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Downloaded from https://academic.oup.com/tas/article/5/2/txab034/6272442 by guest on 18 May 2021 Nutrient intake and influence on markers of oxidative stress in zoo-managed male snow leopards (Uncia uncia) † ‡ †,‡,1 Cayla J. Iske, Jason R. Herrick, and Cheryl L. Morris † ‡ Department of Animal Science, Iowa State University, Ames, IA 50011, USA; and Omaha’s Henry Doorly Zoo and Aquarium, Omaha, NE 68107, USA ABSTRACT: Oxidative stress (OS) results from the glutathione peroxidase [GPx]) and ferric reducing overproduction of reactive species. Nutrient intake antioxidant potential that are protective against can contribute positively or negatively to OS, and OS, and protein carbonyls, thiobarbituric acid re- the lack of established nutrient requirements for active substances, and DNA/RNA damage that most of the exotic species managed in zoos exacer- are indicative of oxidative damage. Weekly copper bates the possibilities for nutrient imbalances that intake (10.1 to 80.2 mg) was negatively correlated potentially could lead to reactive species produc- with DNA/RNA damage (R = 0.44; P = 0.01). tion. The objective of this study was to evaluate Weekly sodium intake (4.4 to 12.7 g) was posi- the influence of nutrient intake and nutritional tively correlated with GPx activity (R = 0.43; P husbandry on markers of OS in male snow leop- = 0.04). More frequent feeding of whole prey (0.3 ards (n = 14) maintained in U.S. facilities (n = 12). to 3 times/wk) was correlated with increased blood Diet samples and husbandry information were SOD activity (R = 0.55; P < 0.01). In conclusion, obtained and snow leopards were immobilized greater dietary copper intake and more frequent once for collection of blood. Samples were ana- feeding of whole prey may reduce OS in snow leop- lyzed for chemical composition (diet and blood), ards. Dietary sodium intake and relationship with antioxidant capacity (blood), and markers of OS GPx activity should be further evaluated to deter- (blood). Correlations between weekly nutrient in- mine benefit or detriment. No cause and effect can takes and markers of OS were analyzed by linear be inferred from our results, but our data suggest regression. Analyzed markers of OS included anti- altering dietary form and nutrient concentrations oxidant enzymes (superoxide dismutase [SOD] and may influence OS in snow leopards. Key words: cat, diet, oxidative stress, snow leopard, whole prey © The Author(s) 2021. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution- NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact email@example.com Transl. Anim. Sci. 2021.5:1-16 doi: 10.1093/tas/txab034 of Nature, and zoo populations have been declining INTRODUCTION over the past 2 decades (Tetzloff et al., 2016). An Snow leopards are listed as an endangered spe- improved understanding of nutrition in zoo-man- cies by the International Union for Conservation aged settings is vital to conserve an assurance population of the species (McCarthy et al., 2017). While the exact cause of the managed population Corresponding author: firstname.lastname@example.org decline is not known, dietary inadequacies could Received August 25, 2020. Accepted April 26, 2021. be a contributing factor to disease and abnormal 1 Downloaded from https://academic.oup.com/tas/article/5/2/txab034/6272442 by guest on 18 May 2021 Iske et al. physiologies, possibly through oxidative stress (OS). institutions, accredited by the Association of Zoos Oxidative stress is defined as a disturbance in the and Aquariums, were used. None of the animals prooxidant–antioxidant balance, in favor of proox- showed any clinical signs of disease or illness and idants or reactive oxygen species (ROS; Sies, 1985). none had documented diseases or illnesses. Detailed Animal diets contain many nutrients with both cat demographics, diet history, fasting days, and pro- and anti-oxidant properties. This includes whole prey feeding frequency were surveyed (Table some trace minerals and vitamins required for 1) for each animal at the time of sample collection physiological function. Excesses of trace minerals through detailed interviews with animal managers. may result in ROS production due to their redox Animals ranged in age from 3 to 16 yr (average 8.5) potential (Tvrda et al., 2014). Antioxidants are em- and weight from 30.5 to 48.9 kg (average 37.9 kg; ployed to reduce and detoxify potentially damaging Table 2). All animals were fed primarily raw, com- prooxidants and defend against oxidative damage. mercially manufactured meat-based diets, for- Antioxidants can be found in the body as enzymes, mulated to meet cat nutrient requirements, with such as superoxide dismutase (SOD) and gluta- supplemental bones and/or whole prey items. Diets thione peroxidase (GPx), which dismutate and neu- and amounts of diets were different for all ani- tralize ROS, or as nonenzymatic compounds in the mals except cats 8 and 9 that were fed identically. diet, such as vitamins A and E, which donate elec- Animals were all managed at different institutions trons to neutralize ROS (Sies, 1997). with exception of cats 7, 8, and 9 that were man- Effect of diet on OS has been evaluated in aged at the same zoo. many of the domestic species, including poultry (Gao et al., 2010), swine (Lauridsen et al., 1999), Sample Collection and cattle (Pedernera et al., 2010), generally fo- Animals were immobilized after an overnight cusing on antioxidant supplementation and dietary fast according to individual institution veterinary trace mineral concentrations. Additionally, evalu- protocols by institution veterinarians using com- ation of dietary influence on OS in obese (Tanner binations of ketamine, medetomidine, butorpha- et al., 2006) and renal insufficient (Yu and Paetau- nol, and midazolam based on each institution’s Robinson, 2006) domestic cats revealed reduction standard medical procedures. Approximately, 5 mL of OS through high-protein diets in the obese cats of fasted blood was collected via venipuncture of and antioxidant (vitamins E and C and β-carotene) a jugular, saphenous, or femoral vein and was col- supplemented diets in renal insufficient cats. The in- lected into 2 separate Vacutainer tubes (Becton, fluence of diet on OS in snow leopards has not been Dickinson and Company, Franklin Lakes, NJ), evaluated. Therefore, the objective of this study was to evaluate dietary nutrient intake and nutritional husbandry influence on markers of OS in male snow Table 1. Relevant husbandry survey questions dis- leopards maintained in U.S. zoos. It was hypothe- tributed to animal managers at each institution sized that dietary nutrients, particularly higher in- during snow leopard sample collections takes of vitamins A and E, would reduce markers Q1. What best describes the male’s diet (if possible provide a of OS and that trace mineral balance would also in- sample copy of a weekly diet): fluence OS. This research has potential to improve a. Single commercial carnivore diet only. diet formulations of exotic cats managed in zoos b. Variety or combination of commercial carnivore diets (in- and to initiate future research evaluating diet and clude products and amounts) OS in relation to phenotypes and physiologies. c. Commercial carnivore diet plus whole prey (rats, rabbits, birds, etc.). d. Whole prey items only MATERIALS AND METHODS Q2. If given whole prey items, what kind (type, size) and how often? Q3. Does this cat have a fast day? If so, how many days per week All animal procedures were approved and what is offered? by each housing institutions’ Animal Care Q4. Using a body condition score index of 1 to 9, what is this cat’s BCS? and Use Committee (IACUC) before animal Q5. What is this cat’s current body weight (include date the weight experimentation. was obtained)? Q6. How often does the male receive enrichment items? Q7. Please provide examples of “preferred” enrichment items: Animals BCS = body condition score. Fourteen male snow leopards (Uncia † Survey was given verbally by the same sample collector for each uncia) from 12 North American zoological institution. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article/5/2/txab034/6272442 by guest on 18 May 2021 Diet and oxidative stress in snow leopards Table 2. Survey data demographics of male snow leopards housed in North American zoos † ‡ Cat Weight, kg Age, yr Diet protein source(s) Weekly as-fed intake, g Weekly DMI, g Fasting days Whole prey frequency 1 43.0 3.5 Beef 7,000 2,400 2 0.4 2 38.5 9.5 Horse 6,000 2,000 1 3.0 3 36.8 7.5 Horse 7,900 2,700 1 1.0 4 44.0 15 Horse 9,500 3,300 0 0.3 5 40.0 7 Pork 9,100 2,800 2 1.0 6 40.9 7 Beef, pork, and horse 6,600 2,100 1 1.0 7 32.3 6 Horse 10,900 3,600 1 1.0 8 30.5 3 Horse 8,200 2,700 1 1.0 9 30.5 11 Horse 8,200 2,700 1 1.0 10 41.0 11 Horse 4,800 1,700 3 3.0 11 41.8 7 Horse 5,400 1,700 1 0.3 12 30.7 5 Beef 6,000 2,100 0 0.5 13 48.9 16 Horse 6,400 1,900 0 2.0 14 32.2 11 Horse 7,000 2,400 2 0.5 The number of days per week that animals were not fed commercially prepared raw meat diets. Frequency was calculated by dividing total number of whole prey items offered per month (30 d) by 4 (wks). one with no additive (blue top) for serum collec- Fatty acid analysis of raw meat diets was con- tion and the other one containing 1.8 mg ethylen- ducted at Iowa State University using gas chro- ediaminetetraacetic acid (EDTA)/mL (purple top) matography (model 3800; Varian Analytical for plasma collection. Blood was processed at 24 °C Instruments, Walnut Creek, CA) to determine fatty according to institution protocol for routine blood acid profiles (Richter et al., 2012). Peak identifica- diagnostics, centrifuged at approximately 1,000 × tion and quantification (Kramer et al., 2008) were g for 10 min to separate serum and plasma, and determined on esterified (Christie, 1972) lipid sam- stored at –80 °C until analyses. Approximately, ples extracted from each diet sample (Folch et al., 900 g of each animal’s daily diet was obtained on 1957). Mineral analyses of raw meat diets were the day of sample collection and stored at –18 °C conducted at Midwest Laboratories (Omaha, NE; until analyses. All collections took place between method 985.01; AOAC, 2006). Diet subsamples February 9 and June 14, 2016. from each institution were sent to Arizona State University for vitamins A (retinol) and E (α-to- copherol) analyses. Vitamins were analyzed via Diet Analyses reverse-phase high pressure liquid chromatography Approximately, 200 g of each diet sample was (HPLC) as previously described (McGraw et al., subsampled, dried at 55 °C, ground in a commercial 2006; Dierenfeld et al., 2009) using an Agilent 1100 blender (Waring blender model 51BL31, Waring, Series (Santa Clara, CA) HPLC system. New Hartford, CT), and analyzed for chemical com- Metabolizable energy (ME) concentrations of position. All chemical analyses were conducted in the diets were estimated using Atwater values (9 kcal/g nutrition laboratory at Omaha’s Henry Doorly Zoo fat, 4 kcal/g protein, and 4 kcal/g carbohydrate) and Aquarium unless otherwise noted and analyzed multiplied by fat, protein, and digestible carbohy- with a coefficient of variance less than 5%. Diets drate content of each diet (NRC, 2006). Atwater were analyzed for dry matter (DM; method 934.01) values, opposed to modified Atwater values, have and organic matter (OM; method 942.05; AOAC, been suggested as more accurate for determinations 2006). Crude protein (CP) was determined using of ME in raw meat diets because of high digest- a Leco Nitrogen/Protein Determinator (method ibility compared with processed and extruded diets 992.15; model TruMacN, Leco Corporation, St. (Clauss et al., 2010; Iske et al., 2016). Digestible Joseph, MI). Fat concentrations were determined by carbohydrate concentrations were calculated using hexane extraction (method 960.39; AOAC, 2000). nitrogen-free extract (NFE) as an estimate with the Total dietary b fi er (TDF) was determined using the following equation: [100 − (% ash + % CP + % fat + Prosky method (Prosky et al., 1994) adjusted for % TDF)]. Though crude fiber is typically used in high-protein samples using quadruple the amount this calculation, TDF is a more accurate measure of protease and double the time for the water bath of dietary fiber (Cho et al., 2001; de-Oliveira et al., after the addition of protease. 2012) and results in a more accurate estimation of Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article/5/2/txab034/6272442 by guest on 18 May 2021 Iske et al. NFE. Due to the additive nature of the calculation above. These values were then converted to gram and reliance on multiple assays along with very low basis that were then multiplied by 1,000, 2,000, carbohydrate and high protein and fat concentra- 3,000, and 4,000 g of DMI to represent the recom- tions of the diets, calculated NFE of some treat- mendations across the various snow leopard weekly ments produced negative numbers, in which case a intakes. value of 0 was used. Plasma samples also were sent to Arizona Husbandry surveys concerning each cat’s cur- State University for vitamins A and E analyses via rent age, weight, diet type, diet feeding amount, HPLC as described above (McGraw et al., 2006; whole prey offered, whole prey feeding frequency, Dierenfeld et al., 2009). Mineral analyses were and number of fasting days were completed by conducted on blood serum samples at Iowa State housing institutions at the time of sampling via University’s Veterinary Diagnostic Laboratory via interview with animal keepers. Fasting days were inductively coupled plasma mass spectrometry defined as the number of days the animals were not (ICP-MS, Analytik Jena Inc., Woburn, MA) with fed their allotment of commercial meat diet only. hydrogen gas. Briefly, samples were diluted 1:20 in Whole prey and enrichment items, such as bones, 1% nitric acid, vortexed rigorously, and analyzed by could be fed on those days. There is no recom- ICP/MS with bismuth, scandium, indium, lithium, mended standard for fasting days, and zoos typ- yttrium, and terbium used as internal standards. ically implement them for respective management considerations or provision of variety in normal Oxidative Stress Markers routine of the animal, in an attempt to mimic the feeding cycle of wild cats. Although these types of Markers of OS were measured in plasma or fasting days do not mimic the gorge/fast feeding serum. Analysis of OS markers included thiobar- strategy of most large felids, they have been used bituric acid reactive substances (TBARS), protein extensively in zoos to provide variety (AZA Tiger carbonyls (PC), DNA/RNA damage, SOD, and Species Survival Plan®, 2016). To account for vari- GPx activity using commercially available assay kits ation in fasting days, weekly dietary intakes were from Cayman Chemical Company (Ann Arbor, MI) used and calculated by multiplying amount of diet and was performed according to the recommenda- fed on feeding days by the number of days cats were tions of the manufacturer (Table 3). Ferric redu- fed per week. Weekly intakes were then multiplied cing antioxidant power (FRAP), an overall total by DM concentration of the diet to give weekly antioxidant capacity assay, was assessed in serum. dry matter intake (DMI). Analyzed dry matter Briefly, the FRAP assay colorimetrically measures nutrient concentrations were then multiplied by the reduction of ferric iron (Fe ) to ferrous iron weekly DMI to yield weekly nutrient intakes on a (Fe ) by the reaction of ferrous-tripyridyltriazine dry matter basis (DMB) [((grams offered per day * complex in relation to antioxidant-based ascorbic d fed/wk) * %DM) * diet nutrient (%)]. Whole prey acid standards (Benzie and Strain, 1996). Samples items, defined as nonliving whole animal carcass, did not require dilution and were expressed in units as determined by survey responses included rabbit, of μM (FRAP value). All assays were run in tripli- rat, fish, guinea pig, chicken, and quail among all cate in a 96-well plate except for PC, which is ana- participating institutions. Whole prey items were lyzed in duplicate. not analyzed for chemical composition because they accounted for less than 10% of each animal’s Statistical Methods diet, based on the frequency of feeding whole prey and size of whole prey items. Therefore, nutrient in- A total of approximately 80 explanatory vari- take was calculated based on commercial raw meat ables were measured and evaluated in the current diet intakes only. Whole prey feeding frequency (d/ study, including individual dietary macronutrients, wk provided) was assessed for correlations with OS. fatty acids, vitamin A, and mineral weekly intakes, as Recommended weekly nutrient intakes were well as blood measures of OS, minerals, and vitamins calculated based on published recommendations A (retinol) and E. Explanatory variables were separ- for domestic cats (NRC, 2006) as specific nutrient ated into categories (proximates, minerals, vitamins, recommendations for snow leopards are not estab- fatty acids, and OS) and analyzed via multiple regres- lished. Calculations were made using published re- sion for their correlation with all response variables commended allowance values (per 4,000 kcal ME/ via the Regression procedure of SAS (SAS Inst. Inc., kg diet) adjusted to a 5,000 kcal ME/kg diet fol- Cary, NC) with OS markers used as response vari- lowing dietary ME determination as described ables. Due to large number of statistical regressions, Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article/5/2/txab034/6272442 by guest on 18 May 2021 Diet and oxidative stress in snow leopards an attempt was made to highlight clinically signifi- cant variables by using a partial R cutoff of 0.40 for significant variables. This was chosen to not only highlight variables with large influence but also as- sess many variables as this is the first research of this nature in snow leopards. Final statistical models in- cluded only significant variables. Age, body weight, frequency fed whole prey, and number of fasting days for each animal were also analyzed via regression analyses. A P-value of 0.05 was considered statistic- ally significant. RESULTS Diet Intake While exact nutritional management of cats varied, all snow leopards were fed raw, commer- cially manufactured beef, horse, or pork-based diets from Nebraska Brand (North Platte, NE), Triple A Brand Meat Company (Burlington, CO), Milliken Meat Products Ltd (Markham, Ontario), or Sustainable Swine Resources (Sheboygan Falls, WI). Protein sources of the commercial diets offered to cats included beef (n = 2), horse (n = 10), pork (n = 1), and combinations of all 3 protein sources (n = 1; Table 2). Leopards were managed with 0 to 3 fasting days each week and were offered 900 to 1,800 g as fed (300 to 600 g DMB) of meat on each non-fasting day. Whole prey offered ranged from once per month to 3 times/wk (Table 2) and ac- counted for less than 10% of the diet on average (by wet weight) due to very small prey items offered by all institutions (rabbit, rat, fish, guinea pig, chicken, or quail). Nutrient concentrations of the commercial diets analyzed are presented in Tables 4 and 5. Initial analysis of vitamin E (α-tocopherol) con- centrations was very low compared with expected values (1.0 ± 0.1 μg/g DMB). Therefore, some diets were analyzed at a secondary laboratory that resulted in different concentrations (107.8 ± 58.4 μg/g). Because of the limited sample size to re- analyze all diets and length of time the diets had been stored, vitamin E was left out of statistical analysis and is not presented. Average ME concen- trations of the diets ranged from 4.5 to 5.9 kcal/g (DMB). Large variations in diet nutrient concentra- tions were measured (Tables 4 and 5), particularly in vitamin A (retinol) and copper, which ranged from 0.05 to 2.4 μg/g DM and 5.4 to 28.6 mg/kg DM, respectively. Weekly DMI of commercial diets ranged from 1,256 to 3,551 g/wk with an average DMI Translate basic science to industry innovation Table 3. Assay kits performed to determine OS in serum and plasma of male snow leopards Assay kit Catalog number Sample Dilution factor Standard curve reporting range Biomolecular measures OS indication Reporting units TBARS 700870 Serum None 0 to 50 µM Malondialdehyde Lipid damage µM PC 10005020 Serum None N/A 2,4-dinetrophenylhydrazine Protein damage nmol/mL DNA/ 589320 Plasma 1:74 10.3 to 3,000 pg/mL 8-hydroxy-2′-deoxyguanosine, DNA or RNA damage pg/mL RNA 8-hydroxyguanosine, 8-hydroxyguanine ‡ || SOD 706002 Plasma 1:5 0.005 to 0.05 Unit/mL Superoxide radical Antioxidant Unit/mL GPx 703102 Plasma 1:20 0.5 to 1.2 initial absorbance NADPH→NADP Antioxidant nmol/min/mL NADP = nicotinamide adenine dinucleotide phosphate; NADPH = nicotinamide adenine dinucleotide phosphate reduced. All kits were purchased from Cayman Chemical Company (Ann Arbor, MI). Assay kit measures Cu/Zn, Fe, and Mn SOD. || One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. Downloaded from https://academic.oup.com/tas/article/5/2/txab034/6272442 by guest on 18 May 2021 Iske et al. of 2,350 g (Table 6). Metabolizable energy intakes 1,983 μg). Weekly mineral intakes also had con- averaged 12,320 kcal/wk but ranged from 6,633 to siderable ranges with the largest variations in in- 18,622 kcals/wk. The large range of DMI resulted take measured for iron (1,321 ± 492.4 mg), copper in weekly nutrient intakes that also ranged widely, (27.6 ± 19.2 mg), and manganese (90.1 ± 36.5 mg; at least 2-fold, for every macronutrient (Table 6). Table 7). On average, CP, fat, and TDF intakes represented Nutrient intakes of snow leopards were com- 56.7%, 32.1%, and 5.4% of the DM, respectively. pared with calorically adjusted recommended Average (± SD) fat (753.2 ± 264.0 g) and TDF nutrient allowances published for domestic cats. (126.4 ± 41.3 g) intakes varied by 108% and 113%, These comparisons indicated that consumption respectively, among cats. Wide ranges in weekly of protein and fat by snow leopards exceeded re- vitamin A intakes were also observed (2,389 ± commended allowances for all cats. Results also Table 4. Macronutrient, ME, and retinol concentrations of commercial raw meat diets fed to male snow leopards (DMB) Cat DM, % OM, % Protein, % Fat, % TDF, % ME , kcal/g Retinol, μg/g 1 34.5 91.1 55.5 32.2 5.5 5.1 1.0 2 34.1 90.5 56.8 25.8 7.0 4.6 2.3 3 33.9 92.7 63.1 26.6 5.4 4.9 1.8 4 35.1 92.2 55.2 33.7 5.1 5.2 1.5 5 30.9 92.8 53.0 37.0 5.1 5.5 0.05 6 32.2 92.5 56.1 33.2 3.8 5.2 0.5 7 32.6 91.1 56.1 33.4 5.5 5.3 0.2 8 32.6 91.1 56.1 33.4 5.5 5.3 0.2 9 32.6 91.1 56.1 33.4 5.5 5.3 0.2 10 34.7 93.0 49.3 43.8 6.3 5.9 2.3 11 30.9 93.9 71.1 21.1 3.4 4.7 2.4 12 34.2 90.8 45.3 38.7 7.3 5.3 0.2 13 30.5 91.8 67.8 19.4 5.2 4.5 2.4 14 33.0 91.7 54.7 34.4 4.3 5.3 0.4 SD 1.6 1.0 7.2 7.2 1.2 0.4 1.0 Average 33.0 92.0 57.0 31.6 5.3 5.1 1.2 Cats 7, 8, and 9 were fed the same diet. Calculated using unmodified Atwater values: 9 kcal/g of fat + 4 kcal/g of CP + 4 kcal/g of NFE. Table 5. Mineral composition of commercial raw meat diets fed to male snow leopards (DMB) Cat S, % P, % K, % Mg, % Ca, % Na, % Fe, mg/kg Mn, mg/kg Cu, mg/kg Zn, mg/kg Zn:Cu 1 0.7 1.3 1.1 0.1 2.1 0.3 671.8 40.2 10.7 111.8 10.4 2 0.7 1.6 1.0 0.1 2.6 0.3 702.9 48.8 11.6 122.1 10.5 3 0.6 1.5 1.0 0.1 1.7 0.4 391.1 23.6 5.4 140.3 26.0 4 0.5 1.4 0.9 0.1 1.6 0.4 356.5 33.0 6.1 137.1 22.5 5 0.6 0.8 1.0 0.1 1.3 0.4 755.6 43.4 28.6 174.8 6.1 6 0.6 1.0 0.1 0.1 1.8 0.5 582.3 42.8 18.1 132.8 7.3 7 0.6 1.2 1.1 0.1 2.0 0.3 609.0 46.1 13.4 120.1 9.0 8 0.6 1.2 1.1 0.1 2.0 0.3 609.0 46.1 13.4 120.1 9.0 9 0.6 1.2 1.1 0.1 2.0 0.3 609.0 46.1 13.4 120.1 9.0 10 0.5 1.2 0.8 0.1 1.4 0.4 375.6 33.3 7.0 124.5 17.8 11 0.7 1.1 1.1 0.1 0.9 0.3 266.0 19.3 6.0 126.7 21.1 12 0.6 1.2 0.8 0.1 1.8 0.3 628.7 35.7 5.6 82.6 14.8 13 0.7 1.3 1.1 0.1 1.7 0.3 548.8 31.5 9.4 145.9 15.5 14 0.7 1.3 1.1 0.1 2.0 0.4 792.9 37.1 10.0 121.1 12.1 SD 0.1 0.2 0.3 0.0 0.4 0.1 158.9 8.9 6.2 20.3 6.2 Average 0.6 1.2 1.0 0.1 1.8 0.3 564.2 37.6 11.3 127.1 13.7 Cats 7, 8, and 9 were fed the same diet. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article/5/2/txab034/6272442 by guest on 18 May 2021 Diet and oxidative stress in snow leopards Table 6. Weekly calculated macronutrient and vitamin intakes of male snow leopards from raw meat diets †,‡ (DMB) || Cat DMI, g OM, g Protein, g Fat, g TDF, g ME , kcals Retinol, μg 1 2,400 2,209 1,346 779.3 132.3 13,997 2,320 2 2,000 1,845 1,158 526.9 143.8 9,440 4,713 3 2,700 2,476 1,686 710.3 144.5 13,139 4,724 4 3,300 3,078 1,843 1,125 170.3 17,499 5,043 5 2,800 2,605 1,489 1,040 142.0 15,313 126.9 6 2,100 1,974 1,197 708.0 81.4 11,160 991.1 7 3,600 3,233 1,991 1,185 194.7 18,622 845.1 8 2,700 2,425 1,493 888.5 146.0 13,967 633.9 9 2,700 2,425 1,493 888.5 146.0 13,967 633.9 10 1,700 1,553 823.9 731.5 105.8 9,878 3,783 11 1,700 1,580 1,197 354.5 56.4 9,296 4,072 12 2,100 1,875 935 799.4 151.5 10,935 396.9 13 1,900 1,780 1,314 375.9 101.0 8,639 4,683 14 2,400 1,151 687 431.8 53.9 6,633 485.7 SD 570.6 589.5 371.7 264.1 41.3 3,475 1,984 Average 2,436 2,158 1,332 753.2 126.4 12,320 2,389 Recommended Allowance/ — 250 112.5 — — 1,250 1,000 g DM Recommended Allowance/ — 500 225.0 — — 2,500 2,000 g DM Recommended Allowance/ — 750 337.5 — — 3,750 3,000 g DM Recommended Allowance/ — 1,000 450.0 — — 5,000 4,000 g DM Weekly macronutrient, energy, and vitamin intakes were calculated from individual raw meat diet analyses and diet records. Cats 8 and 9 were fed the same amount of the same diet. || Calculated using unmodified Atwater values: 9 kcal/g of fat + 4 kcal/g of CP + 4 kcal/g of NFE. Recommended allowance for nutrients was calculated by adjusting the allowance column for adult cats for maintenance in the NRC (2006) to a diet containing 5,000 kcal ME/kg DM. These values are presented from 1,000 to 4,000 g of DM to represent the ranges of snow leopard intake. indicated that all cats consumed at least twice the Concentrations of plasma vitamins and min- concentration of iron as recommended and on erals are presented in Table 8. Plasma vitamin average consumed more than 5 times the recom- concentrations varied widely between cats with mended iron allowance. In contrast, 8 leopards differences of 71% and 112% measured for r etinol failed to meet their adjusted vitamin A allowance (±SD; 4.3 ± 0.8 μg/mL) and vitamin E (11.2 ± based on their individual DMI indicating only 6 4.2 μg/mL), respectively. Plasma mineral concen- leopards consumed adequate concentrations of trations varied from under 20% (calcium and mag- vitamin A for their individual respective DMI nesium) to above 60% for copper (0.06 ± 0.01 mg/ (Table 6). On average, potassium (23.4 g) and dL) and iron (0.2 ± 0.1 mg/dL). magnesium (2.3 g) intakes were 1.5 and 1.9 times greater than the adjusted average recommended Oxidative Stress Markers allowances (15.3 and 1.2 g, respectively) based The activity of antioxidant enzymes (SOD on the average DMI (2,400 g). Average intakes and GPx) and antioxidant potential (FRAP) of phosphorus, calcium, sodium, iron, and man- along with concentrations of DNA/RNA damage, ganese were 3.8 to 6.4 times more than adjusted TBARS, and PC as measured markers of OS in average recommended allowances. Although the blood are presented in Table 9. For SOD and average copper and zinc intakes both met or ex- DNA/RNA damage, assay values fell outside the ceeded average adjusted allowances, 4 individuals range of the standard curve, which should be con- (cats 3, 11, 12, and 14) did not meet the copper sidered during interpretation as there was an in- allowance and 2 individuals (cats 12 and 14) did adequate sample to rerun assays. Average SOD not meet the zinc allowance when considering and GPx activities were 1.9 ± 2.8 units/mL and their individual DMI (Table 7). Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article/5/2/txab034/6272442 by guest on 18 May 2021 Iske et al. †,‡ Table 7. Weekly calculated mineral intake of male snow leopards from raw meat diets (DMB) Cat DMI, g S, g P, g K, g Mg, g Ca, g Na, g Fe, mg Mn, mg Cu, mg Zn, mg Zn:Cu 1 2,400 16.2 31.0 26.3 2.9 51.1 8.3 1,628 97.4 26.1 271.0 10.4 2 2,000 13.7 33.0 19.5 1.7 51.9 5.6 1,434 99.6 23.6 248.9 10.6 3 2,700 14.8 38.9 26.7 2.8 44.9 10.8 1,045 63.0 14.5 374.7 25.9 4 3,300 16.9 45.4 30.3 3.2 53.5 12.7 1,190 110.2 20.4 457.8 22.4 5 2,800 18.0 23.3 28.4 2.4 35.3 10.5 2,121 121.8 80.2 490.6 6.1 6 2,100 13.4 20.6 19.5 2.0 38.8 10.4 1,243 91.4 38.5 283.4 7.4 7 3,600 21.7 43.8 38.0 3.9 71.7 12.0 2,162 163.5 47.7 426.3 8.9 8 2,700 16.3 32.8 28.5 2.9 53.8 9.0 1,622 122.7 35.8 319.7 8.9 9 2,700 16.3 32.8 28.5 2.9 53.8 9.0 1,622 122.7 35.8 319.7 8.9 10 1,700 7.7 20.6 13.5 1.5 24.1 6.7 627.0 55.6 11.7 207.8 17.8 11 1,700 11.0 18.8 18.3 1.6 15.3 5.3 447.6 32.5 10.1 213.1 21.1 12 2,100 11.4 25.1 15.6 1.3 36.4 5.4 1,298 73.7 11.6 170.5 14.7 13 1,900 13.6 25.3 20.4 1.6 32.8 4.9 1,064 61.0 18.3 282.8 15.4 14 2,400 8.7 15.9 14.0 1.4 25.1 4.4 995.8 46.6 12.5 152.2 12.2 SD 570.6 3.8 9.3 9.4 0.8 15.1 2.8 492.4 36.5 19.2 104.6 6.1 Average 2,436 14.2 29.0 23.4 2.3 42.0 8.2 1,321 90.1 27.6 301.3 13.6 Recommended 3.3 6.5 0.5 3.6 0.9 100.0 6.0 6.3 92.5 Allowance/1,000 g || DM Recommended 6.6 13.0 1.0 7.2 1.8 200.0 12.0 12.6 185.0 Allowance/2,000 g DM Recommended 9.9 19.5 1.5 10.8 2.7 300.0 18.0 18.9 277.5 Allowance/3,000 g DM Recommended 13.2 26.0 2.0 14.4 3.6 400.0 24.0 25.2 370.0 Allowance/4,000 g DM Weekly mineral intakes were calculated from individual raw meat diet analyses and diet records. Cats 8 and 9 were fed the same amount of the same diet. || Recommended allowance for nutrients was calculated by adjusting the allowance column for adult cats for maintenance in the NRC (2006) to a diet containing 5,000 kcal ME/kg DM. These values are presented from 1,000 to 4,000 g of DM to represent the ranges of snow leopard intake. Table 8. Concentrations of plasma vitamins and serum minerals in male snow leopards Cat Retinol, µg/mL α-Tocopherol, µg/mL Ca, mg/dL Cu, mg/dL Fe, mg/dL K, mg/dL Mg, mg/dL P, mg/dL Zn, mg/dL 1 3.4 12.7 9.0 0.05 0.1 14.6 2.0 6.3 0.05 2 3.9 9.4 7.9 0.05 0.1 17.4 1.9 5.6 0.05 3 5.1 6.6 8.2 0.04 0.2 14.7 1.9 4.4 0.04 4 4.4 19.4 7.9 0.05 0.1 16.5 2.0 5.2 0.04 5 4.0 7.8 8.8 0.06 0.1 17.1 2.0 5.6 0.05 6 4.6 10.0 8.0 0.05 0.4 14.9 1.8 5.6 0.06 7 3.7 7.0 8.5 0.07 0.6 17.4 2.0 5.1 0.06 8 4.2 5.5 8.8 0.07 0.2 17.7 2.0 5.0 0.05 9 4.5 8.7 8.3 0.06 0.1 13.2 1.9 5.4 0.05 10 4.1 14.6 9.0 0.08 0.1 16.2 1.9 5.8 0.05 11 5.9 10.9 7.5 0.04 0.1 13.7 2.1 4.4 0.07 12 5.1 15.7 8.9 0.07 0.3 17.6 2.0 5.9 0.05 13 2.8 17.2 8.3 0.08 0.1 17.8 2.0 7.0 0.05 14 4.3 11.2 — — — — — — — SD 0.8 4.2 0.5 0.01 0.1 1.6 0.1 0.7 0.01 Average 4.3 11.2 8.4 0.06 0.2 16.1 2.0 5.5 0.05 Due to the small sample size, plasma minerals could not be analyzed for cat 14. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article/5/2/txab034/6272442 by guest on 18 May 2021 Diet and oxidative stress in snow leopards 2,088 ± 1,609 nmol/min/mL, respectively. Analysis DISCUSSION of blood GPx activity in 4 cats resulted in nega- The influence of various nutrients on OS has tive values; therefore, they were not included been well documented in many domestic species in average calculations or statistical analysis. but has not been effectively evaluated in exotic Antioxidant potential (FRAP) averaged 255.6 ± species. Comparison of domestic and exotic ani- 59.5 μM. Markers of OS damage including DNA/ mals leaves room for error due to unique diets and RNA damage, TBARS, and PC resulted in average physiologies; however, these comparisons provide values of 5,414 ± 1,232 pg/mL, 13.8 ± 2.1 μM, an initial baseline for extrapolation. Domestic and 24.4 ± 10.1 nmol/mL, respectively. Intra- and cats are typically used as an adequate model for inter-assay coefficient of variations for SOD, GPx, exotic felids; however, lack of established nutrient FRAP, DNA/RNA damage, TBARS, and PC were requirements specifically for exotic species leads 8.6% and 98.8%, 3.4% and 73.5%, 7.0% and 23.3%, to challenges in diet formulations that could re- 8.1% and 22.7%, 4.3% and 15.3%, and 8.1% and sult in imbalances of certain nutrients, possibly 41.5%, respectively. promoting OS. Results of studies assessing OS Significant correlations with OS markers can be difficult to interpret as physiological re- are presented in Table 10 and Fig. 1. Dietary sponses to OS vary depending on severity, which intakes of copper and sodium were the only nu- itself is ill-defined as “baseline” or “normal” lev- trients correlated with markers of OS. Dietary els of OS are unknown and likely to vary by spe- copper intake was negatively correlated (P = cies. For example, antioxidant enzyme activity 0.01) with blood DNA/RNA damage (R = 0.44) may actually increase in states of “mild” OS as indicating that an elevated intake of copper an adaptation to protect the cell (Halliwell and was associated with less DNA/RNA damage. Gutteridge, 2015). Additionally, many of these Additionally, elevated GPx activity (R = 0.50) studies utilize artificial methods to induce nu- was positively correlated (P = 0.04) with higher trient overloads. Understanding and assessment intakes of sodium. of OS in relation to nutrient intake could be ex- Whole prey items were offered to snow leop- tremely useful for animal management as many ards 0.3 to 3 times/wk (Table 2). More frequent diseases and physiologies, including renal insuf- feeding of whole prey resulted in a positive correl- ficiency (Yu and Paetau-Robinson, 2006) and ation (P < 0.01) with SOD activity (R = 0.55) in sperm quality (Thuwanut et al., 2011), can be in- the blood (Table 10 and Fig. 1B). fluenced by damage to proteins, lipids, and DNA Table 9. Concentrations of markers of OS in blood of male snow leopards †,‡ || $ Cat SOD , U/mL GPx , nmol/min/mL FRAP, µM DNA/RNA Damage , pg/mL TBARS, µM PC, nmol/mL 1 1.7 1,527 242.0 3,265 13.4 14.1 2 10.7 − 177.2 6,316 15.1 26.5 3 0.7 4,422 254.9 6,038 13.9 20.3 4 1.9 4,167 252.1 5,263 11.7 26.7 5 1.8 26.2 225.7 5,627 16.1 34.0 6 0.4 2,654 284.0 4,710 14.2 26.3 7 0.03 − 183.5 3,748 14.5 23.9 8 0.06 3,339 321.7 5,221 15.9 37.8 9 0.00 1,909 187.6 4,141 13.6 18.5 10 5.0 1,559 275.1 4,484 14.4 14.7 11 0.8 1,111 236.7 6,985 9.0 0.8 12 1.4 − 405.7 7,347 12.0 33.3 13 1.3 164.5 279.2 6,618 17.1 27.7 14 1.0 − 252.8 6,028 12.1 36.4 SD 2.8 1,609 59.5 1,232 2.1 10.1 Average 1.9 2,088 255.6 5,414 13.8 24.4 One unit (U) of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. Assay values for cats 1 to 6 and 10 to 14 fell outside the range of the standard curve. || Samples with “−” had negative values for GPx analysis. Assay values for all cats fell outside the range of the standard curve. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article/5/2/txab034/6272442 by guest on 18 May 2021 Iske et al. as a result of OS. The objective of this study was Diet Intake to evaluate the nutrient influence and nutritional All macronutrient intakes exceeded the calor- husbandry on OS markers in male snow leop- ically adjusted nutrient allowances for domestic ards maintained in U.S. zoos. While significant cats. Crude protein and fat intakes represented on correlations do not indicate cause-and-effect re- average 56.7% and 32.1% of the DM, respectively, lationships, they can indicate the influence or im- while TDF intakes represented 3.4% to 7.3% of pact of variables on one another and be applied the DM and averaged 5.4%. The variation in DMI to dietary formulations. Additionally, they can resulted in a range of ME intakes from 6,633 to be further evaluated in relation to phenotypes, 17,499 kcals/wk (948 to 2,493 kcal/d). Daily ME inclusive of presence or absence of clinical dis- requirements for adult exotic cats (NRC, 2006) are ease or illness or reproductive characteristics. 0.75 estimated at 55 to 260 kcal × kg BW . When con- Baseline data are critically important to develop sidering individual metabolic body weights, snow additional research questions that fill knowledge leopards in the current study consumed an ME gaps for species and allow for improvements in 0.75 range of 70.1 to 196.4 kcal × kg BW with an animal management. 0.75 average of 117.3 kcal × kg BW . This was within the range of estimated exotic cat requirements presented in the 2006 National Research Council Table 10. Significant variables from multiple re- (NRC) and provides a narrower range of estimated gression analyses of diet proximates, minerals, vita- daily energy which may be particularly useful to mins, and fatty acids on markers of OS in the blood snow leopard managers. of male snow leopards Dietary vitamin A intakes compared with ad- Marker Variable R Correlation P-value justed recommendations suggested that 8 of the Oxidative damage markers snow leopards did not consume adequate amounts DNA/RNA Copper 0.44 Negative 0.01 of vitamin A, though this does not factor in whole Damage prey consumption that would provide additional Antioxidant markers vitamins to the diet. This was a result of the less SOD Whole prey 0.55 Positive <0.01 frequency than expected measured concentrations of ret- GPx Sodium 0.43 Positive 0.04 inol in the respective diets. Commercial diets are Figure 1. Linear regression of (A) copper (P = 0.01), (B) whole prey (P < 0.01), and (C) sodium (P = 0.04) intakes with markers of OS in the plasma of male snow leopards. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article/5/2/txab034/6272442 by guest on 18 May 2021 Diet and oxidative stress in snow leopards formulated to contain vitamin concentrations that temperature fluctuations are also important as meet or exceed domestic cat recommended allow- continued fat degradation can form more hydrop- ances. For example, Nebraska Brand (Nebraska eroxides to further oxidize fat-soluble vitamins. Packing Inc., North Platte, NE) approximates Additionally, less investigated theories could be vitamin A concentrations of their feline diets to be causing vitamin disappearance, such as microbial between 3.1 and 3.4 μg/g (DM); however, measured vitamin utilization or destruction. Raw meat diets concentrations in the current study only ranged contain high concentrations of a variety of micro- from 0.11 to 1.4 μg/g (DM). organisms (Hellgren et al., 2019). Understanding The less than expected intakes of vitamin the interaction of bacterial species in raw meat A may be partially explained by protein source along with resulting vitamin concentrations could (Lee et al., 2007; Wood et al., 2008). The majority be useful to diet formulations that use raw meat as of snow leopards (n = 10) consumed horse as the the primary ingredient of diets. primary protein source compared with beef (n = 2), Laboratory error and/or variation obviously pork (n = 1), or a combination of all 3 protein occurred in analysis of vitamin E. A variety of sources (n = 1). On average, cats consuming the methods exist for vitamin E determination in foods horsemeat-based diets consumed 2.4 and 5.3 times that include human error. Because of this, the more vitamin A (2,962 μg DM) compared with HPLC method has been recommended for animal those fed beef or pork (1,236 and 559.0 μg, respect- tissue and was the methodology utilized by both ively). Unfortunately, because 10 of the 14 animals analyzing labs in the current study (Desai, 1984). were fed horse-based diets and only 1 animal fed Even using the same methodology, error can still pork, conclusions about the protein source cannot occur and inter-laboratory variation exists. For be made. However, it is interesting to note that this reason, it is important to evaluate results with differences in carotenoid concentrations of grass- expected values and validate laboratory testing fed compared with grain-fed ruminants have been methods. documented and suggest that diet of the animal Plasma vitamin E concentrations in the current prior to harvest plays a critical role in the nutrient study (5.5 to 19.4 μg/mL) were in range with serum composition of the meat (Daley et al., 2010). It is vitamin E concentrations (5.6 to 16.8 μg/mL) re- possible that horses fed more forage compared with ported in leopards (Ghebremeskel and Williams, beef and pork counterparts produce meat that con- 1988; Crissey et al., 2003). Similarly, plasma ret- tains naturally higher concentrations of vitamin inol concentrations in the current study (2.8 to A (Daley et al., 2010). It would be interesting to 5.9 μg/mL) were greater than plasma and serum measure the vitamin concentrations of the source concentrations previously reported for leopards meats prior to commercial production and supple- (Panthera pardus, Uncia uncia; 0.4 to 0.6 μg/mL) mentation with premix formulations. (Ghebremeskel and Williams, 1988; Crissey et al., Another possible explanation for less than 2003) even in the cats that consumed less retinol expected vitamin A concentrations in the snow than recommended. This could support the theory leopard diets was storage conditions and diet that laboratory evaluations of the diets were indeed sample handling. Storage and freezing conditions measured consistently low. Additionally, plasma (Desai, 1984) of commercial raw meat diets in zoos analysis of fat-soluble vitamins is not an ideal need careful consideration and evaluation as they method to evaluate status. Absorption and metab- likely vary drastically among institutions. From olism of key nutrients are highly regulated, and the date of manufacture, diet samples were frozen plasma concentrations may only change in cases of between 30 and 365 d. The freezing process can extreme deficiency or toxicity (Gropper and Smith, be detrimental to fat-soluble vitamins largely due 2013). Measurement of vitamins in storage organs to lipid and fatty acid concentrations in the diet. such as the liver would be the more accurate meas- For example, vitamin A (retinol) concentrations in urement locations for vitamin status; however, it is chicken livers were more than 44% less than fresh difficult to obtain (Albahrani and Greaves, 2016). livers after 90 d of freezing at −18 ° C (Dos Santos Maintenance of plasma mineral concentra- et al., 2009). Fat can breakdown to form hydrop- tions was also evident in our analysis. Average eroxides, which are relatively stable at low temper- copper intake was 27.6 mg compared with the atures, and can oxidize vitamins (Bender, 1992). average adjusted allowance of 14.7 mg; how- The thawing process of frozen meats and storage ever, 4 snow leopards consumed less than the Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article/5/2/txab034/6272442 by guest on 18 May 2021 Iske et al. recommended amount for their respective DMI. (Rossner et al., 2007; Yeh et al., 2008), respectively. Even though the copper intakes varied by nearly Plasma DNA/RNA damage found in the current 160% and some intakes were low, plasma copper study (5,414 pg/mL) was within ranges found in concentrations only varied by 67% (average rats (430 to 6,630 pg/mL; Kadiiska et al., 2005). 0.06 mg/dL) and fell into ranges previously re- Discrepancies observed in plasma OS markers be- ported for domestic cats and cheetahs (0.02 to tween snow leopards, domestic cats, and other spe- 0.1 mg/dL; Dierenfeld, 1993; Vester et al., 2010; cies are likely associated with species differences Depauw et al., 2012). Average plasma calcium that make comparisons and conclusive inferences (8.4), iron (0.2), magnesium (2.0), phosphorus difficult. If wide differences between species exist, a (5.5), zinc (0.05), and potassium (16.1) concentra- dataset of values for an unevaluated species will be tions (mg/dL) also fell into ranges previously re- valuable for future comparisons. ported for exotic and domestic cats (5.6 to 11.6, Of all the nutrients evaluated, only copper 0.02 to 0.1, 0.06 to 0.2, 1.9 to 2.4, 1.5 to 6.5, 0.06 and sodium intakes were correlated with markers to 0.2, and 3.6 to 21.5 mg/dL; Dierenfeld, 1993; of OS, with elevated copper intakes correl- Vester et al., 2010; Depauw et al., 2012). Though ated with reduced DNA/RNA damage and ele- cats consumed 2.6 to 7.6 times the recommended vated sodium intakes correlated with greater iron allowance, plasma iron concentrations fell GPx activity. The role of dietary copper in OS into expected ranges. However, concentration and is not clear as both copper deficiency and tox- balance of minerals should be carefully evaluated icity can lead to OS (Gaetke and Chow, 2003). to avoid toxicity or long-term consequences of in- Copper supplementation (175 mg/kg) well-above creased mineral load. requirements (10 mg/kg) in the diet of growing pigs resulted in a 9.0% reduction in plasma lipid damage compared with control diets with no sup- Oxidative Stress Markers plemental copper, suggesting an antioxidant role Measures of plasma OS vary largely between of copper, though it had no significant effect on species (Maral et al., 1977; Marklund et al., 1982; SOD or GPx (Lauridsen et al., 1999). Reductions Vernet et al., 2004) with differences of nearly 100- in plasma DNA/RNA damage observed in the fold being reported across laboratory and livestock current study with higher copper intakes may re- animals. The activity of plasma SOD in snow leop- sult from reduced superoxide radical formation ards (average 1.9 U/mL) was lower than observed via the catalytic role of copper in SOD (Halliwell in domestic cats (Felis catus; 9.6 U/mL; Marklund, and Gutteridge, 2015). Though this was not dir- 1984). It is interesting to note that the 3 cats housed ectly observed in the current study, it deserves in the same institution had the lowest SOD activity, further investigation. Based on the average in- which could indicate an impact of diet or hus- takes and adjusted nutrient allowances from the bandry on this marker, though consistent differ- NRC, the ideal zinc to copper ratio for cats was ences were not observed for these 3 cats in any other 14.7, while the actual average intake ratio in the markers. Overall, cats have lower SOD activity current study for snow leopards was 13.6. With compared with rats (339.1 U/mL) and humans the evidence that both copper and zinc act as oxi- (29.7 U/mL; Marklund, 1984). Plasma GPx activity dants and antioxidants and are antagonists, this and lipid damage in healthy domestic cats aver- ratio is likely very important when considering aged 6.7 nmol/min/mg protein (334 to 668 nmol/ diet formulations. The ratio has been studied min/mL assuming 50 to 100 mg protein per mL in relation to age-related degenerative diseases plasma; Piyarungsri and Pusoonthornthum, 2016) and OS in humans; therefore, the results of the and 2.3 μM (Todorova et al., 2005), respectively, in present study indicate that it likely has import- previous studies, which are considerably less than ance for snow leopards (Mezzetti et al., 1998; our measured values (2,088 nmol/min/mL and Malavolta et al., 2015). Any future research with 13.8 μM). Some OS markers such as FRAP, PC, cats related to dietary copper should also include and DNA/RNA damage have not been evaluated consideration of zinc. in domestic cats, and thus, other models must be Average sodium intakes were 4 times above re- used for comparison. Concentrations of plasma commended allowances and intakes were well above FRAP (255.6 μM) and protein damage (24.4 nmol/ adequate in all leopards, with higher intakes correl- mL) in snow leopards fell outside ranges docu- ated with increased GPx activity. Little work has mented in human plasma (400 to 1,000 μM; Lotito been done assessing the correlation between dietary and Frei, 2004, 2006) and 9.8 to 19.0 nmol/mL sodium and OS. High sodium diets have resulted in Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article/5/2/txab034/6272442 by guest on 18 May 2021 Diet and oxidative stress in snow leopards increased OS in rats (Hummel et al., 2012); there- influence OS. In partial support of our hypothesis, fore, this should be investigated in cats to assess the sodium and copper intakes influenced GPx activity implications of the observed heightened GPx ac- and DNA/RNA damage in male snow leopards, re- tivity. Caution should be taken, however, to avoid spectively. Very high intakes of iron indicate a need concentrations of dietary sodium that may have un- to carefully evaluate mineral concentrations and desired effects, as leopards were already consuming mineral balance in zoo carnivore diet formulations. more than the recommended allowance in the cur- While higher concentrations may be warranted for rent study. This may be particularly important to growth and reproduction, animals at maintenance consider when working with geriatric, hypertensive, likely do not require the very high concentrations or renal insufficient animals (Elliott, 2006; Jepson of minerals measured in the current study. Contrary et al., 2007). to our hypothesis, vitamin intake did not directly Zoos often provide whole prey items as dietary impact markers of OS. However, a more extensive enrichment. In the current study, all leopards were study is required to address the stability of fat-solu- offered whole prey, but frequency ranged from ble vitamins in raw meat diets including storage and 3 times/wk to once per month (0.3 times/wk). handling to prevent potential degradation and loss. Consumption of whole prey provides a variety Based on the very low estimated intakes observed in of nutrients that may impact metabolism, includ- the present study, supplementation of vitamin A may ing insoluble animal fiber (Depauw et al., 2013). be warranted and should be discussed within man- Alterations in metabolism may provide some ex- agement teams to ensure adequate yet safe intakes, planation of the correlation between increased although replication of analysis in additional labora- frequency of feeding of whole prey and increased tories should also be considered. Based on markers SOD activity. The addition of soluble fiber (apple of OS that we assessed, provision of weekly whole pectin, cocoa fiber, or β-glucan at 5% or 10% of the prey supplementation and higher dietary copper diet) to rat diets resulted in approximately 70% less and sodium intakes may mitigate OS. Replication plasma lipid peroxidation (Sánchez et al., 2011), of this work should be expanded to other cat species while fermentable fiber (source not named) supple- to validate findings and assess species differences in mentation (10 and 20 g/d) significantly increased markers of OS. total antioxidant capacity (Xie et al., 2015) com- pared with diets with no added fiber. Conversely, ACKNOWLEDGMENTS other studies using rats found little to no differences We would like to thank animal care staff at in lipid peroxidation between soluble (barley) and each participating institution, including veterinar- insoluble (cellulose and wheat bran) dietary fiber ians, veterinarian technicians, and animal keepers. types (Belobrajdic et al., 2011). Conclusions on Time and effort to gather diet samples, complete the role of dietary fiber in OS have not always been husbandry surveys, and assist in sample collections agreed (Saha et al., 2017) and have not been evalu- made this project possible. Participating institu- ated in hypercarnivores such as cats. There were no tions included: Utah’s Hogle Zoo, Oklahoma City correlations with markers of OS and TDF intakes Zoo and Botanical Garden, Cheyenne Mountain that ranged from 3.4% to 7.3% of DM. Further re- Zoo, Albuquerque Biological Park, Omaha’s Henry search evaluating the role of whole prey and animal Doorly Zoo & Aquarium, Blank Park Zoo, Bronx fiber is warranted as results indicate some markers Zoo, Woodland Park Zoo, Denver Zoo, Cincinnati of OS may be impacted by whole prey. Furthermore, Zoo and Botanical Garden, San Francisco Zoo, future research should focus on differentiation and and Turtle Back Zoo. analysis of whole prey types, along with specific nu- trient intakes as well as physiological impact from consumption of whole prey. LITERATURE CITED Albahrani, A. A., and R. F. Greaves. 2016. Fat-soluble vita- Conclusions mins: clinical indications and current challenges for chro- matographic measurement. Clin. Biochem. 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Translational Animal Science – Oxford University Press
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