Taurine deficiency in maned wolves (Chrysocyon brachyurus) maintained on two diets manufactured for prevention of cystine urolithiasis

Zoo Biology 25:87–100 (2006)

Research Article

Taurine Deficiency in Maned Wolves(Chrysocyon brachyurus) Maintainedon Two Diets Manufactured forPrevention of Cystine UrolithiasisSara E. Childs-Sanford and C. Roselina Angel�

Department of Animal and Avian Sciences, University of Maryland,College Park, Maryland

This study assesses the long-term effects of an experimental diet vs. acommercially available manufactured diet, intended to reduce clinical diseaserelated to cystinuria, on the taurine status of captive maned wolves. For 13 weeks,two pairs of maned wolves were maintained on the commercially availablemaintenance diet, whereas two individually housed wolves were maintained onthe experimental diet. All six wolves, at the beginning and at the end of the diettrial, had severely decreased plasma concentrations of taurine (as compared tothe normal canine reference range of 60–120 nmol/ml) (National ResearchCouncil [2003] National Academies Press) with average taurine concentrations of16 nmol/ml at the beginning of the study and 3 nmol/ml at the end of the study.There was no statistically significant difference in the taurine concentrationsbetween animals on the maintenance vs. experimental diets. Both diets weresupplemented subsequently with taurine at a concentration of 0.3%. All studyanimals were eventually switched to the taurine-supplemented version of thecommercially manufactured maintenance diet and subsequent samplings werecarried out to monitor plasma taurine concentrations. A final sampling, carriedout approximately 5 months after the initiation of taurine supplementation,showed an average taurine concentration within the target canine reference range(90.25 nmol/ml). There are numerous physiologic (e.g., possible unique metabo-lism and requirements for taurine in this species as compared to other canids) anddietary factors (e.g., effects of the types and concentrations of fiber and protein on

Published online 10 November 2005 in Wiley InterScience (www.interscience.wiley.com).

DOI 10.1002/zoo.20078

Received 12 March 2005; Accepted 2 September 2005

Dr. Childs’ present address is: 137 Brook Way, Ithaca, NY 14850.�Correspondence to: Dr. Roselina Angel, Department of Animal and Avian Sciences, University of

Maryland, College Park, MD 20742. E-mail: [email protected]

rr 2005 Wiley-Liss, Inc.

nutrient availability, taurine metabolism, and enterohepatic circulation oftaurine-conjugated bile salts; impaired taurine synthesis secondary to low cysteineavailability) that could be potential contributors to the development of taurinedeficiency in the maned wolves in this study. Taurine supplementation should beconsidered in maned wolves maintained on diets intended for reduction ofcystinuria-related complications. Zoo Biol 25:87–100, 2006. �c 2005 Wiley-Liss, Inc.

Keywords: cystinuria; plasma taurine; taurine biosynthesis; soybean protein; soluble fiber

INTRODUCTION

The maned wolf is a rare South American canid of which a relatively smallcaptive population is maintained currently in zoological parks across the UnitedStates. In a captive setting, the maned wolf is known for being generally unthriftyand having marginal to poor reproductive success, however, the most serious andwidely recognized disorder afflicting captive maned wolves with an alarmingprevalence is cystinuria [Bovee and Bush, 1978; Bush and Bovee, 1978; Bovee et al.,1981; Mussart and Coppo, 1999]. Cystinuria, which has been shown to have anautosomal recessive mode of inheritance in humans and domestic dogs, ischaracterized by defective transport of the nonessential amino acid cysteine, andthe dibasic amino acids lysine, ornithine, and arginine, through the epithelial cells ofboth the renal tubular and intestinal brush border membranes [Harris et al., 1955;Rosenberg et al., 1965; Casal et al., 1995]. Excess concentrations of cysteine in theurine often result in clinical disease secondary to the formation of cystine uroliths,which, depending on their location, can predispose the patient to complications suchas urinary tract infections and renal insufficiency, as well as cause life-threateningupper and lower urinary tract obstruction [Lindell et al., 1997; Rutchik and Resnick,1997; Joly et al., 1999; Goodyer et al., 2000]. Maned wolves have been plaguedhistorically by complications related to cystinuria that have resulted in considerablemorbidity and mortality [Bovee and Bush, 1978; Bush and Bovee, 1978; Bovee et al.,1981; Mussart and Coppo, 1999]. Basic therapeutic principles in the management ofcystinuria include increasing the solubility of cystine in the urine (by increasing urinepH) as well as reducing urinary cystine concentration, and these objectives can beapproached by urinary alkalinization, administration of thiol-containing drugs, anddietary modification [Joly et al., 1999; Ng and Streem, 1999; Barbey et al., 2000].

In an effort to reduce the incidence of clinical disease from cystinuria in themaned wolf, nutritional modification has been previously researched [Boniface,1998], resulting in the development of a maned wolf diet that was made commerciallyavailable in 1998 and was fed to almost all of the maned wolves in the United States(M. Rodden, personal communication). This diet, which was characterized primarilyby a moderate protein concentration, a low concentration of sulfur-containingamino acids (cysteine and methionine), and a high fiber concentration, resulted in asignificant decrease in the urinary excretion of cystine, but did not result in asignificant increase in the urine pH [Boniface, 1998]. The solubility of cysteine inurine is highly dependent on pH. Low urine pH promotes cystine precipitation andurolith formation, whereas high urine pH results in increased cysteine solubility[Osborne et al., 1989; Dent and Senior, 1995]. In an effort to make furtherimprovements on this diet, an experimental diet was developed that was

88 Childs-Sanford and Angel

demonstrated to significantly raise the urine pH in the maned wolves tested (Childs-Sanford and Angel, unpublished data). In addition, the sodium (Na) concentrationin this experimental diet was decreased based on human studies that demonstratedthat urinary excretion of cysteine is directly correlated with urinary Na excretion,with low Na diets effectively reducing urinary cysteine excretion [Jaeger et al., 1986;Norman and Manette, 1990; Peces et al., 1991; Rodriguez et al., 1995]. In an effort toassess the long-term effects of this experimental diet vs. the commercially availablemanufactured diet on the overall nutritional and health status of the captive manedwolf a long-term diet trial was initiated.

MATERIALS AND METHODS

Animal Husbandry

All animal-related activities were approved by the institutional Animal Careand Use Committee. Seven adult maned wolves were maintained at the NationalZoological Park’s Conservation and Research Center in Front Royal, Virginia.Three of the wolves were housed individually in enclosures with an outdoor area aswell as a concrete indoor enclosure with a den. The other four animals remained inmated pairs, with one pair having an enclosure similar to that of the individualanimals and the second pair having a large outdoor enclosure with access to smalldens constructed of wood. Details regarding den and enclosure parameters havebeen published previously by Brady and Ditton [1979]. One wolf died during the trialdue to a gastric dilatation volvulus. Both pairs became pregnant and gave birthduring the study, however the pups were presumed to be ingested by the dam or siresoon after birth in both instances and it is not known if the pups were alive or not atthe time of their birth. All wolves involved in the study had access to and wereknown to consume invertebrates occasionally as well as small mammalian and avianwildlife in their outdoor enclosures during the course of the study, which spannedfrom December 2001–July 2002.

Diet Trial

There were two diets tested in this study: a commercially manufactured manedwolf maintenance kibble (Mazuri, PMI Nutrition International, St. Louis, MO),referred to in this study as the maintenance diet, and an experimental diet thatdiffered only in the proportion of animal-based to plant-based protein as well as thesodium to potassium ratio (Table 1). Both diets were made using the same extrusionprocess and equipment so that shape and physical characteristics were similar.Approximately 40% of the protein in the maintenance diet was derived from animalsources, whereas the protein in the experimental diet had only approximately 5% ofits protein derived from animal sources. The sodium:potassium ratio was alsodifferent between the two diets. The experimental diet had a lower ratio (0.2%Na:0.8%K) vs. that in the maintenance diet (0.5% Na:0.6% K) (Table 1). For thislong-term study, the four maned wolves that were housed in pairs were assigned tothe original maintenance diet (that was the diet they had been on previously),whereas the three individually-housed wolves were gradually switched to theexperimental diet. Animals were fed once daily in the morning and were each offeredapproximately 800 g of kibble/day. All extraneous items normally added to the diet

89Taurine Deficiency in Maned Wolves

TABLE 1. Formulated ingredient and nutrient composition, as fed, of the experimental diets

Maintenance diet Experimental diet

IngredientMeat Meal 9.58 0.00Low Ash Poultry Meat Meal 2.00 0.00Dried Whey 0.50 0.50Brewer’s Yeast 2.00 2.00Beef Digesta 3.00 3.00Dehulled Soybean Meal 11.81 21.50Corn Gluten Meal 0.00 3.45Soy Protein Concentrate 0.00 0.20Rice Flour 25.00 25.00Corn Flour 9.35 0.00Poultry Fat 6.00 6.00Bleachable Fancy Tallow 6.88 8.47Soy Oil 0.50 0.50Ground Beet Pulp 4.00 11.21Apple Pomace 7.00 7.00Ground Soy Hulls 6.44 2.77Tomato Pomace 2.50 2.50Sodium Chloride 0.35 0.39Lysine 0.00 0.02Other 3.09b 5.49c

Formulated Nutrient Concentrations (analyzed concentrations in parentheses)Protein 18.50 (18.81) 18.50 (18.55)Fat 16.00 (17.2) 16.00 (16.9)Crude fiber 6.5 6.5Neutral detergent fiber 13.77 15.69Acid detergent fiber 9.38 10.01Insoluble fiber (14.8) (16.9)Soluble fiber (3.25) (4.32)Metabolizable Energy, kcal/kg 3530 3530Methionine 0.26 (0.49) 0.27 (0.43)Cystine 0.21 (0.34) 0.22 (0.37)Taurine 0.25 (0.01) 0.25 (0.01)Lysine 0.95 (1.04) 0.98 (1.05)Sodium 0.50 0.20Potassium 0.60 0.80

% of protein in diet from:Animal sources 43.8 5.3Plant sources 56.2 94.7

aPalatability enhancing liquid ingredient with o10% dry matter.bContains (% as fed): dicalcium phosphate, 1.053; calcium carbonate, 0.282; pyridoxine (1%),0.144; choline chloride (70%), 0.141; menadione (2,900 ppm), 0.103; vitamin D3 (7,500 IU/g),0.053; biotin (0.1%), 0.050; vitamin E (500 IU/g), 0.45; vitamin A (27240 IU/g), 0.037; ferroussulfate (31%), 0.030; calcium iodate (60%), 0.025; zinc oxide (72%), 0.022; L-lysine, 0.022;ethoxyquin, 0.018; selenium (0.06%), 0.015; calcium pantothenate (17.6 g/kg), 0.013; folic acid(2%), 0.013; thiamin (10%), 0.012.cContains (% as fed): dicalcium phosphate, 2.713; calcium carbonate, 0.172; pyridoxine (1%),0.142; choline chloride (70%), 0.145; menadione (2,900 ppm), 0.103; vitamin D3 (7,500 IU/g),0.53; biotin (0.1%), 0.05; vitamin E (500 IU/g), 0.045; vitamin A (27240 IU/g), 0.037; ferroussulfate (31%), 0.030; calcium iodate (60%), 0.025; zinc oxide (72%), 0.022; ethoxyquin, 0.018;selenium (0.06%), 0.015; calcium pantothenate (17.6 g/kg), 0.013; folic acid (2%), 0.013;thiamin (10%), 0.012.


at this facility were discontinued except for two mice (approximately 25-g each) perwolf per day. The wolves were maintained on their respective diets for approximately13 weeks.

Animal Procedures

Four weeks before the start of the diet trial (November 1, 2001), animals wererestrained in a squeeze cage and heavily sedated with Telazol (zolazepam plustiletamine, Fort Dodge Laboratories, Inc., Fort Dodge, IA) administeredintramuscularly with a hand syringe. Blood (12ml) was collected from the jugularvein directly into a heparinized syringe. At the end of the diet trial (March 4, 2002), asecond immobilization was carried out and blood was collected in an identicalmanner to the first immobilization.

Sample Processing

Heparinized blood samples were immediately centrifuged at 8,000� g for10min and 2ml of plasma were transferred to a separate centrifuge tube. An equalamount (2ml) of sulfosalicylic acid was added to the plasma and centrifuged at14,000� g for 25min. The supernatant was transferred to a plastic microtainer tubeand frozen at �801C until analysis at the completion of the study.

Taurine Analysis

Samples were analyzed at the Amino Acid Analysis Laboratory, Departmentof Molecular Biosciences, University of California at Davis. Plasma taurineconcentrations were determined by the use of an automated analyzer (Model7300, Beckman Instruments, Palo Alto, CA), which utilizes cation-exchangechromatography and spectroscopic determination of a ninhydrin reaction withamino acids to obtain measured values from a plasma sample. Norleucine was usedas an internal standard to standardize the concentrations of amino acids across time.

Diet Alterations and Subsequent Immobilizations

After the initial (November 2001) immobilizations, the three individually-housed wolves in the experimental group were gradually (over 3 weeks) convertedfrom the commercial maintenance diet to the experimental diet. All animals werekept on the same diets from the start of the trial (December 1, 2001) until March2002 (Fig. 1). As the diet trial progressed, there were reports of decreased appetiteand gradual weight loss of the wolves. Due to these clinical signs the trial wasterminated in March 2002, approximately 4 weeks earlier than originally planned.Based on the taurine results from the November and March samples, which wereobtained after the March immobilization, the decision to add taurine to both of thediets was deemed necessary. Taurine was therefore added to both the experimentaland maintenance diets at a concentration of 0.3% and fed to the wolves beginningMarch 18, 2002. This concentration of taurine was also added to all new batches ofthe commercial maintenance kibble produced and distributed in the United States bythe manufacturer. Iron (iron-polysaccharide complex) was also added to these twodiets at an analyzed concentration of 0.04%. Two subsequent immobilizations werecarried out to monitor the response to taurine supplementation (May 2002 and July2002). Immobilizations, sample processing, and sample analysis were carried out asdescribed previously. Immediately after the May 2002 immobilization, all wolves


were put on the maintenance formula with taurine supplementation (0.3%) as well asmiscellaneous food items normally added by the institution including (per animal)three 25-g mice, 1/4 can (3.3 oz.) Pedigree Completes dog food, 6 grapes, and 30 ccMirra-Coat Liquids (PetAg Inc., Hampshire, IL).

Statistical Analysis

The results of the study were analyzed as a crossover design withheterogeneous variances for the time periods before and after taurine was addedto the diets using the mixed procedures of SAS [SAS Institute, 1999]. Fixed effectswere diet and date of measurement, and wolf ID was a random effect. Pairwisecomparisons using Tukey’s HSD to control for experiment-wise error rate [Ott andLongnecker, 2004] and a contrast comparing average plasma taurine concentrations,before and after addition of taurine to the diet, were carried out. Means wereconsidered significantly different at Po0.05.

RESULTS

Plasma taurine concentrations for individual wolves at the time of eachimmobilization are shown in Table 2, and average plasma taurine concentrationsfrom each immobilization are displayed in Table 3. Plasma taurine concentrations ofall wolves, both at the beginning of the diet trial (November 2001) and at the end ofthe diet trial (March 2002), are severely deficient when compared to the normalcanine reference range of 60–120 nmol/ml (Amino Acid Analysis Laboratory,Department of Molecular Biosciences, University of California at Davis). Althoughall animals demonstrated a decline in taurine concentrations between the first andsecond immobilizations, the decline was not significant (Po0.07) (Table 4). Therewas no significant difference in taurine levels at either immobilization date(November vs. March) between animals on the maintenance diet vs. the experimentaldiet, with average plasma taurine concentrations of 12.08 nmol/ml and 5.16 nmol/mlin wolves on the experimental diet in November and March, respectively, andaverage plasma taurine concentrations of 17.96 nmol/ml and 2.06 nmol/ml in thewolves on the maintenance diet for these same respective months. The average

NOV DEC JAN FEB MAR APR MAY JUNE JULY

Experimental Experimental +3 Maintenance ++5

Maintenance Maintenance +4 Maintenance ++5

_________________________________________________________________________________________________

MONTH

EVENT Nov. 1-2, 2001 December 1, 2001 March 4-7,2002 March 18,2002 May 7, 2002 July 15, 2002 Immobilizations START Immobilizations taurine (0.3%) Immobilizations Immobilizationsand sampel of diet trial and sample added to both and sample collection and samplecollection collection. diets collection

END of diet trial DIET

Exp. Animals1

Control Animals2

_________________________________________________________________________________________________

1 n=2 (Three animals were in this group until January 10th when one animal died.)2 n=4 (Two mated pairs)3 Experimental diet plus taurine at 0.3% (started March 18). 4 Maintenance diet plus taurine at 0.3% (started March 18). 5 Maintenance diet plus taurine at 0.3% as well as miscellaneous food items added by the institution including (per animal) three 25g mice, ¼ can (3.3

oz.) Pedigree® Complete dog food, 6 grapes, and 30cc Mirra-Coat Liquid® (PetAg, Inc., Hampshire, IL 60140) (started May 8).

Fig. 1. Timeline of events during a diet trial testing an experimental vs. a maintenance diet inmaned wolves.


plasma taurine concentration remained at the low end of the domestic canine normalreference range at the May 2002 immobilization, approximately 8 weeks after thediets had been supplemented with additional taurine at 0.3% of diet. At the finalsampling in July 2002, approximately 16 weeks after diet supplementation withtaurine, average plasma taurine for the six wolves was within the target domesticcanine reference range.

TABLE 2. Plasma taurine concentrations (nmol/ml) in six maned wolves

Immobilization date

Wolf

1a 2a 3b 4b 5b 6b

November 2001 16.79 7.37 11.9 10.96 37.43 11.56March 2002 7.88 2.45 1.79 1.89 2.619 1.95May 2002 105.76 21.25 22.74 99.69 24.01 121.07July 2002 146.68 122.65 31.34 32.82 141.65 66.38

aAnimals fed the experimental diet during the diet trial period (November 2001–March 2002).bAnimals fed the maintenance diet during the diet trial period (November 2001–March 2002).

TABLE 3. Changes in plasma taurine concentrations (nmol/ml) in maned wolves associated

with several generalized changes in feeding strategya

Sampling date DietAverage plasma

taurineb (nmol/ml) SEM

November 2001 Maintenancec 18.79h 4.83March 2002 Maintenance/

experimentald4.03i 6.58

May 2002 Maintenance pluse 66.68g 20.28July 2002 Maintenance plus/

miscellaneous itemsf91.18g 20.28

aP-value: diet, 0.7125; supplemental taurine, 0.0003; date, 0.0006. Contrast between plasmataurine values before taurine supplementation was initiated (November 2001 and March 2002)and after taurine was added to the diets (May and July 2002) (P-value5 0.0004 before andafter).bMean of taurine result from each of the six maned wolves involved in the study.cThe maintenance diet was a commercially manufactured diet (Mazuri Feeds, Inc., ManedWolf Maintenance). The formulated taurine concentration in this diet was 0.25%.dDuring the diet trial period (November 2001–March 2002), two wolves were fed theexperimental diet, and four wolves were fed the maintenance diet. There was no effect of diet(experimental vs. maintenance) on plasma taurine concentrations during the diet trial and thusthe mean is for all six wolves in the study (see Table 2).eAfter the March 2002 sampling, the experimental and commercially manufacturedmaintenance diets were modified as follows: taurine added to an analyzed concentration of0.3% and Fe added to an analyzed concentration of 0.04% (two wolves were maintained onthe Experimental Diet to participate in another study).fAfter the May 2002 immobilization, all wolves were put on the maintenance formula withtaurine supplementation (0.3%) as well as miscellaneous food items normally added by theinstitution including (per animal) three 25-g mice, 1/4 can (3.3 oz.) Pedigree Completes dogfood, 6 grapes, and 30cc Mirra-Coat Liquid (PetAg Inc., Hampshire, IL).gMeans differ (Po0.01).hMeans differ (Po0.01).iMeans differ (Po0.01).


DISCUSSION

Compared to normal canine and feline reference ranges for plasma taurine, themaned wolves in this study demonstrated a severe taurine deficiency before the startas well as at the completion of the diet study (November 2001–March 2002).Although normal reference ranges for plasma taurine have not been established forthe maned wolf, the dramatically low concentrations as compared to normalreference ranges for domestic species (canine reference range5 60–120 nmol/ml;feline reference range5 80–120 nmol/ml) (Amino Acid Laboratory, Departmentof Molecular Biosciences, University of California at Davis), likely do represent atrue deficiency. Plasma taurine concentrations o40 nmol/ml are considered evidenceof taurine deficiency in dogs [Backus et al., 2003; Delaney et al., 2003], althoughsome authors suggest that o25 nmol/ml indicates a deficiency [Kramer et al., 1995].Regardless, the plasma taurine concentrations of all six of the maned wolvesinvolved in this study were well below these suggested critical levels.

Taurine is obtained by animals through two methods: diet and biosynthesis.Plants tend to be poor sources of taurine, so taurine is obtained from the dietprimarily through ingestion of foods of animal origin [Spitze et al., 2003]. In thistrial, the experimental and maintenance diets had a significant proportion of theirprotein from plant sources and dietary taurine concentrations may not have beenadequate for the maned wolves. If this was the case, however, a significant effect ofdiet on the plasma taurine concentrations in the wolves would have been expected,because the experimental diet had 95% of its protein derived from plant sources,whereas the maintenance diet had only 60% of its protein from plant sources.Furthermore, the analyzed concentrations of taurine in the diets fed to the wolvesbefore taurine supplementation were typical of average commercial domestic canineextruded dry diets [Backus et. al., 2003]. This would suggest that maned wolves couldpotentially have a dietary requirement for taurine, unlike the domestic dog, in whichno requirement is considered necessary. The taurine requirement for adult catsas published by the National Research Council [2003] is 0.4%, and no requirement islisted for the domestic dog.

Like cats, domestic canids are obligated to conjugate their bile acids to taurine,however, they are not assumed to have a dietary requirement for taurine due to their

TABLE 4. Changes in plasma taurine concentrations (nmol/ml) associated with specific changes

in feeding strategy during and after the diet trial

Sampling date Diet nAverage plasmataurine (nmol/ml) SEM

Initiala Maintenanceb 6 18.79 4.83Finalc Experimentald 2 5.16 1.38Final Maintenance 4 2.06 0.974

aInitial plasma sample taken immediately before the experiment started (November 2001). Allwolves were on the maintenance diet before the start of the experiment.bThe maintenance diet was a commercial diet (Mazuri Feeds, Inc., Maned Wolf Maintenance).cFinal sampling occurred 4 months after the start of the experiment (March 2002).dThe experimental diet was mainly a plant protein-based diet vs. the maintenance diet whichwas a predominantly meat-based diet.


capability for taurine biosynthesis. Biosynthesis of taurine occurs primarily in theliver from cysteine or methionine. The conversion of these sulfur-containing aminoacids to taurine is regulated by the activities of the two rate-limiting enzymes cysteinedioxygenase and cysteine sulfinate decarboxylase [Stipanuk et al., 1992a]. Felids havelow activity of both of these enzymes, especially cysteine dioxygenase, contributingto the essentiality of taurine in this family [Knopf et al., 1978]. Typical canids haveadequate enzyme activity in the liver to synthesize needed taurine under normalconditions [Hayes, 1989]. The presence and level of activity of these hepatic enzymeshave not been studied in the maned wolf. Therefore, it is possible that the manedwolf, like the cat, has a high dietary requirement for taurine based on an inability toadequately synthesis taurine in the liver. If maned wolves, like dogs, do haveadequate hepatic enzyme activity for taurine biosynthesis, the low concentrations ofboth cysteine and methionine in the diets tested in this experiment could potentiallyhave decreased the ability of the wolves to synthesize adequate amounts of taurine.Cystinuric humans and domestic dogs have been documented to have low plasmaand urine taurine concentrations, and it is hypothesized that this is primarily due todecreased synthesis capabilities due to low plasma cysteine concentrations[Martensson et al., 1990; Sanderson et al., 2001a,b]. Furthermore, under conditionsof low substrate availability for the taurine synthesis reaction, formation of alternateproducts may be favored. For example, in rat hepatocytes, glutathione formation isfavored over taurine synthesis during conditions of low cysteine availability[Stipanuk et al., 1992b].

Dietary factors may have also resulted in interference with normal taurinemetabolism. This has been demonstrated in numerous species, including recentevidence published on domestic canids in which dogs maintained on commercialmaintenance diets, especially those containing rice bran or whole grain rice withlamb meal, exhibited low plasma taurine concentrations [Fascetti et al., 2003; Backuset al., 2003]. The two main routes of taurine removal from the tissues include theconjugation of bile acids and fecal excretion. Approximately 95% of conjugated bileacids are normally reabsorbed in the distal ileum via high affinity receptors andreturned to the liver through the portal circulation, completing the cycle ofenterohepatic circulation. Taurine that becomes deconjugated from bile acids or thatis unabsorbed from the diet is lost through fecal excretion. Numerous dietary factorshave been demonstrated to have effects on the enterohepatic circulation and fecalloss of taurine, including the type of dietary protein. In this experiment, soybeanprotein isolate was utilized to supply the majority of the plant-based protein in boththe maintenance and experimental diets. Soybean protein has been demonstrated toresult in increased taurine loss, both through enhancement of the microbialdegradation of bile acids as well as the acceleration of cholecystokinin-mediatedturnover of bile acids [Morris et al., 1994; Kim et al., 1995; Backus et al., 1995].Furthermore, soybean proteins have been shown to have the ability to bind andsequester bile acids within the intestinal tract through the formation of hydrophobicpeptides, thus preventing their reabsorption and recirculation to the liver [Huff andCarol, 1980; Makino et al., 1988; Choi et al., 1989; Benyen, 1990; Iwami et al., 1990;Sugano et al., 1990; Hickman et al., 1992a,b]. Fiber has also been demonstratedto interfere with the enterohepatic circulation of bile acids and thus taurine. Boththe maintenance and experimental maned wolf diets were very high in fiber,primarily soluble fiber. In rats, soluble dietary fiber has been demonstrated


to increase fecal loss of bile salts and decrease hepatic concentrations of taurine ascompared to diets containing insoluble fiber [Ide et al., 1989; Buhman et al., 1998;Ginnett et al., 2003].

Taurine deficiency has been reported in several animal species including cats,dogs, foxes, and exotic felids [Howard et al., 1987; Hayes and Trautwein, 1989;Moise et al., 1991; Sturman, 1992; Kramer et al., 1995; Ofri et al., 1996; Kittlesonet al., 1997]. Taurine, a sulfur-containing b-amino acid, is one of the most abundantfree amino acids in the body and has important functions in virtually all bodysystems. In addition to its role in normal bile salt function, taurine is essential fornormal retinal, cardiac, neurologic, reproductive, immune, and platelet function, andalso functions as an antioxidant [Sturman and Messing, 1990; Hickman et al., 1992b;Chapman et al., 1993; Stapleton et al., 1998; Mankovskaya et al., 2000; Lima et al.,2001; Miglis et al., 2002; Militante and Lombardini, 2002; Schuller-Levis and Park,2003]. Therefore, the effects of a taurine deficiency are likely widespread in multipleorgan systems. The clinically apparent results of taurine deficiency are most welldescribed in the domestic cat, and typically include central retinal degeneration[Hayes et al., 1975; Barnett and Burger, 1980], dilated cardiomyopathy [Pion et al.,1987], and reproductive problems [Sturman et al., 1986]. There were no physicalexamination findings or clinical signs suggestive of central retinal degeneration ordilated cardiomyopathy in the maned wolves involved in this study. Interestingly,however, poor reproduction has been a chronic problem of maned wolves incaptivity in the United States, especially in recent years [M. Rodden, personalcommunication]. For example, in the 2001–2002 breeding season, there were onlytwo puppies born that lived, and ironically, these puppies were born at the only zooin the United States at that time that did not feed the commercial maintenance dietto their maned wolves. In contrast, during the breeding season (2002–2003), aftersupplementation of the diet with taurine, a record 24 puppies were born. Duringpregnancy in mammals, taurine is accumulated in the maternal tissues in preparationfor delivery through the placenta and milk to the fetus and neonate, as taurine is anessential amino acid for the offspring during the perinatal period [Dieter et al., 1993;Aerts and VanAssche, 2002]. Domestic cats with taurine deficiency exhibit increasedfetal resorption and stillbirths, low birth weight of offspring, and decreasedsurvivability of liveborn kittens. In those kittens that survive, there is an increasedincidence of developmental defects including retinal degradation, delayed cerebellarcell division and migration, and abnormal cortical development [Sturman et al.,1986; Sturman, 1988]. The reproductive problems recognized in captive manedwolves in the past have not been thoroughly characterized, but include poor puppysurvival, killing of the puppies by the dam, and reduced fertility. It is therefore highlypossible that a major contributor to the poor reproductive success of captive manedwolves in the United States has been taurine deficiency.

Although plasma taurine concentrations drop to low levels in as quickly asfour weeks in domestic cats maintained on diets deficient in taurine, in both cats anddogs, clinical signs related to the deficiency may take months to years to develop[Pion et al., 1987, 1992; Sanderson et al., 2001a]. Therefore, additional clinicalmanifestations of taurine deficiency in the maned wolves may have developed if thesevere deficiency had continued longer. The severity of the taurine deficiency in themaned wolves may have been exacerbated by the fact that supplemental food itemsnormally added to the maintenance kibble at this facility, which include several prey


items such as mice that are high in taurine, were removed from the diet (with theexception of two approximately 25-g mice/day) for the purpose of the study. Theplasma taurine concentrations of the wolves on the commercial maintenance diet(without taurine supplementation) including these supplemental items has not beenevaluated, although the tremendous improvement in reproductive success in thebreeding season after taurine supplementation in comparison to the poorreproductive success in years before this study, imply that taurine deficiency mayhave existed before this study.

Based on extremely low plasma taurine concentrations and the response ofthese concentrations to taurine supplementation, a taurine deficiency wasdocumented in the maned wolves involved in this study. The findings in this studymay imply that maned wolves have a dietary requirement for taurine, and alsosuggest that diet, especially protein and fiber type and concentration, may have asignificant effect on plasma taurine concentrations in this species. The circumstantialevidence involving improved reproductive success of maned wolves in the UnitedStates after taurine supplementation of the diet supports a role of taurine in the poorreproductive success of the maned wolf, and also may indicate that the clinical effectsof taurine in this species are primarily reproductive. Future research on taurinestatus and the physiology of taurine metabolism in maned wolves is indicated. Basedon the data from this study, it is recommended that all maned wolves maintainedon low protein or otherwise modified diets intended for the prevention of cystinuria-related clinical disease be provided with supplemental taurine at concentrationsrecommended for domestic felids.

CONCLUSIONS

Six maned wolves maintained on two different diets intended for prevention ofcystinuria-related clinical disease exhibited plasma taurine concentrations that weremarkedly lower than canine and feline normal reference ranges. These deficientconcentrations responded within 4 months to taurine supplementation at 0.3% ofdiet by rising into target normal reference ranges. Further research is neededinvolving the physiology of taurine metabolism in maned wolves, as the data fromthis study suggest that taurine metabolism in maned wolves may be different than inother canids. The possibility of a taurine requirement in the maned wolf should beconsidered. Even if maned wolves are physiologically capable of taurine biosynth-esis, the wolves involved in this study may have been unable to do so due to a lack ofsubstrate (cysteine) availability. A marginal to poor sulfur amino acid status due tourine cysteine loss from cystinuria may have been compounded by the feeding ofdiets low in cysteine. Several dietary factors may have interfered with normal taurinemetabolism in the maned wolves involved in this study, mainly the effects of thetypes and concentrations of both protein and fiber on nutrient availability, taurinemetabolism, and enterohepatic circulation of taurine-conjugated bile salts. Theabsence of clinical evidence of dilated cardiomyopathy or central retinal degenera-tion in the taurine deficient maned wolves in this study, in combination with amarked increase in the reproductive success in the United States captive populationafter dietary taurine supplementation, suggest that the clinical consequences oftaurine deficiency in this species may be primarily reproductive. In light ourdocumentation of severe taurine deficiency in all of the maned wolves involved in


this study, we highly recommend that maned wolves fed diets intended for treatmentof prevention of cystinuria-related clinical disease be supplemented with taurine atconcentrations according to NRC recommendations for domestic felids.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the assistance of the animal care andveterinary staff of the National Zoological Park’s Conservation and ResearchCenter, as well as the advice and assistance of Dr. Q. Rogers.

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Taurine deficiency in maned wolves (Chrysocyon brachyurus) maintained on two diets manufactured for prevention of cystine urolithiasis