt worden door de voeding. Bij het samenstellen van de voeding dient men daarnaast ook rekening te houden met de voornaamste pathologiën: ‘metabolic bone disease’, squameuze metaplasie, neurologische symptomen en cachexie. Ander voedingsaspecten waarmee men rekening dient te houden zijn bijvoorbeeld het tijdstip van voeden, soort prooi, grootte van de prooi, de omgeving etc. Ook andere management factoren kunnen gerelateerd zijn aan de voeding of de belangrijkste aandoeningen die door een onjuiste voeding veroorzaakt worden. Verkeerde belichting, slechte waterkwaliteit of toxische stoffen uit de omgeving kunnen oorzaken zijn van een verminderde gezondheid. Over het algemeen zijn de kweekprogramma’s in gevangenschap succesvol in het onderhouden van de amfibieën. Bij het kweken van deze dieren dienen echter te allen tijde verschillende factoren optimaal te zijn om gezonde dieren en reproductie te garanderen. Naast de beschikbare literatuur ontbreekt er nog de nodige informatie; verder onderzoek in de toekomst is nodig om de amfibieën beter te begrijpen.

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INTRODUCTION The amphibian class is a very diverse group of animals. In all corners of the earth they life in different environments and adapt to it in their own way. In the current time however a lot of species are facing extinction. All over the world amphibian declines are seen. Poison dart frogs (Dendrobates spp.) and the Titicaca water frog (Telmatobius culeus) are examples of species that are on the International Union for Conservation of Nature (IUCN) Red List of threatened species (IUCN, 2016). Causes of these declines are disease, habitat loss, pollution, climate change, and others (Angulo, 2008). Some organizations (the Amphibian Ark, the Amphibian Survival Alliance, the Amphibian Conservation Action Plan, and others) are trying to preserve the amphibian diversity by protecting them in the wild or breed species in captivity. This captive breeding has as goal to maintain species and possibly reintroduce them into the wild. Although it has its limitations captive breeding is currently the most successful way of species preservation. Further research however is needed to gain more information regarding the rearing of amphibians. The diversity and special husbandry practices make the amphibians difficult to keep and breed. Nutrition is one of the influential husbandry factors. As we shall see in this review amphibian nutrition is a complicated issue. Different nutrients and factors need to be taken into account to provide the best health and facilitate reproduction. Vitamin A and calcium are two nutrients whom are involved in two of the most occurring pathologies. Metabolic bone disease and lingual squamous metaplasia can be caused by respectively calcium and vitamin A deficiency, but also other nutrients are involved. These are not the only pathologies that can occur; others are also regularly seen, making amphibian nutrition a tough subject. Deficiencies are mostly seen, but excesses can also be observed. These processes result from feed imbalances either from a shortage of nutrients or over supplementation. Feeder-species that are generally given are usually low in nutrients. Calcium and vitamin A tend to be low in most insects. Vertebrate prey on the other hand can cause obesity and hypervitaminosises. Caution should be taken when feeding and one should monitor for symptoms of possible disorders. Reproduction can also be influenced by nutrition. Although the information is scarce, there is some evidence that nutrition is involved in reproductive processes. Overall composing an ideal amphibian diet is difficult and depends on the species. Requirements are mostly unknown and result from extrapolation, thus amphibians should always be monitored correctly. In the future more research on amphibian nutrition is needed to sustain adequate captive breeding programs.

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LITERARY REVIEW 1 Nutrient content of feed

Nutrients in the feed determine the value of a diet. A good diet is important to sustain healthy amphibians. A summary of the most important nutrients for captive amphibians is presented. 1.1 Vitamins

1.1.1 Vitamin A

Vitamin A is also referred to as retinol, but sometimes also retinyl-esters and retinoic acid are considered as vitamin A. Besides retinol β-carotene or pro-vitamin A can as well be a source of dietary vitamin A (Sporn et al., 1976). Amphibians are not able to form either vitamin A or pro-vitamin A form precursors (Densmore et al., 2007). However recently researchers found some evidence that there might be a connection between carotenoids (precursor) and vitamin A (Brenes-Soto et al., 2014). Historically vitamin A is known for its function in vision. Wald (1935) used amphibians in his research and found that vision was in function of vitamin A. He found that for visual excitation 11-cis-retinal was needed in rhodopsin, which is the visual pigment. Besides vision, vitamin A is also believed to play a role in embryogenesis and regeneration (Lewandoski et al., 2009; Clugston et al., 2014).

Figure 1: Overview of vitamin A metabolism (Clugston et al., 2014). Ingestion through retinyl esters, retinol or β-carotene. Oxidized metabolites are excreted. RPE65: Retinal Pigment Epithelium-specific 65 kDa protein, REH: retinyl ester hydrolases, LRAT: lecithin retinol acyltransferase, RDH: retinol dehydrogenases, RRD: retinol reductases, BCM01: β-carotene-15,15’-oxygenase, RALDH: retinal dehydrogenases, CYP26: specific cytochrome enzymes.

Amphibians cannot synthesize vitamin A or other carotenoids (β-carotene). This means that these substances should be available through feeding. Feeder-insects are usually low in vitamin A and therefore should be supplemented (Barker et al., 1998; Finke, 2013; Ferrie et al., 2014; Livingston et al., 2014). For example the house crickets (Acheta domestica) contain inadequate amounts of vitamin A or it's precursors (Brenes-Soto et al., 2014). It is unclear if carotenoids could be a source of vitamin A. If so these nutrients can also be supplied in the feed (Finke, 2002; Brenes-Soto et al., 2014). Vitamin A levels in amphibians can be assessed by examining vitamin A concentrations in the liver (Pessier, 2005). In research on the Common frog (Rana temporaria) and the Leopard frog (Lithobates spp.) the liver proved to be the main storage site (Morton et al., 1949; Futterman et al., 1964). It is mostly stored here as retinyl esters. This is formed after the estrification of vitamin A with long chain acyl-groups (Futterman et al., 1964; Shirakami et al., 2012). Hypovitaminosis A and its effects have been showed initially in Wyoming toads (Anaxyrus baxteri) by Pessier et al. (2005). The deficiency in vitamin A led to squamous metaplasia. Usually the tongue is affected in amphibians, therefore the pathology is referred to as lingual squamous metaplasia or 'short tongue syndrome'. A change of glandular epithelium to squamous epithelium is observed in deficient animals. The shift from mucus-producing cells to keratinized cells leads to a decrease in tongue length and stickiness. This makes it harder for the animals to strike at prey and ingest them. Eventually this may result in starvation

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of the affected animals (Pessier, 2005). In accordance with Pessier (2005) Li et al. (2009) concluded that by enriching a diet with vitamin A among other things health and growth are stimulated through an increase/maintenance of ingestion functions. In Chameleons (Chamaeleo calyptratus) there is some evidence that vitamin A also plays a role in the physiology of the bone (Hoby et al., 2010). By Hoby et al. (2010) it is believed that hypovitaminosis A plays a role in the development of metabolic bone disease (MBD), but further research is needed to provide more evidence for its role in amphibians. Absolute hypovitaminosis can result from a decreased intake of retinol. Besides this a relative shortage is seen when inadequate amounts of unsaturated fatty acids are ingested. These fatty acids assist the transport of vitamin A through membranes. A nutrition that is deficient in unsaturated fatty acids may therefore lead to a relative hypovitaminosis (Uauy et al., 2000) Diagnosis of hypovitaminosis A can be made by determining concentrations of vitamin A in the liver. Pessier (2005) showed that animals who displayed lingual squamous metaplasia contained lower concentrations of vitamin A. When the livers from affected animals were compared with non-affected they contained only 5% of the normal vitamin A values. In deficient animals first the hepatic vitamin A is secreted and transported in the bloodstream. In the American Bullfrog (Rana catesbeiana) it is shown that like in mammals retinol-binding protein facilitates this transport (Shidoji et al., 1977). The circulating vitamin A is a reflection of the amount that is bound to retinol-binding protein. However when the liver vitamin A is perished, vitamin A levels in the circulation drop and this will eventually lead to squamous metaplasia (Quadro et al., 1999). Either topical, oral or intramuscular administration of vitamin A can serve as a correction of the hypovitaminosis. Sim et al. (2010) stated that a topical therapy was more effective in increasing whole body vitamin A than oral supplementation in the African foam-nesting frog (Chiromantis xerampelina). However further research has to be done to determine if the cutaneous absorption is superior to oral administration. When administering vitamin A one needs to avoid over supplementation in order to prevent hypervitaminosis A. Toxic levels have not yet been set but caution has to be taken (Sim et al., 2010). Next to the immediate therapy dietary alternations have to be executed in order to prevent hypovitaminos A in the future (Pessier, 2013). Hypervitaminosis A is seen when amphibians ingest prey that have a high vitamin A content (Crawshaw, 2003) or iatrogenic through supplementation (Clugston et al., 2014). Whole young rodents or livers from mammals are items that usually contain high levels of vitamin A. The clinical signs that can be observed when an animal is 'intoxicated' with vitamin A are weight loss, skin lesions, liver disease and anemia (Crawshaw, 2003; Pessier, 2013). The African clawed frog (Xenopus laevis) is an anuran that can be susceptible to hypervitaminosis A (Crawshaw, 2003). Another effect of hypervitaminosis A could be the development of MBD. Feeding of prey high in vitamin A might increase the risk for MBD. For example a feed consisting of rodents can cause the disorder (Douglas et al., 1994). 1.1.2 Vitamin B

Vitamin B's are needed for neurological functions, mainly vitamin B1 (thiamine) is of interest in amphibians. There is limited knowledge regarding the vitamin B1 functioning and the development of hypovitaminosis B1 in amphibians. However it is suspected that thiamine deficiency is also an entity in amphibians because of its occurrence in other species, for example reptiles (Wright et al., 2001; Donoghue, 2006). Hypovitaminosis B1 can be caused by thiaminase in prey. Especially frozen fish contain this enzyme (National Research Council committee on animals nutrition (NRC), 1993). This thiaminase enzyme causes a decrease of available thiamine in the feed which may result in vitamin B1 deficiency. The deficiency is mostly seen in aquatic species fed a frozen fish diet (Wright et al., 2001). Vitamin B1 plays a role in the neurological functioning and so deficiency can lead to paralysis, tremors, spindly leg syndrome and scoliosis (Wright et al., 2001; Crawshaw, 2003). Thiaminase deficiency, or hypovitaminosis B1, should be considered when fish eating amphibians present neurological signs (Wright et al., 2001). When histology of peripheral nerves is performed a demyelination can be present (Wright et al., 2001; Crawshaw, 2003). Either an injection or feed supplementation and/or changing of the diet can resolve this deficiency. The first one is used in the acute phase, the dietary alternations are a long term measurement (Wright et al., 2001).

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1.1.3 Vitamin D

Baldwin et al. (1980) provided information about vitamin D3 and its link with the calcium metabolism in Rana pipiens (northern leopard frog). They found that when vitamin D3 was present the (re-)uptake of calcium increased; the kidneys and intestines increased absorption of the mineral. Vitamin D3, known as cholecalciferol, could be absorbed from the food (exogenous). It can also be produced in the skin (endogenous). When an animal is exposed to the correct ultraviolet (UV) radiation, mainly UV-B, provitamin D3 (7-dehydrocholesterol) is converted into previtamin D3. After this the liver hydroxylates the previtamin D3 into calcediol (25-hydroxycholecalciferol) (Holick, 2003). This last process is temperature dependent, an optimal isomerization occurs at 25°C. Nevertheless it's also taking place at other temperatures (e.g. 5°C) only at slower rates (Holick et al., 1995). Before reaching it's final and active form, the calcediol should first be processed by the kidney. This leads to calcetriol (1, 25-dihydroxycholecalciferol) which has a main hormonal function in the amphibian, it enables calcium absorption from the food (Holick, 2003).

Figure 2: Amphibian vitamin D3 metabolism (Klaphake., 2010). Different environmental factors and hormones play a role in the vitamin D3 cycle.

Hypovitaminosis D3 could lead to a shortage of calcium. The hypocalcemia will then lead to activation of the parathyroid gland and cause nutritional secondary hyperparathyroidism (NSHP). This is one of the main causes of MBD (Mader, 2006). However to prevent MBD not only sufficient levels of vitamin D3 and/or Ca should be present in the feed. Verschooren et al. (2011) showed that amphibians fed supplemented diets still displayed MBD. They concluded that this was due to a lack of UV-B radiation. In captive management one should take notice of both the diet and the environment in preventing MBD. Care should be taken when supplementing the diet with vitamin D3. The oral requirements for amphibian are largely unknown, which may lead to under or over administration. Not only MBD can develop, adverse effects could also be possible when too much vitamin D3 is ingested (i.e. hypervitaminosis D3) (Tapley et al., 2015). Only one case of hypervitaminosis D3 has been reported by Frye (1992). An ornate horned frog (Ceratophrys ornata) was presented with lethargy, weakness, anorexia and edema/ascites. The fluid retrieved from the coelomic cavity was an acellular transudate containing urea, which might be an indication for kidney disease. A necropsy was also performed and mineralizations were found in the heart and the kidneys. These findings in combination with a history of a vitamin D3 rich led to the assumption of hypervitaminosis D3 (Frye, 1992). When a hypervitaminosis is suspected one should immediately alter the diet to prevent further damage (Densmore et al., 2007). 1.1.4 Other vitamins

Vitamin C and vitamin E both have anti-oxidant properties that protect fatty acids. Especially vitamin E is thought to protect unsaturated fatty acids in carnivores animals. Carnivorous diets usually contain high

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concentrations, thus vitamin E is necessary to stabilize these fatty acids. In turtles vitamin E deficiency might therefore lead to anorexia and subcutaneous nodules due to steatitis (Donoghue, 2006). Nonetheless the effects of vitamin E and others still has to be assessed. 1.2 Minerals

1.2.1 Calcium

According to Stiffler (1993) calcium function in amphibians is comparable with other animals. Muscle contraction and functioning of membranes and enzymes are some of the processes in which calcium is involved. The calcium can be absorbed in multiple ways. It can be absorbed through the skin and gills, the intestines (mostly duodenum) and it can be reabsorbed in the kidney (Kingsbury et al., 1989; Stiffler, 1993). Calcium concentrations in the blood are similar to other animals. They should around 1-2 mmol/l and are important to maintain healthy bone (Stiffler, 1993). Robertson (1977) stated that these concentrations may differ through the seasons, thus abnormal values can be present in healthy amphibians. To maintain these levels in the body calcium needs to be present in the feed. Insects contain usually low amounts of calcium; supplementation is advised (Ferrie et al., 2014). When calcium is supplemented through gut-loading one should take notice that concentrations in the insect may differ depending on the time after supplementation. In research done by Eidhof et al. (2006) evidence was found that in function of time after feeding and concentration of calcium in the feed the Calcium:Phosporus (Ca:P) ratio differed. This means that when either the calcium level is too low or the feed is not given at the appropriate time after supplementing the feeder-insects (too early or too late) the Ca:P ratio could be too low. The lower ratio thereafter could result in deficiency. An ideal Ca:P ratio of 1.5:1 should be pursued to prevent this (Eidhof et al., 2006). Absolute or relative hypocalcemia might lead to the development of MBD. Either a shortage of calcium in the feed or an inverse Ca:P ratio can contribute to the disorder (Wright et al., 2001). Most of the feeder-insects fed to captive amphibians have an inverse Ca:P ratio and therefore amphibians are predisposed for calcium deficiency (Dierenfeld et al., 1995). Arbuckle (2009) showed that this was the case for a lot of feeder-insects commonly used as a feed in captivity. This could be a cause of the high prevalence of MBD in captive populations (Wright et al., 2001). A lot of researchers described the development of MBD in different species, but one of the first cases was described in the African clawed frog (Xenopus laevis) (Bruce et al., 1950). Next to a shortage of calcium, hypercalcemia might also be a possible nutritional incompatibility. Yoshimi et al. (1996) found that when Solomon Islands leaf frog (Ceratobatrachus guentheri ) where fed with a diet that contained high levels of calcium this resulted in hypercalcemia and mineralizations. It must be said that also high concentrations of vitamin-D3 were present in the feed. High dietary concentrations of calcium could also lead to relative deficiencies of other trace elements. According to Donoghue (1998) an interaction between calcium and other elements is presumed. The interaction will lead to a decreased absorption of for example zinc and copper. 1.2.2 Phosphorus

Phosphorus requirements are easily met when feeder-insects are used. Concentrations between 1.5-3.7 g/kg are seen in feeder-species. However because phosphorus concentrations are usually higher than the calcium concentration an inverse Ca:P ratio is present. This is an important factor in the development of MBD. Hence calcium concentrations in feeder-insects should first be increased prior to feeding (Latney et al., 2014). 1.2.3 Other trace elements

In some feeder-insects other trace elements can be deficient. For example in some bloodworms (Chironomidae spp.) low levels of Zinc have been reported. A varied diet, where multiple different feeder-insects are provided are advised to avoid deficiencies of trace elements (Fard et al., 2014)

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1.3 Pigments

Nutritional pigments can play a role in amphibian coloration. The color of the skin is a reflection of chromatophore composure. Three different chromatophores determine the look of the animals: xantophores, iridophores and melanophores. Xanthopores can be influenced by feed, it is to say they can contain carotenoids which can be supplied through food. These carotenoids provide red and yellow pigments to the cells, which lie superficial in the skin (Bagnara et al., 1968). It is considered that amphibians are not able to synthesize carotenoid pigments, therefore the pigment should be supplied through nutrition (Perera et al., 2007). Which carotenoids should be provided depend on the species. Two main carotenoids that have been studied are lutein and β-carotene. They have been identified in the skin of green and orange anurans (Ogilvy et al., 2012a). Dull coloration due to a lack of pigmentation in the skin has been seen in some amphibians held in captivity, for example the Japanese newt (Cynops pyrrhogaster) (Matsui et al., 2002). According to Hill (2006) this would be because of deficient carotenoid amounts in the feed. A change in coloration (see figure 3) could therefore be an indication for a carotenoid deficiency which may result in health problems (Ogilvy et al., 2012a). Integument coloration plays a role in reproduction and is a defense mechanism. According to Sztatecsny et al. (2010) it is used as a form of sexual communication. The colors could also be a defense mechanism against predation. For example green coloration can be used as camouflage, whereas bright colors could serve as a warning signal for predators, e.g. Dendrobatidae (Robertson et al., 2008).

Figure 3: Coloration of two red-eye tree frogs (Agalychnis callidryas) (Ogilvy et al., 2012a). A: frog raised on a carotenoid rich-diet. B: frog raised on a control diet.

Carotenoids also have direct functions apart from the skin coloration. Studies stated that carotenoids could have immunomodulatory (Ewen et al., 2009) and antioxidant roles (Pérez-Rodríquez, 2009). These effects could have positive effects on health and growth by either reducing energy cost for immunity or reducing damage to DNA, proteins and lipids (Ogilvy et al., 2012a). Whether or not β-carotene is also a precursor for vitamin A in amphibians is currently unknown. In other species it is known that β-carotene is a precursor of vitamin A (Olson, 1989) and according to Brenes-Soto et al. (2014) this also seems so for amphibians. Thus supplying carotenoids could have positive effects on health and growth (Yang et al., 2008). For amphibians however it still remains unclear if the transition from β-carotene or other carotenoids to vitamin A is taking place (Densmore et al., 2007). Barush et al. (2012) showed that β-carotene could be converted into retinol in vitro in amphibian tissue. Further research is however needed to determine this in vivo.

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1.4 Protein

Proteins serve mainly as an energy source for amphibian metabolism and building stone for anabolic processes. An important function is the defense against pathogens. For maintaining a functioning immune system energy is needed (Schmid-Hempel, 2003), which may be provided by proteins. The higher the protein content is, the higher the energy supply is and the more energy can be invested in the immune response (Venesky et al., 2012). Venesky et al. (2012) showed that when southern leopard frogs (Rana sphenocephala) tadpoles were fed a diet containing 47.6% of protein immune function was increased compared to a low-protein diet (13.8%). They demonstrated that T-cell function, complement function and Batrachochytrium dendrobatidis (Bd) resistance increased in tadpoles fed a high-protein diet. They concluded that reduced levels of protein may lead to an impaired immune system and might result in disease. Next to these effects higher protein contents can also lead to bigger and more developed tadpoles (Venesky et al., 2012) and higher survival rates (Martins et al., 2013). These bigger tadpoles on its turn led to larger juveniles after metamorphosis (Martins et al., 2013). Martins et al. (2013) showed that Natterjack toads (Epidalea calamita) fed a protein-rich diet (46.2%) were bigger compared to control groups. The bigger size might lead to an increased feeding ability and higher survival rate (Tejedo et al., 2010; Martins et al., 2013). Decreased growth rates could also be seen when the protein content of a feed is too high. The enhanced protein metabolism will require more energy and this will lower the amount of energy available for growth (Martínez et al., 1993). Dietary protein also plays a role in the development of gout. This disorder causes urate crystals to deposit in tissues and joints. In a further stadium it could also lead to kidney disease. These crystals can develop when the excretion of uric acid is not adequate. This could be due to an excessive concentration of protein in the feed, but usually it is caused by dehydration (Donoghue, 1998). 1.5 Fat

Fat is like protein a source of energy, but next to this also supplies some specific nutrients. For example fatty acids and fat-soluble vitamins. A shortage of fat might lead to an impairment of all sorts of function due to a shortage of energy. When lipids are given in excess, in other words the calorie intake is too high, amphibians can develop obesity. The heart and coelomic cavity are places where fat can be stacked (McWilliams, 2008). Excessive fat can also lead to lipid keratopathy. This disease, also known as corneal lipidosis, will lead to haziness of the cornea and thus reduced visibility (Shilton et al., 2001). The reduced sight can have an impact on the hunting abilities of an amphibian and therefore may lead to anorexia and reduced body condition (McWilliams, 2008). High levels of fat in the feed could also lead to secondary deficiencies. This can be caused when for example adult vertebrates are given as solely feed. The higher concentration of fat reduces the relative concentration of other nutrients and can cause relative shortages (Donoghue, 1998). The fat fraction also contains fatty acids, these could be either saturated or unsaturated. Especially the unsaturated fatty acids are important for growth and health of amphibians (Li et al., 2009). Examples of the unsaturated fatty acids are omega-3 fatty acids and omega-6 fatty acids. Unsaturated fatty acids are needed to produce and transport vitamin A into the cell (McDowell, 1989; Uauy et al., 2000). According to Moyad (2005) it prevents pathologies in this membrane transport, as well as in the nervous system. Due to its role in vitamin A transport unsaturated fatty acids could lead to improved health in animals that require high levels of vitamin A (Li et al., 2009). 1.6 Carbohydrates

Amphibians normally only use protein and fat as energy sources, carbohydrates are usually not present in the feed or only in low concentrations (Donoghue, 1998). Abnormal amounts of carbohydrates in the feed could lead to intestinal blockage (McWilliams, 2008). Next to inflammation, infectious diseases, and foreign objects, also carbohydrates can be a cause of diarrhea. This is seen when the amphibians are fed with diets that contain excessive amounts of carbohydrates (Wright et al., 2001; Bertelsen et al., 2003)

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1.7 Fiber

Like carbohydrates, the amount of fiber ingested is also low (Donoghue, 1998). According to McWilliams (2008) high fiber content might lead to intestinal blockage. Next to this insects that have high levels of chitin (insect fiber) might also have a decreased bioavailability. The chitin in the cuticle might be indigestible and so the protein in the cuticle may not be available. The cuticle can also inhibit absorption of nutrients from the insects' gut (Latney et al., 2014). So far fiber seems not necessary in amphibian diets, however more information is needed to confirm this. 1.8 Probiotics

Some researchers showed that amphibians have an endogenous flora in the gastro-intestinal tract. Kohl et al. (2013) described this in the Northern leopard frog (Lithobates pipiens) and Okelly et al. (2010) reported this in the Slimy Salamander (Plethodon glutinous). The role of this microflora remains yet to be unclear (Ferrie et al., 2014). However research is in progress to assess the effect of probiotics on the endogenous flora. Bletz et al. (2013) showed that probiotics could have a positive effect on amphibian health by decreasing sensitivity to disease (chytridiomycosis) through bioaugmentation. According to Antwis et al. (2014) carotenoids can be seen as probiotics. They found that in the Red-eyed tree frog (Agalychnis callidryas) a carotenoid rich diet (black crickets (Gryllus bimaculatus) gut loaded with 5 mg/g carotenoids) led to a different bacterial population. Not only more bacteria were present, also a larger variety of species was present compared to non-supplemented frogs. The larger endogenous species variety in the gut as well as on the skin might lead to a protective/competitive effect against pathogenic bacteria. The protective effect of carotenoids on endogenous flora could be due to its anti-oxidant function. Fraser et al. (2004) showed that some bacteria could use carotenoids to protect their DNA and cell membranes. They can either acquire carotenoids in the gut or on the skin of amphibians and so take advantage of their positive effects; i.e. increased survival and growth (Antwis et al., 2014). 2 Effects of nutrition on health and reproduction

2.1 Health

Nutrition is important in maintaining amphibian health. A couple of nutritional factors have been mentioned, for example proteins and fat are needed to supply energy and vitamin D3 and calcium are important in bone metabolism. In this section some influences of food intake on health are highlighted, as well as the most common pathologies. 2.1.1 Metabolism

The daily energy needs of amphibians are similar to other animals (Donoghue, 1998). Daily requirements depend on multiple factors. Gender, activity and temperature are some factors that determine the normal metabolism. For example in some salamanders the metabolic rate increases with an increase in temperature. This means that an increased temperature will lead to a higher requirement of food. Also when an amphibian is active it logically needs more food. In case of breeding and disease the metabolic rate increases as well. More food is needed to provide sufficient energy for either the developing eggs or immune function (Hadfield et al., 2006; McWilliams, 2008). According to Gatten et al. (1992) metabolic rates are also higher in anurans and in terrestrial amphibians, compared to respectively salamanders and aquatic amphibians. When too much energy is supplied amphibians tend to develop obesity. Usually the excess of calories is provided through preys that contain high concentrations of lipids. Most of the time it is seen when prey that contain high concentration of fat are given as solely feed. Mealworms (Tenebrio spp.) and obese rodents are two examples of species that contain high fat levels (McWilliams, 2008). Poor nutritional condition might result from an insufficient intake of energy. Energy reserves decrease and eventually a state of cachexia can be observed. On necropsy the gonadal fat can be absent or minuscule and histologically an atrophy of bone marrow can be seen (Pessier et al., 2014).

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2.1.2 Integument

The integument of an amphibian has a couple of functions. The thermoregulative function plays a role in the digestion. Either by reflecting or absorbing heat the integument makes sure the correct temperature is maintained resulting in a functioning gastrointestinal tract. It also has a use as sensory organ to catch prey or elude predators. The skin of amphibians has a high sensitivity, with which they can either locate their food or avoid being eaten by predators. Another function regarding nutrition is that the skin also absorbs or secretes electrolytes dissolved in water. This makes the skin a major contributor to the homeostasis of an animal. The skin can take part in the defense against pathogens as well (McWilliams, 2008). In some species it is known that the skin can produce an antibiotic substance that protects against microorganisms (Mangoni et al., 2007). Beside these functions the integument can also serve as food itself. Many amphibians eat their skin after shedding or feed young ones pieces of it. In order to maintain these functions the diet of an amphibian has to be sufficient and well balanced (McWilliams, 2008). Carotenoids for example should be present to have a healthy and colored skin (Frost-Mason et al., 1994). 2.1.3 Immune system

Nutrition can influence the immune system directly or indirectly. For example Parker et al. (2014) stated that by feeding prey with Punica granatum (pomegranate) circulating eosinophils and complement activity increased in the Cane toad (Rhinella marina). Eosinophils are a type of white blood cells and complement activity is another immune defense mechanism; an increase in both might lead to more resistant animals. Pomegranates contain for example tannins and flavonoids that might have these immunomodulatory effect (Lee et al., 2010). Also carotenoids and vitamin A are believed to have a immunomodulating effect (Ewen et al., 2009). Through metabolism or the production of hormones nutrition can also influence the immune system in an indirect manner (McWilliams, 2008). As mentioned a decrease in protein intake can lead to an impairment of immune functioning (Venesky et al., 2012), but in general low energy and inadequate nutrition may decrease immune response. This can be reflected by an increase in morbidity and/or mortality in animals who are in poor overall condition, which reflects the nutritional state (Lee et al., 2006). Garner et al. (2009) showed that juveniles who were in a poor condition were more likely to die because of a Bd infection. Hormones can also influence the immune system. Ledón-Rettig et al. (2009) demonstrated that the couch's spadefoot toad (Scaphiopus couchii) had higher levels of corticosterone in its body when it was underfed. Additional, increased levels of corticosterone may lead to a reduced immune response; this was shown by Rollins-Smith et al. (1997). They showed that when stress hormone (corticosterone) levels were high a decrease of circulating lymphocytes was observed, which is a type of white blood cell. Also new research of Rollins-Smith (2017) suspects a role of nutrition in immunity. She believes that a poor nutritional state might lead to increased levels of glucocorticoids and thereafter an impaired immunity. However she states that this effect of nutritional deficits is only of interest in young amphibians because of their developing immune system (Rollins-Smith, 2017). 2.1.4 Pathologies

Metabolic bone disease According to Wright et al. (2001) MBD is the most occurring nutritional pathology in amphibians. MBD is characterized by demineralization of bone structures. Calcium, vitamin D3 and UV-B radiation play a large role in the bone metabolism. Deficiency of one or more of these factors can lead to MBD (Densmore et al., 2007). Some symptoms that are observed in affected animals are demineralization (radiographic sign), (pathologic) fractures, bone deformities (mandibular, limb, and others) hydrops, subcutaneous edema, cloaca prolapse, weakness and tetany (Wright et al.. 2001; Ferrie et al., 2014). In the normal situation Vitamin D3 is needed to absorb calcium from the gut, thereafter the calcium can be distributed through the body. Vitamin D3 is also provided through feeding, however it has to be processed in order to be active and result in calcium uptake. The skin, liver and kidney convert the vitamin into its metabolic active hormone. This happens in function of UV-B (skin) and temperature (kidney) (Holick, 2003) When either the calcium or vitamin D3 is low in the feed or there is an absence of UV-B radiation, an animal can develop hyperparathyroidism. This means an increase in parathyroid hormone (PTH). One of the functions of PTH is resorption of calcium from the bones. When the parathyroid is stimulated for longer

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periods, great amounts of calcium are being absorbed from the bone, which may result in weakening of the skeletal structures. The process where hypocalcemia caused by nutritional deficiencies leads to a generalized decalcification in function of PTH is called nutritional secondary hyperthyroidism (Holick, 2003). It is stated by Hay et al. (1976) that distribution of 25-hydroxy-cholecalciferol is mediated by lipoproteins in amphibians. This differs from the transport of vitamin-D3 in other vertebrates. Amphibians therefore can develop MBD even when the vitamin-D3, calcium and phosphorus are sufficient, but lipoproteins are occupied or low. This is usually caused by an excessive concentration of fat in the food which compete with vitamin-D3 for the lipoprotein transport function. An amphibian feed consisting out of for example older rodent might contribute to the acquiring of MBD (Hay et al., 1976). Another possible factor that might cause MBD is hypervitaminosis A. Bruce et al. (1950) showed that the ornate horned frog (Ceratophrys ornata) was affected by MBD when fed prey high in vitamin A. These animals were fed a diet consisting of rodents and according to some researchers rodents like mice and rats contain high levels of vitamin A (Bruce et al., 1950; Douglas et al., 1994; Crawshaw, 2003). These high levels interfere with the absorption of vitamin-D3 (Douglas et al., 1994) and might therefore lead to MBD (Bruce et al., 1950). Decalcification can also be caused by renal disease. Normally the kidney plays a role in the calcium and vitamin D3 metabolism; it either reabsorbs calcium or activates vitamin D3 (Baksi et al., 1977; Stiffler, 1993). Impairment of the renal function might therefore lead to deficiency of either calcium or vitamin D3 resulting in MBD. MBD caused by a renal disorder should be suspected when an amphibian is not responding to treatment (Wright et al., 2001). The decalcification of the bone will lead to an impaired function of the skeletal system (Tapley et al., 2015). The weakening of the bones may lead to fractures and deformities (Gagliardo et al., 2010) and these errors could lead to a decrease in feed intake, mobility, reproducing and growth (Tapley et al., 2015). Especially young amphibians after metamorphosis are affected; they have high nutrient demands because of their growth. Gagliardo et al. (2010) showed this in research on the horned marsupial frog (Gastrotheca cornuta). Abnormal posture, abnormal movement, bone deformities, fractures and tetany are some signs an amphibian may present (McWilliams, 2008), but a definite diagnosis is made by performing radiographic examinations. The demineralization of the bone can be seen either general or more locally. In general there is a loss of bone opacity, more localized there can be deformities, (pathologic) fractures, thin cortices and wider marrow cavities. For example the jaws are places where deformities can be seen, especially the mandibula, but also the maxilla and premaxilla can be deformed and demineralized. The fractures are usually seen in long bones (humerus, femur) (see figure 4), however in anurans also pelvic abnormality’s and other fractures are seen (spinal, coracoid) (Wright et al., 2001). Also blood examination can lead to the presumption of MBD, for example the calcium and phospor could be unbalanced. Hypocalcemia and hyperphosphatemia can aid in the recognizing of MBD (Wright et al., 2001; Klaphake, 2010).

Figure 4: Radiographs of two different captive mountain chicken frogs (Leptodactylus fallax) (Tqpley et al., 2015). A: Individual affected by MBD. B: Individual recovered from MBD. Notice that the opacity of the bones is lower in photo A. Although the frog in radiograph B is recovered the deformities in the hind limbs remain.

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The required amounts of either calcium or vitamin D3 are currently unknown and also for UV-B the exact proportions are unclear. However it is known that in captivity both the diet and UV-B radiation should be optimal in order to prevent MBD (Michaels et al., 2014; Tapley et al., 2015). Next to this a therapy can be started to treat the condition. Oral supplements with calcium and vitamin D3 are important measurements, but also calcium gluconate baths or parenteral administering of calcium could be options. The parenteral therapy with calcium gluconate can either be given intravenous, intramuscular or intracoelomically and is advised when acute symptoms (tetany or bloating of the intestines) occur (Wright et al., 2001) The bone can be re-calcified when the correct diet, the required supplements and sufficient UV-B radiation is given. It is essential that the therapy is given until the radiographic image of the bone is normal; it is to say the density is within normal limits. According to Wright et al. (2001) this is a process that can last up to 6 weeks or more. However one should be aware of the fact that deformities may be irreversible and may cause a decreased welfare for the animals (see figure 4) (Tapley et al., 2015). Lingual squamous metaplasia Lingual squamous metaplasia, also called short tongue syndrome, is a result of vitamin A deficiency. Like the name says it is a condition affecting especially the amphibian mouth, however also other mucosae can be affected (reproductive tract, conjunctivae) (Wright, 2006; Pessier, 2013). The disease has been identified in mostly anurans among which the Wyoming toad (Bufo baxteri) and the African foam-nesting frog (Chiromantis xeramplina) (Sim et al., 2010; Pessier, 2013). Symptoms that can occur are lethargy, loss of weight and possibly death because of ingestion problems (Pessier, 2013). Histological a chance of normal mucus-secreting epithelium into squamous epithelium can be seen in the esophagus, cloaca, urinary bladder and especially the oral cavity and tongue (Pessier et al., 2014).

Figure 5: Lingual histologic section of two Wyoming toads (Anaxyrus baxteri), hematoxylin and eosin coloration, 20x (Rodríguez et al., 2014). A: Normal lingual epithelium. B: Severe lingual squamous metaplasia. In picture B there is a decrease of papillae and change of surface epithelium compared to picture A; F: More stratified and squamous epithelium in picture B, M: mild dilation of glandular structures in picture B.

Diagnosis of lingual squamous metaplasia because of low vitamin A levels has been made by examining livers of affected animals. This was done in some Wyoming toads (Bufo baxteri) that had lesions in accordance with squamous metaplasia. Retinol levels of the livers of affected animals were compared with healthy individuals. In the affected toads retinol concentrations were significantly lower than in healthy animals. Other possible diagnostic procedures are wholy body vitamin A in small amphibians or blood samples in larger amphibians (Pessier, 2013). Nevertheless further studies are required to provide guidelines for these methods. By supplementation or alternation of the diet vitamin A deficiency can be prevented. This has been shown by Li et al. (2009) who supplemented the diet of Wyoming toads (Bufo baxteri). Also injections or direct oral and topical supplementation can be a therapy for hypovitaminosis A (Sim et al., 2010; Pessier, 2013). Neurological symptoms Spindly leg and scoliosis are symptoms that can occur either in tadpoles and young metamorphs. Both have been seen in different frog species, for example in the giant monkey frog (Phyllomedusa cf tarsius). It is thought that both can be seen in animals with a vitamin B deficiency; symptoms reduced when vitamin B was supplemented. Dietary measurements are advised in long term management (Wright et al., 2001).

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Paralysis is another entity that might be seen in some anuran species. For example poison frogs (Dendrobates auratus) are affected by a flaccid paralysis of especially the hind legs, that might eventually result in the death. After histological examination a demyelination of nerves was seen in the affected frogs. An exact cause has not been identified, however Wright et al. (2001) suspect that vitamin B deficiency's, hypocalcemia or infectious agents or toxins might cause the neural demyelination (Wright et al., 2001). Obesity Obesity can be seen in animals that ingest excessive amounts of energy. This can be seen for example after feeding prey that have high concentration of fat. Amphibians also tend to eat as much as possible, eating as much prey as they can acquire. The building up of fat reserves is a physiological process in the wild to deal with for example hibernation and reproduction. In captivity these processes are mostly of no interest, therefore an excess of prey might also lead to an increased energy intake. The diet should be adjusted to the animal's metabolic rate (Wright et al., 2001). Cachexia Cachexia is seen when an animal does not acquire its energy needs. This starvation results in lipolysis and muscle loss (Merkle et al., 1988). Dehydration, skin defects, reduced activity, and reduced fecundity are seen in amphibians with cachexia (Grably et al., 1981; Merkle, 1990; Wright et al., 2001). The process of starvation resulting in cachexia has been described in the African clawed frog (Xenopus laevis) by Merkle (1990). The different phases finally led to a body-fat decrease of almost 100% in combination with muscle atrophy and reduced ovarian functioning. Adjusting of the diet according to the energy needs is required to resolve the cachexia. However at first the energy supplementation should be higher in order to let the amphibian gain weight. Possible husbandry stressors should be avoided and adequate feeding regimes (correct prey and timing) should be provided to prevent ongoing starvation (Wright et al., 2001). Overpopulation could be a possible stressor according to Narayan et al. (2015). The nutritional stress (resource shortages etc.) could lead to higher glucocorticoid levels, lower body condition scores and decreased reproduction (Narayan et al., 2015). Gastric overload For example horned frogs (Ceratophrys spp.) tend to ingest many or large prey, which might result in gastric overload. It is a distention of the stomach that can lead to life-threatening symptoms. The higher intra-abdominal pressure can lead to dyspnea and hypovolemic shock. There might also be a putrefaction of the gastric content, which might cause secondary consequences by toxins in the bloodstream. It is an emergency situation and a therapy should be executed as fast as possible. The materials need to be retrieved either directly through the oropharynx with a forceps, lavage with a gastric feeding tube, through esophageal endoscopy or through a celiotomy in combination with a gastrotomy. Supportive therapy should be given during or after these operations (i.e. corticosteroids and fluids) (Wright et al., 2001). Impaction Impaction is the thickening of food contents in the gastrointestinal tract. An intake of foreign objects or debris might lead to gastric impaction and/or intestinal obstruction. Symptoms that are observed are abdominal bloating and lethargy. After a diagnosis (radiographically (possibly with a contrast-study) or ultrasound) a treatment can be started. The material can be diluted through oral fluids or with a feeding tube or it can be removed through a celiotomy in combination with a gastrotomy and/or entrotomy (Wright et al., 2001). 2.2 Reproduction

In breeding programs it is important that as many amphibians as possible are 'produced', in order to sustain captive breeding programs. Nutrition is an important factor to provide the correct nutrients and energy to reproduce offspring. The effects of nutrition could particularly be regarding females or males, but there are also some general views. For example when selecting an animal for reproduction it should be in a good nutritional state. This means that the appearance of such an animal should be comparable with a healthy, wild amphibian and also it's body weight should be similar. When animals are in a good condition, they usually have enough energy reserves to cope with the breeding itself or the periods of anorexia that can accompany the breeding (Wright et al., 2001).

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Energy is required to reproduce; if more energy is available for reproduction it will be more successful. In amphibians it is suspected that fitness depends on the energy that is stored in fat bodies, muscles, gonads or the liver (Fitzpatrick, 1976; Komoroski et al., 1998; Villecco et al., 1999). For example if an Asian bullfrog (Rana tigrina) was supplied with more energy their fecundity increased (Girish et al., 2000). Also in the African clawed frog (Xenopus laevis) it is believed that food influences ovarian function. Shortages in food supply led to regression of the ovaries (Alexander et al., 1935). This is also seen in studies by Merkle (1990) on the African clawed frog (Xenopus laevis). A cachectic state, or a shortage of energy, also led to ovarian regression. According to Merkle (1990) a 70% decrease of ovarian mass can be observed when an animal was starved. This depletion of energy might interfere with the development of eggs and therefore might lead to reduction of fecundity (Wright et al., 2001). Thus captive amphibians should be fed a correct diet in order to let oogenesis take place. Stress might as well be a factor that influences egg quality or clutch size. Stress can result from for example disease, but regarding nutrition it can also be due to a lower position in the hierarchy. Therefore subordinate animals might have higher stress levels because of food shortages and therefore a lower fecundity (Green, 2002). Also body condition and survival ability are believed to be influenced by nutrition in the mountain yellow-legged frogs (Rana muscosa). A correlation between overabundance of prey and higher survival rates is suspected (Pope et al., 2001). A difference between female and male energy allocation is stated by some researchers (Halliday et al., 1988; Wells, 2007). Females are believed to invest more energy in the development of gametes and less in breeding activity (Wells, 2007), whereas males tend to invest more in breeding activity then in spermatogenesis (Halliday et al., 1988). A reason for the different energy allocation could be that the female fecundity is more dependent on the production of gametes and that this process is more energy consuming than spermatogenesis (Halliday et al., 1988; Wells, 2007). A common perception is however that larger amphibians have a greater energy storage overall (Wells, 2007). Martins et al. (2013) saw in their research that a high-protein diet led to larger Natterjack toad (Epidalea calamita). The bigger size can on its turn lead to larger clutches and bigger eggs in females (Tejedo, 1992a) and to higher mating success in males (Tejedo, 1992b). Also bigger Siberian tree frogs (Rana amurensis) could produce more eggs (Solomonova et al., 2011). Which according to Wells (2007) could be caused by higher energy storage. Immunity may also be involved in the process of reproducing. When northern cricket frogs’ (Acris crepitans) immunity was compromised hatching rates decreased (McCallum et al., 2007). This means that reproduction could be less successful when the immune function is compromised by inadequate nutrition, by for example a low-protein content (Venesky et al., 2012). Another aspects of reproduction could be dietary vitamin A. It plays a role in embryogenesis, for example in limb development. This process can be compared to limb regeneration, in which vitamin A is also required (Lewandoski et al., 2009). In the African clawed frog (Xenopus laevis) it is shown that embryos need vitamin A for development. However excessive amounts of vitamin A should also be avoided; eye or limb malformations could occur (Weissman, 1961; Kraft et al., 1995). Carotenoids can also have effects on amphibian reproduction. First the coloration can be either a signal of deficiency (Ogilvy et al., 2012b) or an indication of male quality (Richardson et al., 2010). McGraw (2005) showed that females preferred brightly colored males for mating, in other words color can determine reproductive success (Ogilvy et al., 2012b). Second carotenoids could have positive effects on egg quality and offspring. Also in the egg carotenoids could have an antioxidant effect and so lead to protection of a growing embryo (Blount et al., 2000). Dugas et al. (2013) showed this effect in Strawberry Poison Frogs (Oophaga Pumilio). An increase in developing offspring was seen in anurans where carotenoids were supplemented due to an increase in egg quality. However when supplemented fewer clutches were produced, according to Dugas et al. (2013, 2016) due to parental care for the developing tadpoles. Other issues that can be taken into account regarding reproduction are the amount fed and the timing of feeding. In wild living amphibians an increase in feeding is seen in periods prior to breeding. In captivity one should therefore provide unlimited feed two months before breeding. After the breeding season the feeding should be returned to normal/restricted levels to prevent obesity. This increased intake of feed supplies the energy needed for reproduction (Browne et al., 2007).

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3 Means of feeding and supplementing

3.1 Feeding

Amphibians can either be omnivores or carnivores. Usually the feeding switches to a complete carnivorous food regimen after metamorphosis if they weren't already completely carnivorous (salamanders and caecilians). The carnivores can be fed with either invertebrate (feeder-insects) or vertebrate prey dependent on their size. The bigger the amphibian is, the more they tend to feed opportunistic; they eat a larger variety of prey (Wright et al., 2001). Commonly used feeder-insects for captive amphibians are crickets (Gryllus spp.) and mealworms (Tenebrio spp.). Fish and mice are examples of vertebrate prey items used for captive amphibians (McWilliams, 2008). Amphibians are considered carnivores, however there is evidence that some species either eat plants accidentally (Silva et al., 1989) or have enzymes that can digest plant material (Oshima et al., 2002). Besides these means of feeding cannibalism is sometimes observed in carnivore amphibians. This usually occurs when different sized animals are put together (larger ones eat the smaller ones). This could be prevented by housing the large amphibians together and apart from the smaller ones (McWilliams, 2008). Dermatophagy is a process that also occurs in the amphibian class. Why the animals eat their own skin remains to be unknown, but it is thought by Frye (1991) that it is a way to recycle proteins in the epidermis. Dermatophagy has been reported in numerous cases, Weldon et al. (1993) gave an overview. For example multiple toad species (Bufonidae) show this behavior (Weldon et al., 1993). A general concept when feeding is that the feeding should mimic the natural situation. This could be done by either replicating the amphibians' natural environment or by making the feeding a challenge, for example feed at the appropriate time, in the correct environment and using moving prey of the appropriate size. This will enhance natural behaviors (climbing, foraging and others) and will lead to a healthy physical condition (Wright et al., 2001; Campbell-Palmer et al., 2006; McWilliams, 2008; Ferrie et al., 2014). Invertebrate preys are most frequently used in amphibians, however some animals also eat vertebrate prey. Which prey they prefer depends on their gape width, but usually amphibians feed opportunistic (Wright et al., 2001). 3.1.1 Invertebrate prey

In a natural environment an amphibian usually eats a large variety of invertebrate species. By doing this it makes sure a complete diet is maintained. Next to the fact that in captivity the invertebrates that are offered are frequently deficient in multiple nutrients (McWilliams, 2008) (see table 1 and 2), the diet is usually also unvaried compared to the wild. This may lead to nutritional and behavioral deficiencies (Slight et al., 2015). Table 1: Summary of Appendix A and B: Main nutrient contents of multiple feeder-species.

Species Protein (crude) Fat (crude) Calcium Phosphorus Ca:P Vitamin A Vitamin D3

Crickets 58.5-64.9% 9.8-22.8% 0.1-1.3% 0.8-1% 0.13-1.63:1 0.0-471 0.0 Cockroaches 47.4-78.8% 20-31.2% 0.1-0.2% 0.4-1% 0.18-0.3:1 0.0-386 482 House flies 85.8% 8.3% 0.3% 1.6% 0.19-0.21:1 0.0 434 Mealworms 36.4-67.7% 17.7-31.1% 0.0-0.1% 0.6-1.4% 0.06-0.09:1 0.0-811 0.0 Soldier flies 35.1% 28.1% 1.9% 0.0% 2.62:1 0.0 100-200 Superworms 43.1-68.1% 14.3-39.3% 0.0-0.1% 0.0-0.7% 0.08-0.13:1 41 - Fruit flies 52.1-70.1% 10.5-12.6% 0.1-0.8% 1.1-2.7% 0.10-0.28:1 2.2 - Woodlice 41.2% 11.5% 14.38% 1.22% 11.79:1 - - Aquatic invertebrates

52.1-78.75%

3-10.7%

-

-

-

-

-

Protein, fat, calcium and phosphorus shown in % of dry matter. Vitamin A and Vitamin D3 shown in IU/kg.

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Table 2: Recommendations for adult amphibians based on requirements for other species (fish, rats).

Nutrient Recommendation

Protein (%) 30-60

Fat (%) 40-70

Calcium (%) 0.6 (rat1)

Phosphorus (%) 0.3 (rat1)

Ca:P 1.5:1

Vitamin A (IU/kg) 2914 (fish2)

Vitamin D3 (IU/kg) 1111 (rat1)

Protein and fat requirements are based on research in amphibians (McWilliams, 2008). Ca:P ratio should be ideally 1.5:1 (Eidhof et al., 2006). 1: NRC, 1995. 2: NRC, 1993.

When feeder-insects are used as feed they should be alive and they should be a good source of nutrients. Next to these conditions one should take into account that the feed might by contaminated with pathogens or other substances. For example mycotoxins, bacteria, viruses and even antibiotics can be present in the feed and can be potentially harmful for the animals (Ferrie et al., 2014). Thus when rearing feeder-insects contaminants should be avoided in the feed and the prey should be held in a hygienic environment (Pessier et al., 2010). Another way of providing feeder-species is by collecting them from outdoors. Leaf litters or field sweepings could be a source for insects. However caution should be taken when brightly colored species are collected, because of a risk for toxicities. Another risk of toxicity could be feeder-insects that have been contaminated with herbicides or pesticides. One can avoid this by not collecting in areas where these products have been used. Also in wild collected insects there is a risk for infection with multiple pathogens (Wright et al., 2001). 3.1.2 Vertebrate prey

When feeding large amphibians one could opt using vertebrate prey. Fish, rats and mice could be possible feed. The tissues mostly contain sufficient amounts of nutrients. The bones and liver contain for example enough calcium and vitamins. However the disadvantages should be mentioned. It could be that the predator becomes the prey when the vertebrate prey is still alive. Also when a vertebrate is obese this could result in excessive levels of saturated fat and hypervitaminosis of vitamins soluble in fat. Another disadvantage could result from feeding young vertebrates. These could be deficient in for example fat-soluble vitamins and calcium, which may lead to shortages of nutrients and its consequences (Douglas et al., 1994; Donoghue, 1998; McWilliams, 2008). Adult vertebrates however usually provide sufficient amounts of nutrients. Their bones supply calcium, phosphorus and magnesium, the kidney and liver provide vitamins and minerals, zinc is supplied by the pancreas and iodine is supplied by the thyroid glands (Donoghue, 1998). A remark should be made regarding the storage of dead vertebrate feeding materials. Dead rodents or frozen fish can become rancid when stored inadequately. The rancid feeding materials might lead to steatitis, which on its turn can lead to discomfort for the amphibians. Rancid food should be avoided or vitamin E and selenium can be supplemented or injected when an animal ingests rancid food to prevent steatitis (Wright et al., 2001).

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3.2 Supplementing

Most of the feeder-insects used as amphibian feed are deficient in vitamins and/or minerals (Barker et al., 1998). To provide sufficient amounts of nutrients the invertebrates need to be supplemented either by dusting or gut-loading (Livingston et al., 2014). Other feed could also be enriched in order to provide the correct amounts of nutrients. Frozen fish for example can be supplemented with vitamin B1 and E (McWilliams, 2008). 3.2.1 Dusting

Dusting is referred to as coating of the feeder-insects with nutrients one wants to supplement with (Ferrie et al., 2014). Li et al. (2009) provides a guideline for dusting crickets. Effectiveness of the method decreases as the time between dusting and consumption increases. In house crickets (Acheta domestica) up to 50% of the dust can be lost within 90 seconds of administering. The loss of the powder can be explained due to the grooming or movement of the insects (Li et al., 2009). Although this is a simple method to supplement the insects it does not provide the same amounts compared to gut-loaded insects. Next to that, the exact amount that is ingested by the amphibian is unknown; it is difficult to estimate the amount of substance on the prey (Ferrie et al., 2014). Other disadvantages of this method are a rapid loss of the dusting substance from the insect (50% in the first minutes) and no usability for aquatic species or amphibians in humid environments (Li et al., 2009). 3.2.2 Gut-loading

Gut-loading means that the insects are supplemented with nutrients by adding these to the insects' diet. After supplementing the insects with for example vitamin A or calcium, they should be consumed within a certain time span. Feeding freshly gut-loaded insects is advised (Ogilvy et al., 2012a). When feeding the nutrients to the insects some factors should be taken into account: the nutrients need to be tasteful and easy to consume and sufficient concentrations should be reached within the insects. When these conditions are met gut-loading is the method of choice for supplementing (Ferrie et al., 2014). 4 Other husbandry related factors

4.1 Water

Water intake occurs either through the skin or indirect through ingestion of prey. There is no direct oral intake of water (Hillman et al., 2009). The amphibian integument is an important organ in preserving water homeostasis. The skin has a good vascularization and also has a thin stratum corneum. These characteristics make the skin permeable to water and other substances (Campbell et al., 2012). Water can be absorbed or lost through the skin, as well as salt and other electrolytes, e.g. calcium and phosphorus (Kingsbury et al., 1989; Wongdee et al., 2013). The loss of water through the skin is in function of environmental factors (Hillman et al., 2009). Urinating can also be a way of water excretion. These processes keep an osmotic balance within the environment (McWilliams, 2008; Hillman et al., 2009). In captive environments hydration usually is met by using municipal water. To use this water for amphibians some recommendations are provided by Odum et al. (2011) to preserve the health of captive species. Some of the measures that can be used are avoiding cleaning products with ammonia or phosphates and avoid standing water to prevent intoxication. For example phosphor and fluoride in the water can play a role in the pathogenesis of MBD (Shaw et al., 2012). When an amphibian is dehydrated a problem with the excretion of uric acid occurs. The uric acid crystals can be deposited in the joints and soft tissues. This phenomenon is also known as gout (McWilliams, 2008). Water hardness and water pH can also influence amphibian health. Hard water can for example cause skin damage and abnormal pH can cause bacterial growth (Wright et al., 2001). 4.2 Enrichment

An amphibian has the need for activity, challenges and variety (Seidensticker et al., 1998). Through enrichment of the environment this could be achieved (McWilliams, 2008). Besides improving the environment also the feed could assist in enrichment for the animals. As mentioned captive amphibians are

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usually fed with invertebrate feeder-species. To stimulate the natural behavior and health of amphibian species one could feed live prey. Natural behavior could even be more enhanced when these prey are interacting with the environment. This could be done by placing the insects under materials like plants, leafs or sand, which may result in more natural hunting conditions (Campbell-Palmer et al., 2006). 4.3 Ultraviolet (UV)

UV radiation is an important factor in the bone metabolism. Especially UV-B radiation is needed in order to maintain a normal calcium metabolism. To absorb calcium from the gut vitamin D3 is needed, which is also taken from the feed. However in order to be active this vitamin D3 first has to be processed by the body. This is where UV-B plays a significant role. Before the vitamin D3 can be hydroxylated by the liver it first has the be converted from provitamin D3 to previtamin D3 in the skin. This happens when the skin is exposed to UV-B radiation and is caused by photolysis. Thereafter vitamin D3 can further processed by the liver and the kidney in order to perform its hormonal role. Deficiencies of any of these factors will lead MBD (Holick, 2003). The UV-B is usually provided by the sun, however in captivity one should take measures to match the radiation with the amphibian's needs. The wavelengths of UV-B required lie between 280 and 315 nm , one should provide this in the spectrum of UV given to an amphibian (Wright et al., 2001) (Holick, 2003). Apart from the spectrum of the light bulbs also the temperature has to be taken into account. The places where the most UV-B radiation is present should be accompanied with the highest temperature. This is to stimulate the natural behavior of the amphibians, they can self-regulate their temperature. Not only areas where they can bask should be supplied, also retreats should be placed where they can escape the UV radiation (Tapley et al., 2015). This should also be deep shade because in light shade there is still exposure to UV by reflection and diffusion (Turnbull et al., 2005). Excessive exposure to UV may lead to adverse effects. Erythema, increased mucus production, corneal defects and more general effects like stress, immunosuppression, gene mutations and cell death can be seen (Licht et al., 1997; Wright et al., 2001; Blaustein et al., 2003). 4.4 Other lightning factors

The rhythm of lightning and feeding should be accustomed to the species. In diurnal species food should be given in the beginning of the day when there is a lot of light and nocturnal ones should be fed at the end of the day when the light intensity is decreasing (Wright et al., 2001). When the light is too intense it might also suppress feeding behavior. For example species that live under dense vegetation in the forest might be unwilling to eat because of exposure to excessive light. One could adjust the light levels or provide shade to cope with this problem (Wright et al., 2001). 4.5 Temperature

Amphibians should have the possibility self-regulate their body temperature. Temperature is important in for example maintaining a healthy bone metabolism (Holick et al., 1995). Recent research also states that increasing the temperature might be an effective therapy against Bd. Chatfield et al. (2011) showed that an increased temperature (30°C) could be an additional option in the treatment of the fungal infection.

4.6 Disease

Disease could result in an alteration of feed intake. It will lead to an increase of energy utilization by triggering the immune response (Schmid-Hempel, 2003), this means that more energy intake via nutrition is required. However in some cases a diseased animal will have a reduced ingestion of feed because of the disease. For example Venesky et al. (2009) showed that when amphibian tadpoles were infected with Bd they ingested less food compared to tadpoles that were not infected.

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4.7 Toxicities

Toxicities can either occur through feeding or can be due to contact with the environment. A lot of substances can cause toxicity, some are already mentioned. In this chapter examples are given of substances that can be toxic as well. Metals Copper (Cu) intoxication is an example of an environmental toxicity. In the southern leopard frog (Rana sphenocephalus) it decreased embryonic survival (Lance et al., 2012). Other metals that can cause toxicity are lead, aluminum, zinc and others. These substances are sometimes used as building materials for cages and such, however because of their toxic characteristics they should be avoided (Densmore et al., 2007). Nitrogen products Ammonia and nitrite could also be possible contaminants of amphibian enclosures. Especially the water quality should be monitored to prevent adverse effects. Possible causes of water pollution could be a high population density or an impaired filtration system (Densmore et al., 2007). High concentrations of ammonia might lead to a decreased immunity, whereas increase of nitrite leads to methemoglobinemia (Wright et al., 2001). Prevention and treatment of this toxicity bears on water changing and preventive measurements (e.g. prevent overpopulation) (Densmore et al., 2007). Chemicals Chemicals like cleaning products and pesticides may also result in toxicity (Densmore et al., 2007). Detergents and disinfectants that contain for example chlorine and iodine can cause skin lesions and possibly death (Crawshaw, 1992; Wright et al., 2001). After utilization of these products a thorough washing of the environment is advised (Densmore et al., 2007). Pesticides like carbamates and organophosphates can cause neurological signs and are perhaps lethal (Sparling et al., 2001). There have been reports of other possible consequences of pesticide toxicity, but the neurological signs are most frequently seen (Densmore et al., 2007). Oxalate Oxalate toxicity has only been seen once, in animals that fed on insects the ingested plant materials that contained high concentrations of oxalate. A couple of waxy frogs (Phyllomedusa sauvagii) died after eating crickets that fed on oxalate-producing plants. Oxalate intoxication can lead to the deposition of renal calculi and this may result in kidney disease. Symptoms that were seen were lethargy, ascites and edema; eventually the disease resulted in death (Wright et al., 2001).

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DISCUSSION In general amphibians are a very diverse group and for a lot of species requirements are unknown. A common advice would be the further study on different species in order to assess their needs. Next to this further research also has to be done on which species are endangered and thus should be included in captive-breeding programs. In the future breeding of more amphibians will be necessary because of the ongoing declines. Sufficient funding should also be available to supply the necessary means to establish breeding colonies. Because of the great variety one could opt for the use of model species to assess requirements that can be used for other species. For example the western clawed frog (Xenopus tropicalis) for research on vitamin A metabolism (Clugston et al., 2014). However caution should be taken for deficiency or excess and one should monitor for health disorders. Another problem is that the amphibians that are almost extinct are understudied (Ferrie et al., 2014). Species-specific information for these threatened species are mostly unknown. Further studies in the future are advised to provide guidelines for the endangered species to keep them in captivity. An example of a critically endangered species that requires further research is the Titicaca water frog (Telmatobius coleus) (Angulo, 2008). Currently some research is taken place on Bolivian anurans and the Titicaca water frog in particular (Telmatobius coleus) (Muñoz, 2013). Nonetheless more studies are needed to understand these frogs. Next to the research it is needless to say that also the factors that threaten these species should be limited or avoided. Efforts should be taken to avoid for example habitat loss, pollution or disease (Angulo, 2008). When guidelines are set and amphibians can be reared and kept in a good health, the amphibians are excellent for captive breeding programs. The small body size, high fecundity, and low maintenance costs are some of the advantages of amphibians regarding captive breeding (Tapley et al., 2015). The fact that there are already some successful amphibian captive breeding stories is a promising fact for the future. A study executed by Griffiths et al. (2008) stated that 62% of these programs had very good results. No requirements or maximum levels of for example vitamin A are known for maintaining an adequate vitamin A metabolism. Also further research on supplementation is needed; which form of vitamin A is the best to provide adequate levels of vitamin A (Sim et al., 2010; Clugston et al., 2014). Next to this it has to be assessed if β-carotene can be processed to form vitamin A (Ferrie et al., 2014). However there is already some evidence that this transition is not taking place in amphibians. For example in tissue from the Cuban tree frog (Osteopilus septentrionalis) and the Cane toad (Bufo marinus) and in living Tiger salamanders (Amblystoma tigrinum) there was no conversion from β-carotene to retinol (Collins et al., 1983; McComb, 2010). Nevertheless further research is needed whether or not β-carotene can be processed into vitamin A in amphibians. So far most diagnoses of lingual squamous metaplasia caused by hypovitaminosis A have been made by determining liver concentrations of vitamin A (Pessier, 2013). However full body vitamin A concentrations (small dead animals) or blood samples (larger animals, possibly alive) can be used in the future if correct guidelines are set. When treating hypovitaminosis A, injections, per oral or topical solutions can be used. However one must take care of iatrogenic hypervitaminosis A when supplementing this to amphibians. Hypervitaminosis A can also lead to problems in developmental stages (limb and eye malformations). But also regarding tadpoles requirements are unknown and it is likely there are species differences. For example between the Common Indian toad (Bufo melanostictus) and the Skipper frog (Rana cyanophlyctis). The first one developed epidermal deformities when vitamin A excess was present, where the latter one remained untouched (Jangir et al., 1994). Only limited information is available of the vitamin A metabolism. Clugston et al. (2014) states that in the future research regarding the vitamin A metabolism should be performed on the Xenopus genus. This because of the fact that it has already been used in previous research; manipulation and maintenance of the species has been well documented. It can also serve as a model species in different research areas (Robert et al., 2011; Berg, 2012). Next to this genome sequence has been made of the western-clawed frog (Xenopus tropicalis), which might also have future study purposes concerning vitamin A metabolism (Hellsten et al., 2010). By mostly researching anurans and more specific the Xenopus genus, the vitamin A metabolisms in caudata and gymnophina are not well known (Clugston et al., 2014). Further research is needed in order to see if they have a comparable vitamin A metabolism.

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If amphibians are sensitive to reactive oxygen species one could op using vitamin E as anti-oxidant nutrient. Next to this also other nutrient contents could have anti-oxidant affects. For example vitamin C, carotenoids and flavonoids can be used in combination with vitamin E to prevent oxidative damage and steatitis (Carini et al., 1998; Facino et al., 1998; Krinsky, 2005; Durukan et al., 2006). Nonetheless these effects still have to be assessed in amphibian species. Calcium is also an important nutrient and the metabolism is more or less well described. Like vitamin A metabolism however future studies still need to examine whether or not it is the same in all amphibians. Next to this also requirements and maxima should be determined in order to maintain healthy amphibians. Calcium concentrations in the blood could be an indicator for MBD. Hypocalcaemia is an abnormality that is usually present in case of MBD, thus it could be used as a form of diagnosis. Standard concentrations in the blood are however not available and should be investigated. The phosphorus levels are also involved in MBD and calcium metabolism. The Ca:P ratio plays an important role; an increased phosphorus might lead to relative hypocalcaemia. For some species the ideal ratio still has to be assessed (Forzán et al., 2017). In others however it is known that the ratio should lie around 1:1.5 (Eidhof et al., 2006). In general the same principles apply to the other nutrients as well; there is a shortage of information. For example requirements, interactions and sometimes even their function needs to be investigated in the future. For now Ferrie et al. (2014) provided some recommendations making use of requirements for other (model) species (National Research Council (U.S.) (NRC), 1993; NRC, 1994; NRC, 1995; NRC, 2006). However at all times the captive population should be monitored in order to provide an accurate insight on their health condition. The requirements for fish, poultry, cats, dogs, and rats can however be used as guidelines to assess a nutritional base. Assessing the health in captive population is subject to difficulties. It is hard to evaluate small animals and next to this there is no standard procedure for reviewing amphibian's health (Pessier et al., 2014). Appetite and body condition score could be parameters to monitor overall health (Wright et al., 2001; Green, 2002). However records of consummation have to be kept and body condition score might be a subjective value. For the pathologies, even the most occurring ones, more research still is required. One could for example gain new insight in MBD. Another cause of MBD might be secondary renal hyperparathyroisidm because of renal failure. For example the Harlequin frog (Atelopus varius) displayed MBD disease along with a renal disease, namely polycistic nephropathy (Pessier et al., 2014). Renal disease leading to increased phosphorus levels might be the underlying cause of the MBD. However the disorders were only concurrent in a few individuals and a link has not yet been made. Thus the role of kidney disease in the development of MBD has to be examined in the future. Magnesium might also play a role in the development of MBD, but a link still has to be established. Klaphake (2010) suspects this because of magnesium's role in the calcium metabolism in mammals. But further research is necessary to provide evidence for this. In the Northern leopard frog (Rana pipiens) an increase in alkaline phosphatase activity was seen after supplementing exogenous magnesium (McWhinnie et al., 1971). Whether or not this is associated with MBD in amphibians is currently unknown. The role of nutrients regarding other amphibian pathologies can be the subject to future research. For example the influence of the diet on brown skin disease could be further investigated. This disorder has been reported in the Puerto Rican crested toad (Peltophryne lemur) and could be related with nutritional deficits (Crawshaw et al., 2014). Reproduction in general is also an understudied area in the amphibian class, but it is very important to ensure the production of offspring in captive breeding programs. The information that is available is only regarding a few species and regarding a couple of parameters. The studies however state that there is definitely an effect of nutrition on reproduction. For example the quality of an egg and male fertility can be influenced by the diet. The knowledge needs to be expanded to have a view on reproduction comparable with other vertebrate species. An idea would be to extrapolate from other animals in the search for new areas of research. An example could be the influence of nutrition on sexual hormones, although this is also an understudied area in other animals (Allen et al., 2004). Like in the wild a variety of prey species in captivity is advised to provide optimum nutrient content in combination with an increased activity and welfare (i.e. natural behavior) (McWilliams, 2008). However a diet consisting of multiple different feeder-insects can be difficult to achieve. A varied diet that approaches the natural diet might be unavailable due to insufficient funds or import restrictions. Some studies state however that in axolotls the variety of the diet doesn't play a role. They found that single-prey diets had no

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disadvantages compared to mixed-diets. Growth and natural behavior were maintained or even enhanced on a single-prey item (Mehrparvar et al., 2013; Slight et al., 2015). Further research is needed to find out if this also plays a role in other species within the amphibian class. Another aspect is the amount that has to be fed. In captivity the amount is an estimation based on leftovers and the condition of the animals, it is largely an empirical process (Green, 2002). Accurate records can be kept to get an insight on the amounts of feed ingested. Also appetite can be monitored, which might be a sign for nutritional deficiencies or disease (Wright et al., 2001). In the future standard metabolic rates might also be a guideline for assessing quantities of food. This is known in for example the African clawed frog (Xenopus laevis) (Hey, 1949), but still has to be assessed in other species. If we consult the available literature there are no specific requirements for just amphibians, extrapolation from other species can be a solution to estimate nutrient contents required. If we compare the amount of nutrients in the feed with the standard requirements for the amphibian we see that most of the feeder species are deficient (see table 1 and 2). We have to note some shortages in these assumptions. The exact requirements for amphibians are mostly unknown and even if there is information then it's only for a couple of species or nutrients. Thus caution has to be taken when comparing nutrients in the feed with requirements for other species. For some prey-species information is lacking (see appendix I). Next to this the information is variable; There are different presentations of concentrations, inter-species differences, and intra-species differences. Overall the conclusion can be made that research on amphibians needs to continue to establish correct requirements and also efforts should be made to find an ideal prey item. Currently when feeding one should feed a varied diet to have positive effects of multiple feeder-insects. For example feed a specie that has a good Ca:P ratio combined with a specie that contains the necessary protein. If a varied diet is not possible one should opt supplementing a certain species. For example crickets are frequently used, but have an off-balanced Ca:P ratio. Preferably gut-loading could be used to increase calcium levels (Arbuckle, 2009). Also other nutrient levels could be enhanced through supplementation, for example vitamin A. When assessing the needs of a captive individual or group one should take notice of the natural environment the animal evolved or survived in. For example in animals that normally live in environments with low resources, toxicities could take place when nutrients are given in excess (McWilliams, 2008). Husbandry factors are also important in maintaining captive amphibians. Together with nutrition they play a role in the prevention of diseases and amphibian welfare. In the current review the emphasis lies on nutrition rather than husbandry in total. Therefore it is just a short summary of what is known regarding amphibian husbandry. In conclusion, amphibians are a very diverse group of animals with diverse requirements. More research is needed to provide species specific information. However standard guidelines and extrapolation should give an estimation of what a diet should consist of in order to provide good health and reproduction. In the future more study into amphibian nutrition is definitely needed to sustain the declining populations. There is however some evidence that the current husbandry practices are successful; more than half of the captive breeding programs seem to sustain healthy amphibians (Griffiths et al., 2008). There are nonetheless still opportunities for improvement.

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APPENDIX APPENDIX I: Main nutrient contents of multiple feeder-species.

Crude protein and crude fat content shown in % of dry matter or g/kg. Calcium and phosphorus content shown in % of dry matter or mg/kg. Vitamin A and vitamin D3 presented in IU/kg. Nd= No detection. 1 Finke, 2015, 2 Barker, 1997, 3 Ferrie et al., 2014, 4 Finke, 2002, 5 Bernard et al., 1997, 6 Oonincx et al., 2012, 7

Finke, 2013, 8 Pennino et al., 1991, 9 Fard et al., 2014.