HEMATOLOGIC, PLASMA BIOCHEMISTRY, AND SELECT NUTRIENT VALUES IN CAPTIVE SMOOTH DOGFISH (MUSTELUS CANIS)

Author(s): Meredith E. Persky , D.V.M., J. Jason Williams , Ph.D., P.A.S., Renae E. Burks , R.V.T., Michelle R. Bowman , D.V.M., Jan C. Ramer , D.V.M., Dipl. A.C.Z.M., and Jeffry S. Proudfoot , D.V.M. Source: Journal of Zoo and Wildlife Medicine, 43(4):842-851. 2012. Published By: American Association of Zoo Veterinarians DOI: http://dx.doi.org/10.1638/2012-0002R1.1 URL: http://www.bioone.org/doi/full/10.1638/2012-0002R1.1

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Journal of Zoo and Wildlife Medicine 43(4): 842851, 2012 Copyright 2012 by American Association of Zoo Veterinarians

HEMATOLOGIC, PLASMA BIOCHEMISTRY, AND SELECT NUTRIENT VALUES IN CAPTIVE SMOOTH DOGFISH (MUSTELUS CANIS)
Meredith E. Persky, D.V.M., J. Jason Williams, Ph.D., P.A.S., Renae E. Burks, R.V.T., Michelle R. Bowman, D.V.M., Jan C. Ramer, D.V.M., Dipl. A.C.Z.M., and Jeffry S. Proudfoot, D.V.M.

Abstract: The Indianapolis Zoo maintains a large collection of smooth dogsh (Mustelus canis). During the rst several years of captivity, there was a period of high mortality in adult, wild-caught sharks in the collection. Smooth dogsh with supercial abrasions would rapidly succumb to infection and death, regardless of the treatment approach. Although the sharks did successfully produce viable offspring, there was an elevated pup mortality rate, with 0% of the pups reaching 1 yr of age during the same period of high mortality in adult sharks. This poor response to captivity prompted interest in the physiologic response of these animals to illness. The objective of this investigation was to establish a preliminary data set of hematologic and plasma chemistry reference intervals, along with select nutrient parameters specic to wild-caught adults maintained in prolonged captivity (i.e., greater than 22 mo). Blood samples were collected from 20 clinically healthy, male (n 10) and female (n 10) dogsh sharks at the Oceans facility at the Indianapolis Zoo. Although gender differences in mortality rate were not apparent, complete blood cell counts, plasma biochemical proles, and select nutrient analyses were performed and analyzed accordingly. Statistically signicant differences (P  0.05) specic to sex were determined for parameters including packed cell volume (PCV), absolute and relative ne eosinophilic granulocytes, relative percentage of coarse eosinophilic granulocytes, globulins, the albumin/globulin ratio, total protein, phosphorus, iron, selenium and copper. White blood cell counts appear to be lower in this species compared to other captive elasmobranchs. Further research into appropriate hematology standards including nutritional parameters appears warranted. Key words: Elasmobranch, hematology, Mustelus canis, plasma biochemistry, serum minerals, smooth dogsh shark.

INTRODUCTION
Smooth dogsh (Mustelus canis, order Carcharhiniformes, family Triakidae) are a demersal species of elasmobranch widely distributed throughout the western Atlantic Ocean.8,9 They migrate between the coasts of New England and South Carolina in response to changes in water temperature and feed primarily on teleosts and crustaceans.8,31 The smooth dogsh is currently listed as near threatened by the International Union for Conservation of Nature and has been proposed as an addition to the species management list for the National Oceanic and Atmospheric Association Fishery Service. For the rst few years, this species was held at the Indianapolis Zoo, adult wild-caught individuals died from septicemia after seemingly supercial wounds, despite various intensive treatment strategies.
From the Indianapolis Zoological Society, 1200 West Washington Street, Indianapolis, Indiana 46222, USA (Persky, Williams, Burks, Bowman, Ramer, Proudfoot). Present address (Persky): Zoo Miami, 12400 Southwest 152nd Street, Miami, Florida 33177, USA. Correspondence should be directed to Dr. Persky (meredithpersky@gmail.com).

Collection animals successfully reproduced; however, offspring tended to be short lived, with a 100% pup mortality rate over the rst 3 yr postexhibit opening. The poor neonatal survivorship combined with a high rate of infection and adult mortality provoked interest in the immunologic capacity of these animals under captive conditions. Peer-reviewed literature specic to hematologic and biochemical standards of the smooth dogsh has focused primarily on wild-caught animals sacriced for research purposes, making interpretation of bloodwork during this period difcult.4,28,30 There was also concern related to nutrition and its potential impact on a seemingly poor immunologic response. Previous literature has suggested that primitive elasmobranchs may possess a limited enzymatic antioxidant system, which may in turn increase their dependence upon dietary antioxidants to counter cellular damage from oxidative stress.3234 In order to test this hypothesis against Indianapolis Zoo collection animals, serum levels of both enzymatic and dietary antioxidants were investigated in healthy adults. By documenting complete blood counts (CBCs), plasma biochemical proles, enzymatic antioxidants, and nutritional metabolites, this

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PERSKY ET AL.CBC, CHEMISTRY, AND NUTRIENT VALUES OF DOGFISH

843

research aims to provide reference data to improve the preventative health care and nutritional management of this species in captivity.

MATERIALS AND METHODS

Wild-caught adult dogsh sharks, 10 males and 10 females, weighing an average of 3.8 kg, were housed at the Oceans facility of the Indianapolis Zoo for a minimum of 22 mo prior to phlebotomy. All sharks were maintained under identical husbandry parameters, including diet, light cycle (9hr photoperiod) and water temperature in an approximately 68,800-L indoor saltwater concrete touch tank. The dietary prole consisted of multiple frozen feeder sh, including male capelin (Mallotus villosus), high- and low-fat Atlantic herring (Clupea harengus), farm harvested rainbow trout (Oncorhynchus mykiss), whole shrimp (Penaeus setiferus), and lobster tail (Panulirus homarus). All sharks were given access to an identical diet at all times; however, verication of consumption for dietary ingredients specic to individual animals was not possible. Mazuri shark/ray II vitamins (5MD8, Mazuri Exotic Animal Feeds, PMI International, St. Louis, Missouri 63166, USA), were also included in the diet as a component of feeder sh at 1.5 g per 2.3 kg of sh fed. Visual assessments as reported by husbandry staff indicated an approximate 40 60% rejection of the vitamin tablet on a consistent basis due to the animals ability to separate the tablet from feeder sh. Water quality was analyzed on a weekly basis and was found to be consistent with standards specic to the Indianapolis Zoo (salinity 3035 ppt, pH 7.58.5, ammonia [NH3/NH4] 0 mg/L, nitrates [NO3] ,25 ppm). Sharks were individually identied prior to each blood draw by conrming transponders (Trovan Electronic Identication Devices Ltd., Burnsville, Minnesota 55337, USA) positioned in the epaxial musculature of each specimen. Animals were manually restrained in dorsal recumbency, just below the surface of the water, for venipuncture. Approximately 12 ml of blood was collected from the caudal vein using a 12-ml syringe and a 20-gauge, 3.0-cm needle (Becton Dickinson, Franklin Lakes, New Jersey 07417, USA).42 Samples were transferred to Microtainer tubes containing dry EDTA, Vacutainer lithium heparin, liquid EDTA, and Vacutainer trace element serum tubes (Becton Dickinson). Sample collections occurred within a 5-day period, with a maximum of seven sharks being processed on the

same day. This permitted the completion of CBC analysis within 5 hr of initial blood collection, as recommended based on the limited stability of leukocytes in EDTA.2,17 All white blood cell (WBC) counts and WBC differentials were performed by the same laboratory technician to maintain consistency. Hematocrit tubes containing whole blood were centrifuged at approximately 10,400 rpm (13,000 g) for 6 min to allow the large immature red blood cells to separate from the plasma layer within the microhematocrit tube.2 PCVs were recorded by standard microhematocrit method. WBCs were differentiated based on morphology using Wright Giemsa stain (Colorant Selon Wright Giemsa, EMD Chemicals Inc. Gibbstown, New Jersey 08027, USA) and named using methods described by Arnold.1,2 Cell classications included neutrophils, monocytes, lymphocytes, coarse eosinophilic granulocytes (CEGs), ne eosinophilic granulocytes (FEGs), basophils, granulated thrombocytes (GT), and nongranulated thrombocytes. Because the clinical signicance of GTs is unknown, the WBC count and the leukocyte differentials were determined by both exclusion (WBC) and inclusion (WBC-GT) of these cells. Whole blood was diluted 1:100 in a Natt and Herrick solution that was modied to mimic the physiologic state of marine elasmobranchs.2,26 The osmolality was increased by adding 13.5 g/L NaCl and 31.5 g/L urea to the preltered stock solution.2 The total WBC counts (including and excluding the GTs) were determined rst by tallying all nonerythrocyte cells per grid of an improved Neubauer hemacytometer (Baxter Healthcare Corporation, Scientic Products Division, McGaw Park, Illinois 60085, USA), as it is difcult to differentiate thrombocytes and similarly sized lymphocytes.35,39,44 The WBC thrombocyte tally (T-WBC) was multiplied by the nal factor (110) for the number per microliter. The nal WBC count was then obtained by multiplying the T-WBC by the percentage of thrombocytes from the blood smear, where all thrombocytes were tallied as part of the leukocyte differential. The absolute value of all thrombocytes was then subtracted from the T-WBC and reported as the WBC count. The WBC-GT count was determined by subtracting the absolute value of the nongranulated thrombocytes from the T-WBC. Leukocyte differentials were determined (excluding all thrombocytes) for the WBC count; the differential for the WBC-GT counts included GTs.

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Lithium heparin samples were centrifuged at 1,030 g for 5 min to separate the cells and 1-ml aliquots were stored at 208C until shipped on dry ice for analysis, within 3 wk of collection. Specimens were analyzed at North Carolina State University Diagnostic Laboratories, NCSU College of Veterinary Medicine (Raleigh, North Carolina 27606, USA). The plasma chemistry analyses were performed on a Roche Cobas Integra 400 Plus (Roche Diagnostics, Indianapolis, Indiana 46256, USA). All biochemistry samples were run on the same day, with the same calibration. Serum tubes containing samples for vitamin and mineral analysis were also centrifuged at 1,030 g for 5 min; 1-ml aliquots of plasma were stored at 208C until shipped on dry ice for analysis to the Diagnostic Center for Population and Animal Health at Michigan State University (East Lansing, Michigan 48910, USA). All minerals excluding iron were run on the Agilent inductively coupled plasma mass spectrometer model 7500 CE (Agilent Technologies, Center Valley, California 95051, USA). Iron was analyzed on the Olympus AU800 Chemistry Analyzer (Olympus America, Center Valley, Pennsylvania 18034, USA). The Waters high-performance liquid chromatograph with photodiode array detection was used to determine concentrations of atocopherol and ascorbic acid (Waters Corporation, Milford, Massachusetts 01757, USA). The liquid EDTA tubes collected for enzymatic antioxidant testing were centrifuged at 1,030 g for 5 min. The plasma was discarded and the volume (milliliters) of remaining RBCs was estimated. The RBCs were suspended in 0.9% saline at 3 times the estimated RBC volume, in a 15-ml conical centrifuge tube. The conical tube was mixed by gentle inversion and spun at 1,030 g for 5 min. The supernatant was discarded. The RBC washing, centrifugation, and decanting of the supernatant was repeated. The RBCs were lysed by adding cold, deionized water at four times the estimated RBC volume. The samples were stored at 808C until shipped on dry ice for analysis to Luminos LLC (Ann Arbor, Michigan 48108, USA). Free and total glutathione was determined using the Luminos DetectXTMTM Glutathione Fluorescent Detection Kit K006-F1 (Interactive Assay Solutions, Ann Arbor, Michigan 48108, USA) according to product specications. Free and total glutathione values were calculated from a net standard curve using a 4-parameter logistic curve-tting computer program, with glutathione disulde values calculated as exactly half of the

difference between the total and free determinations. A 1:80 dilution of each sample was evaluated for superoxide dismutase utilizing a Dojindo SOD Assay Kit (Dojindo Moledular Technologies, Inc., Rockville, Maryland 20850, USA). All superoxide dismutase values were determined from the mean standard curve using a 4-PL curve-tting computer program. Dilutions (1:2) of each sample were evaluated for glutathione reductase activity using a modied version of the Luminos DetectXTM Glutathione Reductase Activity Kit K009-F1 (Interactive Assay Solutions). The standard range for the modied assay was 0.400.025 mU/ml. Reported glutathione reductase values were determined from the net standard curve using a 4-PL curve-tting computer program. Both dilutions of each sample read below the lowest standard or zero. Data were imported from a computerized spreadsheet (Excel, Microsoft Corporation, Redmond, Washington 98052-6399, USA) into a statistical software program XLStat (Addinsoft USA, New York, New York 10001, USA) for analyses. Mean, standard deviation, and minimummaximum intervals were determined, assuming a normal distribution, for each hematology (Tables 1, 2) and plasma chemistry analyte (Table 3). Means and standard errors were calculated for micronutrient parameters (Table 4) and circulating enzymatic antioxidants (Table 5). A two-tailed t-test for independent samples was utilized to test for signicance based on gender. Values of P  0.05 were considered statistically signicant.

RESULTS
All animals were clinically healthy at the time of blood collection. Distributions (mean, standard deviation, minimum, maximum) of CBC and plasma chemistry analytes are reported in Tables 13. Lymphocytes were the dominant leukocyte in the WBC differential count (Table 1). When included in the differential count, GTs were the dominant cell type observed in female sharks, with lymphocytes being the second most common leukocyte (Table 2). GTs were the second most prevalent leukocyte in the male sharks, with the lymphocyte remaining the most common WBC observed (Table 2). CEGs were the fewest cells seen. There were no basophils observed in this study. Gender differences were observed in both sets of reported CBC values (Tables 1, 2). PCV was higher in the male sharks. Statistically signicant differences observed in Table 1 (excluding GT cells) and in Table 2 (including GT

PERSKY ET AL.CBC, CHEMISTRY, AND NUTRIENT VALUES OF DOGFISH

845

Table 1. Mean, SD, minimummaximum, and signicance level for complete blood cell count (excluding thrombocytes) values from captive male (?) vs. female (/) smooth dogsh sharks (M. canis). n 20.a
Measure Mean 6 SD Signicance (P) Minimummaximum

/ PCV (%) ? PCV (%) / WBC (per ll) ? WBC (per ll) / neutrophils (%) ? neutrophils (%) / neutrophils (per ll) ? neutrophils (per ll) / lymphocytes (%) ? lymphocytes (%) / lymphocytes (per ll) ? lymphocytes (per ll) / monocytes (%) ? monocytes (%) / monocytes (per ll) ? monocytes (per ll) / ne eosinophilic granulocytes (%) ? ne eosinophilic granulocytes (%) / ne eosinophilic granulocytes (per ll) ? ne eosinophilic granulocytes (per ll) / coarse eosinophilic granulocytes (%) ? coarse eosinophilic granulocytes (%) /coarse eosinophilic granulocytes (per ll) ?coarse eosinophilic granulocytes (per ll)
a

24.2 29.7 15,330 14,300 22.0 24.9 3,366.9 3,373.7 33.1 35.4 4,909 5,281.8 8.1 11.3 1165 1,675.2 33 22.2 5,289 3,129.3 3.8 6.2 600.1 840

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

4.1 2.4 4,086.8 6,436.7 5.0 7.1 1,039.9 1,313.3 13.7 8.0 1,833.1 3,588.3 4.9 4.4 744.4 1,079.5 10 5.6 2,917.7 1,559.5 1.1 3.5 321.9 435.5

0.002 0.67 0.31 0.99 0.65 0.77 0.14 0.23 0.008 0.05 0.05 0.18

1830 2733 10,50023,900 6,90028,500 1228 1340 1,2844,628 1,7945,945 1863 2251 2,0888,428 2,27714,535 016 517 02,492 3453,990 1652 1533 1,71212,428 1,4496,765 26 315 2321,434 2491,710

PCV, packed cell volume; WBC, white blood cell.

cells) included the absolute and relative values of FEGs, with female sharks having higher values than the male sharks. In Table 1 (excluding GT cells), male smooth dogsh had a higher percentage of CEGs compared to female conspecics. The plasma biochemistry results are displayed in Table 3. Measured values for two plasma biochemical parameters, creatinine (,2 mg/dl) and lactate dehydrogenase (,10 IU/L), were below detection limits for all 20 animals. Statistically signicant gender differences were observed when evaluating total protein and globulins with reported values being higher in the male sharks. Conversely, phosphorus was higher in the female sharks sampled. Plasma micronutrient data are presented in Table 4. Serum a-tocopherol (lg/ml), ascorbic acid (mg/ml), molybdenum (lg/ml), cobalt (ng/ ml), manganese (ng/ml), and zinc (lg/ml) did not differ according to gender (P  0.05). Enzymatic antioxidant data are presented in Table 5. Frozen EDTA erythrocyte lysates specic to male and female specimens were analyzed for free and total glutathione, glutathione reductase, glutathione disulde, and superoxide dismutase. No difference (P  0.05) specic to enzymes was deter-

mined according to gender in any assay for any metabolite.

DISCUSSION
The function and quantication of elasmobranch cells is poorly represented in the literature and has contributed to a lack of standardization within the nomenclature of elasmobranch hematology.2,5,11,20,21,24,44 In particular, the role of GTs and eosinophilic granulocytes remain unknown, as they do not have denite mammalian counterparts.2,7,20,44 This has led to controversy over including GTs as part of the WBC differential.2,27,37,44 The authors of this paper have followed the protocols established by Arnold for performing WBC counts and differentials in an attempt to assist in the standardization of hematology techniques in elasmobranchs.1,2 The methodologies used in this study of smooth dogsh were adopted from those described in the sandbar shark, Carcharhinus plumbeus, allowing comparisons between species.2 Basophils were absent in both studies.2 Basophils appear to be rare or absent in many sharks previously studied.5,7,27,41,45 Lymphocytes were the most abundant cell type in both sharks (Table 1) except when GTs

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JOURNAL OF ZOO AND WILDLIFE MEDICINE

Table 2. Mean, SD, minimummaximum, and signicance level for complete blood cell count (including GTs) values from captive male (?) vs. female (/) smooth dogsh sharks (M. canis). n 20.a
Measure Mean 6 SD Signicance (P) Minimummaximum

/ ? / ? / ? / ? / ? / ? / ? / ? / ? / ? / ? / ? / ? / ?

PCV (%) PCV (%) WBC-GT (per ll) WBC-GT (per ll) neutrophils (%) neutrophils (%) neutrophils (per ll) neutrophils (per ll) lymphocytes (%) lymphocytes (%) lymphocytes (per ll) lymphocytes (per ll) monocytes (%) monocytes (%) monocytes (per ll) monocytes (per ll) ne eosinophilic granulocytes (%) ne eosinophilic granulocytes (%) ne eosinophilic granulocytes (per ll) ne eosinophilic granulocytes (per ll) coarse eosinophilic granulocytes (%) coarse eosinophilic granulocytes (%) coarse eosinophilic granulocytes (per ll) coarse eosinophilic granulocytes (per ll) granulated thrombocytes (%) granulated thrombocytes (%) granulated thrombocytes (per ll) granulated thrombocytes (per ll)
a

24.2 29.7 21,390 20,220 16.8 17.8 3,502.4 3,553 25.9 32 5,513.3 6,764.9 5.4 5.8 1,138.9 1,089.2 22.5 12.9 4,939.8 2,414.2 3.1 3.2 660.7 632.2 26.3 28.3 5,634.9 5,766.5

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

4.1 2.4 4,190.6 8,781.8 5.6 3.3 1,014.7 1,482.4 9.7 6.8 2,245.1 4,396.7 3.0 2.6 655.4 538.9 9.2 4.8 2,604.1 1,223.7 1.5 2.5 326.9 655.5 7.3 8.3 1820.5 3,069.8

0.002 0.71 0.63 0.93 0.12 0.43 0.75 0.86 0.009 0.01 0.92 0.90 0.57 0.91

186 30 2733 14,90028,800 7,50037,600 727 1423 1,4074,896 1,2755,764 1548 2246 2,9409,648 2,62517,296 111 29 2882,376 3001,880 738 117 1,40710,944 2624,454 16 19 2161,206 1892,358 1442 1340 2,4508,232 1,65010,218

PCV, packed cell volume; WBC, white blood cell; GT, granulated thrombocyte.

were included (Table 2).2 Lymphocytes have been the most common WBC observed in other elasmobranch species.2,7,44 GTs were the second most prevalent cell type observed when included in the total WBC count and differential in male sharks but were the dominant leukocyte found in female sharks (Table 2). The relative percentage of GTs in smooth dogsh was slightly higher than values reported in the sandbar shark.2 Further research is necessary to determine the function and clinical signicance of GTs to elasmobranch health. Smooth dogsh had lower WBC counts than the sandbar shark.2 Smooth dogsh also appear to have lower WBC counts than other captive elasmobranch species, including brown sharks (C. plumbeus), lemon sharks (Negaprion brevirostris), and nurse sharks (Ginglymostoma cirratum), although different leukocyte nomenclature is used.40,41 WBC counts appear lower in clinically healthy, captive smooth dogsh compared to other captive elasmobranchs, regardless of including GTs in the

WBC count.2,41 Although the WBC counts are lower, they do not appear to be leukopenic, which could account for the rapid demise in debilitated animals of this species. Data from clinically compromised smooth dogsh may now be compared to these baseline data and could provide some further insight into the initially high mortality rate associated with supercial abrasions in these animals. When evaluating the plasma biochemical parameters, the creatinine kinase and aspartate aminotransferase values appear low.18,40 This may be secondary to species-related differences or variation in sample collection technique, because these samples were obtained from manually restrained animals in captivity, as opposed to trawlers utilized to sample wild-caught animals. In addition, animals were not sacriced for this study. Glucose values also appeared lower in the smooth dogsh, presumably because of minimal handling time required to obtain samples from captive sharks.18 The remaining chemistry panel is

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Table 3. Mean, SD, minimummaximum, and signicance level for plasma biochemical values from captive male (?) vs. female (/) smooth dogsh sharks (Mustelus canis). n 20.
Measure Mean 6 SD Signicance (P) Minimummaximum

/ ? / ? / ? / ? / ? / ? / ? / ? / ? / ? / ? / ? / ? / ? / ? / ? / ? / ? / ?

glucose (mg/dl) glucose (mg/dl) urea nitrogen (mg/dl) urea nitrogen (mg/dl) phosphorus (mg/dl) phosphorus (mg/dl) calcium (mg/dl) calcium (mg/dl) total protein (g/dl) total protein (g/dl) albumin (g/dl) albumin (g/dl) globulin (g/dl) globulin (g/dl) albumin/globulin ratio albumin/globulin ratio aspartate aminotransferase (IU/L) aspartate aminotransferase (IU/L) creatine kinase (IU/L) creatine kinase (IU/L) sodium (nmol/L) sodium (nmol/L) potassium (nmol/L) potassium (nmol/L) sodium/potassium ratio sodium/potassium ratio chloride (mmol/L) chloride (mmol/L) bicarbonate (mmol/L) bicarbonate (mmol/L) anion gap (mmol/L) anion gap (mmol/L) osmolality (mOsm/kg) osmolality (mOsm/kg) creatinine (mg/dl) creatinine (mg/dl) lactate dehydrogenase (IU/L) lactate dehydrogenase (IU/L)
a

99.8 6 8.2 107.8 6 9.2 993.8 6 23.1 981.1 6 12.7 5.4 6 0.9 4.5 6 0.4 16.8 6 0.6 17 6 0.5 2.4 6 0.3 3 6 0.4 0.5 6 0.1 0.5 6 0.1 1.9 6 0.2 2.5 6 0.4 0.3 6 0.1 0.2 6 0.0 10.6 6 6.8 12.1 6 5.4 6.2 6 4.0 7.3 6 1.2 255.3 6 3.8 255.4 6 2.0 4.0 6 0.5 4.3 6 0.5 64.6 6 7.7 60.2 6 7 254.6 6 6.1 253 6 3.5 6.6 6 1.4 9.4 6 4.2 0.2 6 0.5 0.4 6 0.8 843.1 6 12.7 838.6 6 5.6 , 2a , 2a ,10a ,10a

0.06 0.15 0.02 0.47 0.001 0.82 0.00027 0.01 0.59 0.41 0.94 0.25 0.20 0.48 0.06 0.64 0.32

88115 96123 9691,027 9661,002 3.16.1 3.95 15.918 16.217.8 1.82.7 2.53.5 0.30.6 0.40.6 1.52.3 2.13.1 0.20.3 0.10.3 325 523 213 59 249260 253259 3.14.6 3.75.1 58.680.1 50.268.9 245261 249260 48 617 01.2 01.9 823.2860.1 829.7845.4

Values too low to measure; out of range.

similar to previously reported plasma or serologic chemistry values in elasmobranchs.18,40,41 Data concerning nutritionally specic blood parameters, including antioxidant enzyme expression in elasmobranchs, are also relatively limited.10,3234 Of primary interest to this research were those metabolites suggested to relate specically to antioxidant capacity and immune response. Circulating ascorbic acid (vitamin C) and d-atocopherol (vitamin E) are both signicant immune defense components in various sh species with deciencies of vitamin C demonstrated to affect iron-related blood parameters.12,15,16,19,23,34,46,47

Both vitamin C and E were elucidated in dogsh serum at signicant concentrations (Table 4); however, direct comparison to other elasmobranchs was difcult because of a lack of preexisting species-specic data sets. This present investigation does support research from previous efforts in that serum concentrations of vitamins C and E appear to be present in greater quantities in M. canis than what is typical for other species of sh, with no difference observed due to sex.10,14,34 Signicant differences specic to gender were determined for several parameters (Table 4). Mean serum selenium (ng/ml) was signicantly

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Table 4. Mean, SE, minimummaximum, and signicance level for serum nutrient values from captive male (?) vs. female (/) smooth dogsh sharks (Mustelus canis). n 20.
Measure Mean 6 SE Signicance (P) Minimummaximum

/ ? / ? / ? / ? / ? / ? / ? / ? / ?

a-tocopherol (lg/ml) a-tocopherol (lg/ml) selenium (ng/ml) selenium (ng/ml) ascorbic acid (mg/dl) ascorbic acid (mg/dl) copper (lg/ml) copper (lg/ml) molybdenum (lg/ml) molybdenum (lg/ml) iron (lg/dl) iron (lg/dl) cobalt (ng/ml) cobalt (ng/ml) manganese (ng/ml) manganese (ng/ml) zinc (lg/ml) zinc (lg/ml)

12.3 9.3 230 347 0.43 0.49 0.81 0.59 0.80 0.74 71 32 71.6 73.1 13.7 6.1 0.9 1.0

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

2.38 0.99 13.27 51.64 0.05 0.03 0.03 0.02 0.10 0.11 11.93 4.88 13.77 8.15 4.16 1.51 0.03 0.10

0.26 0.04 0.32 0.01 0.69 0.01 0.93 0.10 0.30

6.8632.48 5.1814.85 176292 168695 0.190.70 0.350.70 0.660.94 0.490.75 0.201.20 0.301.50 22158 1161 24.9180.7 47.0123.8 2.845.7 2.118.5 0.71.1 0.51.4

lower and copper (lg/ml) and iron (lg/dl) significantly higher in female vs. male sharks respectively. Iron is a primary metabolite involved in lipid peroxidation reactions and is important to oxidative phosphorylation.45 Iron can be absorbed into the system via both the gill membrane and the intestinal mucosa, with deciencies typically represented by depressed erythrocyte counts and stunted growth rates.45 Copper, although an important cofactor in various enzyme systems including cytochrome oxidase, superoxide dismutase, and lysyl oxidase, has a demonstrated toxicity in elasmobranchs at greater than 2.0 mg kg1 dry weight in the diet.40 Toxicity has been shown to cause damage to gills in channel catsh and necrosis in the liver and kidney.45 In addition, the requirement for copper

is dependent upon the concentrations of water borne metabolic antagonists such as zinc, Fe, and molybdenum, which can interfere with Cu absorption.45 Selenium is an essential nutrient for both aquatic and terrestrial animals, acting as an integral component in the antioxidant system of glutathione peroxidase (GP),45 the primary function of GP being the protection of cell membranes from oxidative damage by neutralizing hydroperoxides.45 Typical deciency symptoms include reduced growth rate, anorexia, and subsequent mortality.45 It should be noted that project specimens were not sacriced for this research and thus tissue levels of specic metabolites were not determined. In certain cases, tissue concen-

Table 5. Mean, SE, minimummaximum, and signicance level for enzymatic antioxidant values from captive male (?) vs. female (/) smooth dogsh sharks (Mustelus canis). n 20.
Measure Mean 6 SE Signicance (P) Minimummaximum

/ ? / ? / ? / ? / ?

glutathione reductase (mU/ml) glutathione reductase (mU/ml) superoxide dismutase (U/ml) superoxide dismutase (U/ml) superoxide dismutase (U/mg Hb) superoxide dismutase (U/mg Hb) glutathione total (lM) glutathione total (lM) glutathione free (lM) glutathione free (lM)
a

TLMa TLMa 31.3 6 3.8 28.3 6 3.6 181.7 6 52.7 145.8 6 36. 4 88.1 6 16.3 68.5 6 11.3 72.6 6 11.9 59.9 6 10.3

0.57 0.58 0.34 0.43

14.949.4 12.145.4 27.8454.7 26.9295.8 34.6187.9 20.7144.2 30.6140.7 13.2126.0

TLM, values too low to measure; out of range.

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trations of micronutrients may be a more meaningful gauge of actual metabolic status.22,25 In regards to enzymatic antioxidants, the obtained data demonstrated both the presence and absence of important enzyme systems including superoxide dismutase and both total and free glutathione. Catalase activity was not determined because of previous literature supporting the absence of this enzyme in primitive sh species, including the spiny dogsh (Squalus acanthias).29,33,38 A feature of note in the present study was the lack of measureable glutathione reductase. Glutathione reductase is a virtually omnipresent enzyme, having been elucidated in numerous species.6,13,36,43 Values represented as below detectable limits (Table 5) were determined in all 20 animals, regardless of gender. Samples were analyzed in triplicate to verify the lack of activity utilizing nicotinamide adenine dinucleotide phosphate and nicotinamide adenine dinucleotidedependent assays capable of detecting glutathione reductase at concentrations 2,500 times lower than what is typical in human serum. Many features of elasmobranch metabolism, including fewer isoforms of enzymes and proteins, have been attributed to their primitive evolutionary position.3 Our data set appears to support the hypothesis that smooth dogsh rely, at least to some degree, on an antioxidant structure that does not utilize typical coenzyme systems to ameliorate oxidative cellular damage.

ogy Laboratory at the North Carolina State University College of Veterinary Medicine, Dr. Russ Hart, and the staff from Luminos LLC for their efforts with enzyme determination, Gretchen Cole, D.V.M., Dipl. A.C.Z.M., Ellen McBride, R.V.T., Jennifer Niederlander, R.V.T., the aquarists at the Indianapolis Zoo, and Wm. Kirk Suedmeyer, D.V.M., Dipl. A.C.Z.M. This project was sponsored and funded by the Indianapolis Zoo.

LITERATURE CITED
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CONCLUSIONS
This research established preliminary baseline values for hematology, plasma chemistries, selected trace minerals, and fat-soluble vitamins in 20 clinically healthy, captive smooth dogsh shark, M. canis, at the Indianapolis Zoo. Gender differences (P  0.05) were observed among PCV, absolute and relative FEGs, relative percentage of CEGs, globulins, the albumin/globulin ratio, total protein, phosphorus, iron, selenium, and copper. WBC counts appear to be lower in this species compared to other captive sharks.2,18 This baseline information may be used to monitor the health status and nutritional requirements of captive populations of smooth dogsh and ultimately result in improved nutritional parameters and greater fecundity and pup survival rates within this species in captive settings. Acknowledgments: The authors thank Jill Arnold, M.S., M.T. (A.S.C.P.), for assisting with methodologies, the staff of the Clinical Pathol-

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46. Wedemeyer, G. 1968. Stress-induced ascorbic acid depletion and cortisol production in two salmonid shes. Comp. Biochem. Physiol. 29: 12471251. 47. Xue, G., G. Yu, T. Hirata, M. Sakaguchi, and J. Terao. 1998. Antioxidative activity of carp blood plasma on lipid peroxidation. Biosci. Biotechnol. Biochem. 62: 201205. Received for publication 2 January 2012