Full text of DOI:10.1111/j.1939-165X.2002.tb00289.x

Plasma Biochemistry Reference Values of
Wild Bonnethead Sharks, Sphyrna tiburo
Craig Harms, Trisha Ross, Al Segars
Background — Elasmobranchs (sharks, skates, and rays) are of commercial, sport, research, and exhibit importance, however,
blood chemistry reference values have been determined for few of these species. Objectives — The purpose of this study was to
establish plasma biochemistry and PCV reference values for wild bonnethead sharks (Sphyrna tiburo). Methods — Heparinized
blood samples were collected from 24 bonnethead sharks at the time of capture in trawl nets off the coast of South Carolina and
Georgia. Weight, length, PCV, total solids (TS, by refractometry), and plasma biochemical analyses were done using standard tech-
niques. Wilcoxon rank-sum and Kendall tau b tests were used to compare values by animal size, boat and sex; 1-way ANOVA was
used to compareTS and total protein (TP) concentrations. Results — Median (quartiles; minimum-maximum) values were as fol-
lows: PCV 22% (22%, 26%; 17-28%),TS 6.3 (6.0, 6.8; 5.8-7.5) g/dL, total protein 2.9 (2.7, 3.4; 2.2-4.3) g/dL, albumin 0.4 (0.4, 0.4; 0.3-0.5)
g/dL, globulins 2.6 (2.3, 3.0; 1.9-3.8) g/dL, sodium 282 (279, 285; 273-292) mmol/L, potassium 7.3 (6.4, 7.9; 5.7-9.2) mmol/L, chloride
290 (285, 296; 277-304) mmol/L, total CO2 3 (2, 4; 0-5) mmol/L, calcium 16.8 (16.2, 17.4; 15.8-18.2) mg/dL, phosphorus 8.8 (7.5, 10.0;
5.9-12.7) mg/dL, urea nitrogen 1004 (986, 1028; 944-1068) mg/dL, creatinine <0.1 mg/dL, glucose 184 (165, 191; 155-218) mg/dL,
aspartate aminotransferase 42 (33, 66; 15-132) U/L, lactate dehydrogenase <5 U/L, creatine kinase 82 (47, 233; 18-725) U/L, and
osmolality 1094 (1078, 1111; 1056-1139) mOsm/kg. No differences based on sex were detected.TS and total TP values were related
by the fitted line TS = (1.006ϫTP) + 3.318. Conclusions — Values reported here will be useful for evaluating the health status of
bonnetheads in wild and captive research conditions and in exhibits. (Vet Clin Pathol. 2002;31:111-115)
Key Words: Bonnethead, elasmobranch, plasma biochemistry, shark, Sphyrna tiburo
———◆———
Treatment of wild animals held in captivity and health
assessment of the same animals in the wild is sometimes
hampered by scarcity of clinical pathology reference val-
ues. For captive wild animals, confinement, environmen-
tal conditions, and nutrition may result in clinical pathol-
ogy values different from those of their wild counter-
parts. During investigations of anthropogenic alterations
of the wild environment there may be no baseline refer-
ence values that can be used to assess responses of
affected species. Blood chemistry of teleost fishes, partic-
ularly those common in aquaculture and to a lesser
degree some ornamental species, has received some
Bonnethead sharks are members of the hammer-
head shark family, Sphyrnidae.10,11 In bonnetheads, the
lateral expansion of the head is less pronounced than
that in the great hammerhead (Sphyrna mokarran) and is
more of a flattened spade shape, leading to the other
common name, the shovelhead. Bonnetheads are the
smallest member of the genus, rarely reaching more
than 1.5m in length, and are considered more sluggish
than other Sphyrna species. They consume primarily
crabs and other invertebrates. Because of their size, dis-
position, unique cranial conformation, and close phylo-
genetic relation to more notorious members of the
Sphyrnidae, they make popular exhibits in public
1-4
attention. Elasmobranchs (sharks, skates, and rays)
have received less attention. Although there are >500
species of elasmobranchs, many of which are of com-
mercial, sport, research, and exhibit importance, blood
chemistry reference values have been determined for
relatively few of these species.5-9 In many instances, these
reference values are based on small sample sizes (<10),
include a limited panel of analytes, or both. Here, we
report PCV and 20 plasma biochemistry reference values
established from 24 wild bonnethead sharks (Sphyrna
tiburo) sampled at the time of capture.
aquariums12 and interesting research subjects for com-
15,16
parative anatomy and physiology.
Bonnetheads also
have emerged as important indicators of environmental
toxicology of heavy metals17 and endocrine disrup-
tors.18,19 Bonnethead sharks are found in shallow tropical
10,11
to warm temperate waters.
In the western Atlantic,
they range from North Carolina (less commonly north
to New England) to southern Brazil. Identical or closely
related species are found worldwide.
In the course of trawl censusing of sea turtles off the
From the Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Center for Marine Sciences and
Technology, Morehead City, NC, and the Environmental Medicine Consortium, College of Veterinary Medicine, North Carolina State University,
Raleigh, NC (Harms); the College of Veterinary Medicine, North Carolina State University, Raleigh, NC (Ross); and the South Carolina Department
of Natural Resources, Beaufort, SC (Segars). Corresponding author: Craig A. Harms, DVM, PhD, Department of Clinical Sciences, College of
Veterinary Medicine, North Carolina State University, Center for Marine Sciences and Technology, 303 College Circle, Morehead City, NC 28557
(craig_harms@ncsu.edu).
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Plasma Biochemistry of Bonnethead Sharks
Table 1. Bonnethead shark (Sphyrna tiburo) PCV and plasma biochemistry reference values.*
Parameter
n
Median
24 (0.24)
6.3 (63)
2.9 (29)
0.4 (4)
2.6 (26)
0.14
Lower Quartile
22 (0.22)
6.0 (60)
2.7 (27)
0.4 (4)
2.3 (23)
0.13
Upper Quartile
26 (0.26)
6.8 (68)
3.4 (34)
0.4 (4)
3.0 (30)
0.17
Minimum-Maximum
17-28 (0.17-0.28)
5.8-7.5 (58-75)
2.2-4.3 (22-43)
0.3-0.5 (3-5)
1.9-3.8 (19-38)
0.12-0.19
PCV, % (L/L)
21
20
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
Total solids, g/dL (g/L)
Total protein, g/dL (g/L)
Albumin, g/dL (g/L)†
Globulins, g/dL (g/L)
A/G ratio
Sodium, mmol/L
282
279
285
273-292
Potassium, mmol/L
7.3
6.4
7.9
5.7-9.2
Chloride, mmol/L
290
285
296
277-304
Bicarbonate, mmol/L†
3
2
4
0-5
Anion gap, mmol/L
–5.8
–7.6
0.1
–15.7-7.5
Calcium, mg/dL (mmol/L)
Phosphorus, mg/dL (mmol/L)
Urea nitrogen, mg/dL (mmol/L)
Glucose, mg/dL (mmol/L)
Aspartate aminotransferase, U/L
Lactate dehydrogenase, U/L
Creatine kinase, U/L
Osmolality, mOsm/kg
*Standard units (SI units)
16.8 (4.2)
8.8 (2.8)
1004 (358)
184 (10.2)
42
16.2 (4.0)
7.5 (2.4)
986 (352)
165 (9.2)
33
17.4 (4.3)
10.0 (3.2)
1028 (367)
191 (10.6)
66
15.8-18.2 (3.9-4.5)
5.9-12.7 (1.9-4.1)
944-1068 (337-381)
155-218 (8.6-12.1)
15-132
<5
<5
<5
<5-11
82
47
233
18-725
1094
1078
1111
1056-1139
†Includes values below the reliable limit of detection of the analyzer.
20
coast of South Carolina and Georgia, bonnetheads
of tail, to the nearest 1 cm) prior to being returned to the
ocean. Sharks with any external wounds (generally
shark bites sustained in the net) were excluded from
sampling. The sex of the sharks was determined by ex-
ternal examination based on the presence or absence of
claspers. Bonnetheads were manually restrained, and
blood was collected from the caudal vein using 3-mL
syringes fitted with 22-ga by 1.5-inch (0.7ϫ40 mm) nee-
dles or directly intoVacutainer tubes (Becton Dickinson,
Franklin Lakes, NJ, USA). Lithium heparin was used as
the anticoagulant. Samples were centrifuged at ambient
temperature for PCV and total solids (TS) determination
(5 min, 14,000g, Micro-capillary Centrifuge, Model MB,
International Equipment Co, Needham Heights, MA,
USA), and for harvesting plasma (5 min, 1000g, Adams
Sero-Fuge CT 1600, Clay Adams Co, Parsippany, NJ,
USA). TS were determined by refractometry (RHC-
200ATC refractometer, Westover Scientific, Woodinville,
WA, USA).
were frequently caught. This situation afforded the op-
portunity to determine blood and plasma biochemistry
values of clinically normal wild bonnetheads and to
compare these values by sex and by size.
Materials and Methods
Bonnetheads were collected in July 2001 by 2 fishery-
independent trawlers (boats A and B) operating in near-
shore waters between Winyah Bay, SC (33.18°N,
79.15°W), and St. Augustine, Fla (29.86°N, 81.26°W), in
water 15-40 feet (4.5-12.2 m) deep. Water temperature
ranged from 27.9°C to 29.7°C. Trawlers used 20-m 4-
seam nets with 20-cm mesh and without turtle exclud-
ers.Trawl duration was limited to 30 minutes. When the
net was emptied on deck, sea turtles received the high-
est priority for processing; sharks were not sampled if
sea turtles were present in the trawl. Blood was collect-
ed from sharks within 3-5 minutes of being brought on
board. Sharks were counted, weighed (to the nearest 0.1
kg), and measured (straight length from tip of nose to tip
Plasma was stored in cryovials in liquid nitrogen on
board and was maintained at –20°C on shore until
processed in the laboratory. Plasma chemistry analyses
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Veterinary Clinical Pathology
Vol. 31 / No. 3 / 2002

Harms, Ross, Segars
were performed by automated bichromatic spectropho-
tometry (albumin, calcium, phosphorus, total protein
[TP], urea nitrogen, bicarbonate and glucose concentra-
tions, and aspartate aminotransferase [AST], lactate
dehydrogenase [LDH], and creatine kinase [CK] activi-
ties) and ion selective electrode (sodium, potassium, and
chloride concentrations) on a Roche/Hitachi 912 Clinical
Chemistry System (Roche Diagnostics, Indianapolis,
Ind, USA). Globulins concentration, albumin/globulin
(A/G) ratio, and anion gap ([Na+ +K+] – [Cl- + bicarbon-
ate]) were calculated. Plasma osmolality was measured
using a freezing point osmometer (Advanced Micro-
Osmometer, Model 3MO, Advanced Instruments Inc,
Needham Heights, Mass, USA).
Statistical analyses were performed with a commer-
cial software package (JMP 3.0.1, SAS Institute, Cary,
NC, USA). Data were tested for normality by the
Shapiro-Wilk W-test. Data for weight, length, and AST
and CK activities were not normally distributed; there-
fore, all summary statistics were reported as median,
lower (25%) and upper (75%) quartiles, and minimum-
maximum values, and nonparametric methods were
used for comparisons, except for correlations of TS and
TP concentrations. A Wilcoxon rank-sum test was used
to compare values by sex, boat, and presence of hemol-
ysis, and the Kendall tau b was used to examine correla-
tions by weight and length. A 1-way ANOVA was used
to determine the correlation between TS and TP values.
Statistical significance was set at P <.05. Data were
reported in both standard and SI units.
7.5
7.0
6.5
6.0
2.0
2.5
3.0
3.5
4.0
4.5
Total protein (g/dL)
Figure 1. Bonnethead shark total protein (TP) and total solids (TS)
concentrations were significantly correlated (1-way ANOVA, n = 24,
R2 = .88, P<.0001). The equation for the fitted line is TS = (1.006
ϫTP)+3.318.
tau b=–.42, P=.005; weight: tau b=–.44, P=.006). However,
CK activity was significantly higher in bonnethead plas-
ma collected on boat A than in that collected on boat B
(boat A: median [minimum-maximum] 215 [75-725] U/L;
boat B: 46 [18-68] U/L; Wilcoxon rank sum, P < .0001).
Phosphorus (boat A: 9.4 [7.3-12.7] mg/dL; boat B: 8.2 [5.9-
10.4] mg/dL; P = .049) and osmolality (boat A: 1110 [1088-
1139] mOsm/kg; boat B: 1077 [1056-1094] mOsm/kg; P =
.0001) also were significantly higher in plasma from
bonnetheads collected by boat A. Lighter and shorter
bonnetheads were collected on boat A (2.9 [2.0-5.0] kg,
90 [80-105] cm) compared with boat B (7.9 [3-10] kg, 114
[89-124] cm) (P = .0008 for both weight and length). TP
and TS concentrations were significantly linearly corre-
lated (R2 = .88, P < .0001) and were related by the fitted
line TS = (1.006ϫTP) + 3.318 (Figure 1).
Results
Twenty-four bonnetheads were collected, of which 13
were male, 7 were female, and 4 were of unrecorded sex.
Median weight and length (quartiles, minimum-maxi-
mum) were 3.2 (2.8, 7.2; 2.0-10.0) kg and 94 (88, 114; 80-
124) cm, respectively. PCV and plasma chemistry values
were tabulated (Table 1). In all cases, creatinine concen-
tration was <0.1 mg/dL and therefore was not included
in the Table. All values for albumin and most for bicar-
bonate (n=21) fell below the reliable limit of detection of
the chemistry analyzer (1.0 g/dL for albumin, 5 mmol/L
for bicarbonate). The analyzer-determined values were
used to calculate A/G ratio and the anion gap, respec-
tively, for these samples, while recognizing the inherent
uncertainty beyond simply indicating very low concen-
trations. Slight hemolysis was noted in 9 of 24 samples
but there was no significant difference between samples
with and without hemolysis for any measured analyte.
No significant differences in weight, length, or
blood values were evident between sexes. Only CK
activity differed according to animal size, with smaller
bonnetheads having higher CK values (length: Kendall
Discussion
The collection method employed in this study, ie, trawl-
ing followed by physical restraint of sharks out of water,
would be expected to cause some alterations in blood
chemistry values from those of sharks in a normal
swimming pattern. However, for collecting blood from
wild sharks, some capture stress, either by net or by
hook and line, is unavoidable. Blood collection of captive
fish is not much different, usually involving net capture
and out-of-water sampling. In captivity, the options exist
for chemical sedation prior to removal from water, or
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Plasma Biochemistry of Bonnethead Sharks
placement of indwelling catheters to permit blood col-
osmolality. Differences in bonnethead plasma CK val-
ues and osmolality between the 2 boats suggested pos-
sible differences in handling, although they could also
have reflected differences in the populations sampled.
In a capture and transport study on dusky sharks, both
CK and osmolality values increased with time in sharks
subjected to hook and line capture followed by close
confinement for transport.8 Smaller bonnetheads, which
came predominantly from boat A, tended to have high-
er plasma CK values. Higher CK values could have re-
sulted from more struggling by smaller individuals,
which was the subjective judgement of one of us (T. R.).
Potassium values were higher than those reported pre-
viously for sharks5 and could be related to capture
stress8 (see Haulena et al26 for a contrary observation) or
difficult venipuncture. However, no differences were
noted between boats nor were differences detected
based on the presence or absence of slight hemolysis.
The higher TS values determined by refractometry
as opposed to TP concentration determined by the
chemistry analyzer agreed with the findings in a previ-
ous report6 but differed from the 2ϫ to 3ϫ higherTS val-
ues suggested in that report. In the present study, TS
concentration differed from TP concentration by a near-
ly constant amount, with a slope close to 1, reflecting a
stable contribution of urea nitrogen and trimethylamine
oxide to the TS value.
lection while the fish remains in water. However, chem-
ical sedation with tricaine methanesulfonate causes
increases in PCV, blood glucose and potassium concen-
trations, and urinary electrolyte loss in teleosts,21 and
placement of indwelling catheters requires prior
surgery.22 Specifying the collection method and utilizing
practical methods are key to establishing useful blood
chemistry reference intervals.
Electrolyte concentrations and osmotic balance dif-
fer considerably between elasmobranchs and teleosts.23,24
Marine teleosts maintain plasma osmolality values
approximately 33% that of seawater through a combina-
tion of a relatively impermeable skin barrier and active
transport of electrolytes across the gills. Marine elasmo-
branchs osmoconform, or nearly so, having plasma os-
molality slightly hyperosmotic to seawater, but they still
maintain electrolytes at considerably lower concentra-
tions than those of the surrounding seawater. These
fishes maintain these low concentrations through high
plasma urea and trimethylamine oxide concentrations,
while excreting excess electrolytes through the rectal
gland.23 Urea is essential to normal physiologic function
in marine elasmobranchs.24 Although urea is the prima-
ry nitrogenous waste product, it is also actively reab-
sorbed in the renal tubules to support plasma osmolali-
ty. Thus, reduced urea concentrations may reflect either
hepatic disease (decreased urea production) or renal
disease (increased urea loss).
Blood chemistry values in this study were generally
consistent with existing published blood chemistry val-
ues for other sharks, although strict comparisons are
difficult because of variations in species, sampling con-
ditions, sample sizes, and laboratories.5-9 Anion gaps in
trawled bonnetheads were low, even negative, as com-
pared with values reported for 2 freshwater teleost
species, hybrid striped bass and red pacu (median
[quartiles]: 17.2 [15.8, 20.6] mmol/L and 5.1 [6.9, 9.0]
mmol/L, respectively),1,25 because of chloride ion con-
centrations that were usually higher than sodium ion
concentrations. Bicarbonate contributed little to anion
gap values, which were <5 mmol/L, consistent with a
previous report for dusky sharks, Carcharhinus obscurus.8
Although anion gap values were not specifically report-
ed in other shark blood chemistry reports, many shark
species may have a positive anion gap based on a sodi-
um ion concentration that exceeds that of chloride.5,6,9
However, the higher chloride ion concentrations report-
ed here have been observed previously in bonnetheads7
and dusky sharks.8
Virtually no work has been published relating
changes in blood chemistry values to disease in any
elasmobranch species.6 Values reported here will be use-
ful for establishing those relationships and evaluating
the health status of bonnetheads in wild and captive
research conditions and in exhibits. ◊
Acknowledgments
The authors thank Greg Lewbart, David Whitaker, Bruce Stender,
the crews of the trawlers, and the staff of the Clinical Pathology
Laboratory at the NCSU-CVM.This project was supported by the
South Carolina Department of Natural Resources and the state of
North Carolina. ◊
©2002 American Society forVeterinary Clinical Pathology
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