A paucity of research exists investigating influence of diet on oxidative stress (OS) in zoo-managed animals. Many zoo-managed species have unknown nutrient requirements and OS can be used as a measure to evaluate dietary nutrient concentrations and narrow dietary recommendations. The overall dissertation objective was to evaluate influence of diet and nutrient intakes on markers of OS in zoo-managed animals through three objectives: 1) evaluate influence of five commercial gut-loading diets on resulting live cricket nutrient composition and impact on markers of OS in two zoo-managed toad species 2) evaluate dietary nutrient intake and nutritional husbandry influence on markers of OS in male snow leopards maintained in U.S. zoos and 3) assess the impact of three dietary vitamin E concentrations on OS, maze learning performance, and anxiety behaviors in feeder rats. Results demonstrated that 1) potassium and high protein to fat ratios increased markers of OS in toads 2) OS is influenced by copper and sodium intakes and whole prey consumption in male snow leopards 3) dietary vitamin E intakes five to 22 times above nutrient requirements are positively correlated with increased OS and are not beneficial for feeder rat welfare in relation to maze learning performance. This novel research in zoo-managed species provides some of the first data of its kind evaluating influence of dietary vitamin and mineral concentrations and ratios in relation to OS. The results presented provide some additional clarity to nutrient target ranges for future research in amphibians, felids, and rodents via an approach that has not been taken in exotic animal nutrition. Additionally, results provide valuable information to animal managers for improved health and advancements in husbandry to ensure success of assurance populations.

Nutrition affects a myriad of processes in the body including OS, defined as an imbalance between pro-oxidants and antioxidants, in favor of pro-oxidants. Both nutrition and OS can affect many physiologies and phenotypes such as animal growth, reproduction, learning, and behavior. For zoo-managed species this is a critical issue because specific nutrient requirements for a majority of exotic species are not known. To address the overall dissertation objective, two research chapters (2-3) were completed using species managed in zoo breeding programs with challenges affecting their sustainability that are potentially linked with nutrition and OS (toads and snow leopards). Chapter 4 utilized a unique zoo-managed model (feeder rats) to evaluate controlled concentrations of one dietary antioxidant (vitamin E) to evaluate influence on maze learning performance and anxious behaviors that could be potential indicators of animal welfare.

In Chapter 2, two toad species were utilized to evaluate OS when fed five different, live cricket, dietary treatments (crickets fed different diets (gut-loading diets)). With more than 50% of known amphibian species threatened with extinction, assurance populations are vital to sustain wild populations via reintroduction programs; however dietary nutrient deficiencies may be hindering amphibian health in zoos. The objective of Chapter 2 was to evaluate, for the first time in amphibians, correlations of dietary nutrients (proximates, vitamins A and E, and minerals) and markers of OS. We hypothesized that live crickets would differ in nutrient composition based on the diet they were fed, and toads fed crickets containing higher concentrations of vitamins A and E would have lower levels of OS. The hypotheses were partially supported as crickets did vary in nutrient composition (particularly protein, fat, calcium, and carotenoids) and nutrient intake did influence glutathione peroxidase (GPx) activity in toads. Contrary to our hypotheses, vitamin E did not impact markers of OS; however, GPx activity did decrease with increasing dietary retinol but this requires further investigation to determine positive or negative OS impacts. Additionally, higher cricket protein:fat ratios and potassium concentrations were correlated with increased GPx activity. Results of Chapter 2 indicate dietary imbalances in vitamins and minerals and very high protein to fat ratios (above 10.0) are correlated with increases in markers of OS. Results indicate that crickets should be monitored for low fat concentrations to prevent excessively high protein:fat ratios that could affect fat-soluble vitamin absorption. Evaluation of ideal ratios requires further investigation through controlled dietary studies. Our results also suggest the use of some products (Vita Bug®) for gut-loading, requires specific supplementation of nutrients (calcium) for amphibian health.

In Chapter 3, the objective was to evaluate the influence of dietary nutrient intakes and nutritional husbandry on markers of OS in male snow leopards maintained in U.S. zoos. Snow leopards are an endangered species and zoo populations have been declining over the past two decades. Dietary inadequacies may be a contributing factor to disease and abnormal physiologies affecting zoo-managed snow leopards, possibly through OS. Improvement of nutritional understanding in zoo-managed settings is likely a vital component of managing this assurance population. Diets of each cat were collected and analyzed as well as weekly feeding amounts to yield weekly nutrient (proximates, minerals, vitamins A and E, and fatty acids) intakes. It was hypothesized that dietary nutrients, particularly higher intakes of vitamins A and E, would reduce markers of OS and that trace mineral balance would also influence OS. Our hypothesis was not supported as dietary vitamin intakes did not directly impact OS. In support of our hypothesis, dietary minerals (copper and sodium) were correlated with alterations in OS in male snow leopards. Reduction in oxidative DNA/RNA damage was correlated with increases in copper intakes; however, some snow leopards consumed less copper than recommended, highlighting the need to carefully evaluate concentrations in exotic cat diets. Very high intakes of some minerals such as iron also support the need to carefully evaluate mineral concentrations and mineral balance in zoo carnivore diet formulations. Additionally, the use of supplemental whole prey positively influenced superoxide dismutase (SOD) activity. These results suggest alteration of diet, specifically with attention to mineral premixes and addition of whole prey, may be beneficial for improving snow leopard OS and potentially improving the sustainability of zoo-managed snow leopard populations.

Chapter 4 utilized growing feeder rats (raised to be fed to other animals) to achieve objectives of assessing the impact of specific dietary vitamin E concentrations on OS markers, maze learning performance, and anxious behaviors. While rats are not an exotic species, they do have published nutrient requirements allowing for dietary manipulation to assess target concentrations. Additionally, they can be useful as models for other zoo-managed species. We hypothesized that rats fed higher concentrations of dietary vitamin E would have suppressed OS levels, superior maze learning performance, and fewer anxiety behaviors. Oxidative stress did appear to be influenced by dietary vitamin E; however, not as anticipated because SOD activity was lowest and concentrations of PC highest in rats fed highest dietary vitamin E concentrations. Our hypothesis was not supported by results of Chapter 4, which demonstrated that feeding excessive concentrations of dietary vitamin E, did not impact rat maze learning performance. Diets containing the lowest concentrations of dietary vitamin E, resulted in more anxiety behaviors in rats and thus did support our hypothesis. Conclusions from Chapter 4 indicated that dietary vitamin E intakes 5-22 times above nutrient requirements were not beneficial for 13-ek old feeder rat maze learning performance and OS markers. Results indicate the potential risks of feeding super doses of vitamin E and can aid in supplementation decisions in diet formulation for exotic animal and feeder animal diets.