Safety and efficacy of a novel iron chelator (HBED; (N,N'-Di(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid)) in equine (Equus caballus) as a model for black rhinoceros (Diceros bicornis).

While iron overload disorder (IOD) and related disease states are not considered a common occurrence in domestic equids, these issues appear prevalent in black rhinoceroses under human care. In addressing IOD in black rhinos, altering dietary iron absorption and excretion may be the most globally practical approach. A main option for treatment used across other species such as humans, is chelation therapy using iron-specific synthetic compounds. As horses may serve as an appropriate digestive model for the endangered rhinoceros, we evaluated the potential use of the oral iron chelator N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid (HBED) in horses for safety and efficacy prior to testing in black rhinoceros. Health and iron digestibility and dynamics were assessed in horses (n = 6) before, and after treatment with HBED (50 mg/kg body weight) for 8 days using a crossover design with serum, faecal and urine collection. A preliminary pharmacokinetic trial was also performed but no trace of HBED was found in serially sampled plasma through 8 h post-oral dosing. HBED increased urinary iron output in horses compared to control by 0.7% of total iron intake (p < 0.01), for an average of 27 mg urinary iron/day, similar to human chelation goals. Blood chemistry, blood cell counts and overall wellness were not affected by treatment. As healthy horses are able to regulate iron absorption, the lack of change in iron balance is unsurprising. Short-term HBED administration appeared to be safely tolerated by horses, therefore it was anticipated it would also be safe to administer to black rhinos for the management of iron overload.

Species and individual rhinoceros affect the bacterial communities, metabolites, and nutrient composition in faeces from Southern black rhinoceros (Diceros bicornis minor) and Southern white rhinoceros (Ceratotherium simum simum) under managed care.

Gut microbiota are essential to nutrient metabolism and the maintenance of hindgut health. The characterization of faecal bacterial communities from healthy individuals is important for the establishment of baseline data that can be compared to periods of gut dysbiosis. Diet is a key determinant of the faecal microbial community structure and generation of volatile fatty acids, a main energy source for the host. While rhinoceroses are herbivores, black rhinoceroses are browsers and white rhinoceroses are grazers. The objective of our study was to characterize and compare diets, faecal bacterial communities, nutrients and metabolites between and amongst Southern white rhinoceroses and Southern black rhinoceroses (n = 3 rhinos/species) managed at Disney's Animal Kingdom® . Faecal bacterial communities were similar between individual white rhinos and dissimilar between species and individual black rhinos. Faecal butyrate and propionate molar proportions and concentrations were greater in black rhinos than white rhinos, whereas lactate was greater in white rhinos. The Shannon diversity, total operational taxonomic units, and relative abundance of Firmicutes were greater in white than black rhinos. The relative abundance of Proteobacteria in faeces from black rhinos was 3-fold greater than from white rhinos. One black rhino had a greater relative abundance of Verrucomicrobia (7.45 ± 1.31%) than all other individual rhinos (0.01-1.37%). White rhinoceroses demonstrated similar abundances of bacterial phyla and communities between one another and by individual, while black rhinoceroses were more dissimilar by individual. The dissimilarities between black rhinos were suspected to be due to total diet consumption variability, including browse diversity, and lack of direct contact. In contrast, the white rhinos commingled (i.e. nose-to-nose contact) and consumed similar amounts of hay, pellets and training items. These results suggest that species-specific diets and the individual contribute to differences in faecal bacterial communities, nutrients and metabolites between black and white rhinos housed at the same institution.

A modified diet to support conservation of the Atala hairstreak butterfly (Eumaeus atala Poey).

Raising insects in a laboratory for release into the wild is a common conservation practice, but maintaining breeding colonies year-round can be limited by seasonal food availability. Food availability is particularly challenging for insects which depend on specific host plants. For example, our early efforts to rear the imperiled Atala hairstreak butterfly (Eumaeus atala Poey) resulted in colony failure during winter due to lack of food. To overcome this barrier, we developed a modified freeze-dried host plant diet to support the colony. The diet consisted of reconstituted freeze-dried leaves and stems from fresh-growth coontie (Zamia integrifolia), the host plant for the Atala butterflies. We fed larvae less than 9 mm on this freeze-dried diet and transferred them to live coontie plants after they were more than 9 mm. We reared a colony of Atala butterflies using these methods for 859 days, resulting in more than 3400 animals released into the wild. Comparing colony counts during that time period to the 548 days we reared them without modified freeze-dried diet showed a clear benefit in using freeze-dried diet. A growth trial (N = 40) of larvae fed on only freeze-dried diet compared to larvae fed on fresh coontie cuttings found no significant difference in larval or pupal development between groups (p = 0.71 and p = 0.47, respectively). We, therefore, conclude that the freeze-dried diet provided an appropriate alternative for Atala colonies when fresh growth from the host plant is unavailable, and we recommend use of this technique for raising other host plant-dependent insect species of conservation concern.

Analysis of the distal gut bacterial community by 454-pyrosequencing in captive giraffes (Giraffa camelopardalis).

The objective of this study was to characterize the structure of the fecal bacterial community of five giraffes (Giraffa camelopardalis) at Disney's Animal Kingdom, FL. Fecal genomic DNA was extracted and variable regions 1-3 of the 16S rRNA gene was PCR-amplified and then sequenced. The MOTHUR software-program was used for sequence processing, diversity analysis, and classification. A total of 181,689 non-chimeric bacterial sequences were obtained, and average number of sequences per sample was 36,338 -± 8,818. Sequences were assigned to 8,284 operational taxonomic units (OTU) with 95% of genetic similarity, which included 2,942 singletons (36%). Number of OTUs per sample was 2,554 ± 264. Samples were normalized and alpha (intra-sample) diversity indices; Chao1, Inverse Simpson, Shannon, and coverage were estimated as 3,712 ± 430, 116 -± 70, 6.1 ± 0.4, and 96 ± 1%, respectively. Thirteen phyla were detected and Firmicutes, Bacteroidetes, and Spirochaetes were the most dominant phyla (more than 2% of total sequences), and constituted 92% of the classified sequences, 66% of total sequences, and 43% of total OTUs. Our computation predicted that three OTUs were likely to be present in at least three of the five samples at greater than 1% dominance rate. These OTUs were Treponema, an unidentified OTU belonging to the order Bacteroidales, and Ruminococcus. This report was the first to characterize the bacterial community of the distal gut in giraffes utilizing fecal samples, and it demonstrated that the distal gut of giraffes is likely a potential reservoir for a number of undocumented species of bacteria.

Near infrared reflectance spectroscopy (NIRS) analyses of nutrient composition and condensed tannin concentrations in carolina willow (Salix caroliniana).

Iron overload disorder has been described in a number of zoo-managed species, and it has been recommended to increase the tannin composition of the diet as a safe way to minimize iron absorption in these iron-sensitive species. The goal of this study was to examine the potential of near infrared reflectance spectroscopy (NIRS) as a rapid and simple screening tool to assess willow (Salix caroliniana) nutrient composition (crude protein: CP; acid detergent fiber: ADF; neutral detergent fiber: NDF; lignin, gross energy: GE) and condensed tannin (CT) concentrations. Calibration equations were developed by regression of the lab values from 2 years using partial least squares on n = 144 NIRS spectra to predict n = 20 independent validation samples. Using the full 2-year dataset, good prediction statistics were obtained for CP, ADF, NDF, and GE in plant leaves and stems (r(2 ) > 0.75). NIRS did not predict lignin concentrations reliably (leaves r(2) = 0.52, stems r(2) = 0.33); however, CTs were predicted moderately well (leaves r(2) = 0.72, stems r(2) = 0.67). These data indicate that NIRS can be used to quantify several key nutrients in willow leaves and stems including concentrations of plant secondary compounds which, depending on the bioactivity of the compound, may be targeted to feed iron-sensitive browsing animals.

Nutrition and health in amphibian husbandry.

Amphibian biology is intricate, and there are many inter-related factors that need to be understood before establishing successful Conservation Breeding Programs (CBPs). Nutritional needs of amphibians are highly integrated with disease and their husbandry needs, and the diversity of developmental stages, natural habitats, and feeding strategies result in many different recommendations for proper care and feeding. This review identifies several areas where there is substantial room for improvement in maintaining healthy ex situ amphibian populations specifically in the areas of obtaining and utilizing natural history data for both amphibians and their dietary items, achieving more appropriate environmental parameters, understanding stress and hormone production, and promoting better physical and population health. Using a scientific or research framework to answer questions about disease, nutrition, husbandry, genetics, and endocrinology of ex situ amphibians will improve specialists' understanding of the needs of these species. In general, there is a lack of baseline data and comparative information for most basic aspects of amphibian biology as well as standardized laboratory approaches. Instituting a formalized research approach in multiple scientific disciplines will be beneficial not only to the management of current ex situ populations, but also in moving forward with future conservation and reintroduction projects. This overview of gaps in knowledge concerning ex situ amphibian care should serve as a foundation for much needed future research in these areas.

Challenges with effective nutrient supplementation for amphibians: A review of cricket studies.

Over the last 25 years, numerous studies have investigated the impact of insect supplementation on insect nutrient content. In light of recent nutrition related challenges with regards to zoo amphibians fed an insect based diet, this review attempts to comprehensively compile both anecdotal and published data in the context of practical application on this topic. Insects, primarily crickets, used for amphibian diets historically demonstrate low concentrations of key nutrients including calcium and vitamin A. Commonly used practices for supplementation involving powder dusting or gut loading have been shown to improve delivery of calcium and vitamin A, though often not reaching desired nutrient concentrations. The large variety of factors influencing insect nutrient content are difficult to control, making study design, and results often inconsistent. Formulation and availability of more effective gut loading diets, combined with a standardized protocol for insect husbandry and dietary management may be the most effective way to supplement insects for use in amphibian feeding programs. Ideally, the nutritional improvement of feeder insects would begin at the breeder level; however, until this becomes a viable choice, we confirm that supplementation of crickets through both gut-loading and dusting appear necessary to support the nutritional health of amphibians and other insectivores in managed collections.

Leaping forward in amphibian health and nutrition.

The Epidemiology Working Group, a subgroup of the participants of the Disney's Animal Kingdom Workshop on "Ex situ Amphibian Medicine and Nutrition," identified a critical need to design and implement approaches that will facilitate the assessment and evaluation of factors impacting amphibian health. In this manuscript, we describe and summarize the outcomes of this workshop with regards (a) the identified gaps in knowledge, (b) identified priorities for closing these gaps, and (c) compile a list of actions to address these priorities. Four general areas of improvement were identified in relation to how measurements are currently being taken to evaluate ex situ amphibian health: nutrition, infectious diseases, husbandry, and integrated biology including genetics and endocrinology. The proposed actions that will be taken in order to address the identified gaps include: (1) identify and quantify major health issues affecting ex situ amphibian populations, (2) identify and coordinate laboratories to conduct analyses using standardized and validated protocols to measure nutritional, infectious diseases, genetic, and hormonal parameters, (3) determine in situ baseline distribution of parameters related to amphibian health, and (4) establish an inter-disciplinary research approach to target specific hypotheses related to amphibian health such as the effects of population genetics (e.g., relatedness, inbreeding) on disease susceptibility, or how environmental parameters are related to chronic stress and hormone production. We think is important to address current gaps in knowledge regarding amphibian health in order to increase the probability to succeed in addressing the issues faced by in situ and ex situ amphibians populations. We are confident that the recommendations provided in this manuscript will facilitate to address these challenges and could have a positive impact in both the health of in situ and ex situ amphibian populations, worldwide.

Vitamin A values of wild-caught Cuban tree frogs (Osteopilus septentrionalis) and marine toads (Rhinella marina) in whole body, liver, and serum.

Recent issues surrounding captive amphibians are often nutritionally related problems, such as hypovitaminosis A. Although supplementation of frogs with vitamin A is a topic of investigation, the underlying issue is understanding vitamin A metabolism in amphibian species. To develop a range of "normal" vitamin A concentrations for captive amphibians, baseline vitamin A concentrations must be established in wild amphibian species. In this study, two species, Cuban tree frogs (Osteopilus septentrionalis; n = 59) and marine toads (Rhinella marina; n = 20) were collected from the wild as part of an invasive species control program at Zoo Miami, Miami, Florida. Serum, liver, and whole body samples were analyzed for vitamin A content. The Cuban tree frogs showed higher concentrations on average of vitamin A in serum (82.8 ppb), liver (248.3 IU/g), and whole body (5474.7 IU/kg) samples compared with marine toads (60.1 ppb; 105.3 IU/g; 940.7 IU/kg, respectively), but differences were not significant (P = 0.22). What can be considered "normal" values of vitamin A concentrations across different amphibian species requires further investigation. Although all amphibians collected in this study appeared healthy, a larger sample size of animals, with known health histories and diets, may provide stronger evidence of normal expectations.

Management strategies of iron accumulation in a captive population of black rhinoceroses (Diceros bicornis minor).

During routine health screens for black rhinoceroses (Diceros bicornis minor) in a captive setting, serum iron and ferritin were analyzed as well as total iron binding capacity and total iron saturation. Trends for ferritin and percent iron saturation showed steady increases since 2003 in four of four animals (three males; one female) with two animals (one male; one female) consistently showing higher elevations over conspecifics. The historical diet had been comprised of a commercial or in-house complete pelleted feed; several species of fresh browse, Bermuda grass, alfalfa and timothy hays, as well as enrichment and training items (apples, carrots, sweet potatoes, and a small amount of leafy greens and vegetables). In 2009, one of the three male rhinoceroses showed a threefold increase in ferritin and concurrently exhibited clinical signs of lethargy, decreased appetite, and disinterest in training. The lone female showed a twofold increase; she also became reproductively acyclic in the prior year. The male was immobilized for examination and phlebotomy. During the same time period, a new version of the complete pelleted feed, with a reduced amount of iron, was introduced. Subsequent to the diet change, the male's ferritin levels have consistently declined, and the female started cycling again. Even with these corrective steps to reduce iron levels, levels of iron saturation remained high, and ferritin levels were still above 1,500 ng/ml. Therapeutic phlebotomy was instituted via a rigorous training program that allowed phlebotomies over a 30-min time frame. This was possible because of a long-term training program for the animals, consistent training personnel, routine collection of samples on a monthly basis, and general comfort level of the animals in the restraint chute. The results of this integrated approach showed some significant improvements and an overall positive impact on the animals.

Changes in serum calcium, phosphorus, and magnesium levels in captive ruminants affected by diet manipulation.

A combination of low serum calcium (Ca), high serum phosphorus (P), and low serum magnesium (Mg) has been observed in individual captive ruminants, primarily affecting kudu (Tragelaphus strepsiceros), eland (Taurotragus oryx), nyala (Tragelaphus angasii), bongo (Tragelaphus eurycerus), and giraffe (Giraffa camelopardalis). These mineral abnormalities have been associated with chronic laminitis, acute tetany, seizures, and death. Underlying rumen disease secondary to feeding highly fermentable carbohydrates was suspected to be contributing to the mineral deficiencies, and diet changes that decreased the amount of starch fed were implemented in 2003. Serum chemistry values from before and after the diet change were compared. The most notable improvement after the diet change was a decrease in mean serum P. Statistically significant decreases in mean serum P were observed for the kudu (102.1-66.4 ppm), eland (73.3-58.4 ppm), and bongo (92.1-64.2 ppm; P < 0.05). Although not statistically significant, mean serum P levels also decreased for nyala (99.3-86.8 ppm) and giraffe (82.6-68.7 ppm). Significant increases in mean serum Mg were also observed for kudu (15.9-17.9 ppm) and eland (17.1-19.7 ppm). A trend toward increased serum Mg was also observed in nyala, bongo, and giraffe after the diet change. No significant changes in mean serum Ca were observed in any of the five species evaluated, and Ca was within normal ranges for domestic ruminants. The mean Ca:P ratio increased to greater than one in every species after the diet change, with kudu, eland, and bongo showing a statistically significant change. The results of this study indicate that the diet change had a generally positive effect on serum P and Mg levels.

A comparison of oral and topical vitamin A supplementation in African foam-nesting frogs (Chiromantis xerampelina).

Vitamin A is essential for a variety of functions, including cellular differentiation, morphogenesis, growth, vision, immune response, and reproduction. A captive population of African foam-nesting frogs (Chiromantis xerampelina) with a known history of vitamin A deficiency had higher than expected incidence of sudden death, bacterial osteomyelitis, and stunted growth. Due to the high prevalence and untreatable nature of the diseases in the population, euthanasia of the population was recommended. Before euthanasia, the population was entered into a study to compare oral dietary supplementation of vitamin A to topical treatment with water-miscible vitamin A palmitate (AQUASOL A Parenteral, Mayne Pharma Inc., Paramus, New Jersey 07652, U.S.A.). Eighty-four frogs, weighing 2-7 g, were divided into a control and three treatment groups of 21 frogs per group, with normalized weight distribution. The control group received standard daily nutrition of crickets dusted with a supplement containing 342,000 international units (IU) vitamin A/kg. The treatment groups consisted of oral supplementation with crickets dusted with a fortified supplement containing 822,510 IU vitamin A/kg; topical vitamin A palmitate 50 IU every other day; and topical vitamin A palmitate 50 IU once a week. After 30 days, all frogs were euthanized, and 12 frogs from each group were analyzed for whole-body vitamin A levels. The control and treatment groups 1, 2, and 3 had average whole-body vitamin A levels of 1371.4 IU/kg (SE 284.4), 908.7 IU/kg (SE 186.5), 6385.9 IU/kg (SE 675.9), and 3521.8 IU/kg (SE 575.1), respectively. These results suggest that oral supplementation using a product high in vitamin A may be ineffective at raising whole-body vitamin A levels above those achieved with standard nutrition. Topical administration of vitamin A on an every other day and once a week dosing schedule achieved levels 4.5- and 2.5-fold higher than standard nutrition, respectively.

Influence of a contaminated fish diet on germline expanded-simple-tandem-repeat mutation frequency in mice.

Herring gulls (Larus argentatus) in polluted areas on the North American Great Lakes were previously shown to have elevated germline mutation frequencies at minisatellite DNA loci. Airborne or dietary contaminants likely caused induced mutations, but the importance of each exposure type was unknown. Follow-up experiments with lab mice determined that air pollution significantly induced germline mutations; however, an evaluation of mutations induced by the diet of herring gulls has not yet been conducted. To address this issue, we fed mice a high-fish diet (58% wet mass) of the most common prey species for herring gulls nesting in Hamilton Harbour, a polluted industrial area on Lake Ontario. We bred the mice and screened pedigrees for germline mutations at expanded-simple-tandem-repeat (ESTR) DNA loci. Mutation frequencies were compared to those in a reference group that was fed fish from Atlantic Canada, and a control group that was fed commercial chow. Germline mutation frequencies were highest in mice fed contaminated fish, but were only marginally or not significantly affected by diet treatment. Statistical power to detect differences among treatment groups was low, and the effect of diet may have more clearly emerged if larger sample sizes were available. Levels of organic pollutants in the fish from Hamilton Harbour were higher than those from Atlantic Canada, but their ability to induce ESTR mutations is unknown. Our findings suggest that a contaminated fish diet may contribute to the elevated germline mutation frequencies observed previously in gulls at this site, but air pollution is likely a more important route of exposure.

An approach to feeding high-percentage fish diets to mice for human and wildlife toxicology studies.

Experimental feeding of sport fish to rodents has been an important tool for the study of biological effects induced by a contaminated fish diet. Most rodent feeding studies have used low-to-moderate levels of tissue from large fish species incorporated into diets fed to rats and have given little consideration to issues of diet palatability or nutrition. There are currently no rodent diet models suitable for assessing the risk to human populations of diets very high in daily fish content or to wildlife species consuming high percentages of whole, small-bodied fish. In this study, we describe an approach to feeding mice high percentages (up to 50%) of homogenized, whole fish using Atlantic herring (Clupea harengus) as a test species. We created a novel gel diet medium for mice that contains a variety of nutritional supplements and is flexible in terms of the fish percentage that can be incorporated. In choice trials, mice preferred 30 and 35% fish gels to their regular commercial dry chow, indicating that the gel diet medium was palatable. In a longer feeding trial, mice ate 35% fish gel for 12 days and 50% fish gel for 12 days (total of 24 consecutive days) and did not differ in body mass compared to age- and sex-matched controls. We conclude that our fish-based gel diet is suitable for rodent feeding trials in toxicology studies that examine dose responses to fish consumption and risk in human and wildlife populations among which daily fish intake is very high. Our general approach may also be applicable for feeding mice materials other than fish.