Division of Zoo Animals, Exotic Pets and Wildlife, Vetsuisse Faculty, University of Zurich, Switzerland Antioxidant Status of Faeces of Captive Black Rhinoceros (Diceros bicornis) in Relation to Dietary Tannin Supplementation M. Clauss 1,8 , N. Pellegrini 2 , J. C. Castell 3 , E. Kienzle 3 , E. S. Dierenfeld 4 , J. Hummel 5 , E. J. Flach 6 , W. J. Streich 7 and J.-M. Hatt 1 Addresses of authors: 1 Division of Zoo Animals, Exotic Pets and Wildlife, Vetsuisse Faculty, University of Zurich, Switzerland; 2 Department of Public Health, Human Nutrition Unit, University of Parma, Italy; 3 Institute of Animal Physiology, Physiological Chemistry and Animal Nutrition, LM University of Munich, Germany; 4 Department of Animal Health and Nutrition, Saint Louis Zoo, St. Louis, MO, USA; 5 Zoological Garden of Cologne, and Institute of Animal Science, Animal Nutrition Group, University of Bonn, Germany; 6 Zoological Society of London, Whipsnade Wild Animal Park, Dunstable, UK; 7 Leibniz-Institute for Zoo and Wildlife Research (IZW), Berlin, Germany; 8 Corresponding author: Tel.: ++41 44 6358376 ; fax: ++41 44 6358901; E-mail: mclauss@vetclinics.unizh.ch With 2 figures and 1 table Received for publication March 16, 2006 Summary In context with the frequent observations of excessive iron (Fe) storage in captive black rhinoceroses (Diceros bicornis), it has been suggested that both an excessive dietary Fe content and a lack of dietary Fe-chelating substances, such as tannins, is the underlying cause. Therefore, studies on the effects of tan- nin supplementation to captive diet are warranted. Six captive rhinoceroses were fed their normal zoo diet (N), and a similar diet supplemented with either tannic acid (T, hydroly- sable tannin) or quebracho (Q, condensed tannins), and the total antioxidant capacity (TAC) was measured as mmol Trolox equivalents per kg fresh faeces. The TAC values on diets N (1.24 ± 0.39 mmol/kg fresh faeces) and T (1.34 ± 0.33 mmol/kg fresh faeces) were similar, but signifi- cantly higher on diet Q (2.32 ± 0.61 mmol/kg fresh faeces). In contrast to expectations, faecal TAC increased with increasing faecal Fe, possibly as a result of the fact that the faecal Fe content was positively correlated to the proportion of con- centrate feeds in the diet, which also contain antioxidants, such as vitamin E, in addition to Fe. Increased antioxidant status caused by the use of tannin substances could have a beneficial effect on animal health, but if tannins should be incorporated in designed diets, other tannin sources, such as grape pomace should be tested. Introduction Captive black rhinoceroses (Diceros bicornis) are prone to excessive iron storage (Smith et al., 1995; Paglia and Dennis, 1999; Dierenfeld et al., 2005), which has been linked to various disease syndromes in this species. As compared to the forages these animals consume in the wild, diets in captivity are high in iron (Castell, 2005) and low in iron chelators, such as tannins (Wright, 1998). Therefore, the excessive iron storage observed could be a consequence of the high amounts of iron that is not restricted in its availability. Accordingly, there is substantial interest in the consequences of adding tannin sources to captive diets on the health of the animals (Clauss, 2003). A major problem in studies that introduce a dietary substance with the aim of improving the long-term effects of the overall ration is the choice of an adequate parameter for the documentation of a measurable influence of that substance. Ideally, such trials should be performed on a long-term basis with a large number of animals, which can all be bled regularly, and whose overall long-time health performance can be evaluated. However, with a species like the black rhino, such conditions are very difficult to meet. On the one hand, animal numbers at one facility will hardly be enough for a statistical evaluation, but on the other hand, it will be difficult to find facilities with a similar commitment to animal training, proficiency in bleeding the animals and readiness to perform controlled feeding trials on identical diets. Using eight animals from three different zoos, it could be shown that the inclusion of tannins to their regular diets influenced the production of salivary tannin-binding proteins (Clauss et al., 2005) but had no measureable influence on iron absorption as measured by conventional balance trials (Clauss et al., 2006b). It had been planned, in addition, to evaluate iron parameters and antioxidant status in regular blood samples from these animals, as the feeding of tannic acid had resulted in an enhanced antioxidant status in juvenile roe deer (Capreolus capreolus) (Clauss et al., 2003). However, as it soon became evident that regular bleeding would not be possible as planned in advance with the different zoos, we looked for an alternative parameter for measuring any potential influence on the antioxidant status. Consequently, a published protocol for the measurement of antioxidant status in human faeces (Garsetti et al., 2000) was adopted, as access to freshly defaecated faeces was not a limiting factor in the study. In the study in humans, antioxidant capacity had been correlated with the intake of polyphenol-containing substances (coffee and red wine); therefore, we hypothesized that the antioxidant status in rhinoceros faeces should increase with tannin supplementation. Additionally, as hydroxyl radical formation in faeces was correlated with faecal iron content in studies with humans and rats (Erhardt et al., 1997; Stone et al., 2002), we expected a negative correlation between faecal iron content and antioxidant status. U.S. Copyright Clearance Center Code Statement: 0931–184X/2006/5306–0319 $15.00/0 www.blackwell-synergy.com J. Vet. Med. A 53, 319–322 (2006) Ó 2006 The Authors Journal compilation Ó 2006 Blackwell Verlag, Berlin ISSN 0931–184X